crystals
Article
A Personal History of Using Crystals and
Crystallography to Understand Biology and
Advanced Drug Discovery
Tom L. Blundell
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK;
tlb20@cam.ac.uk
Received: 10 July 2020; Accepted: 27 July 2020; Published: 5 August 2020






Abstract: Over the past 60 years, the use of crystals to define structures of complexes using X-ray
analysis has contributed to the discovery of new medicines in a very significant way. This has been in
understanding not only small-molecule inhibitors of proteins, such as enzymes, but also protein or
peptide hormones or growth factors that bind to cell surface receptors. Experimental structures from
crystallography have also been exploited in software to allow prediction of structures of important
targets based on knowledge of homologues. Crystals and crystallography continue to contribute to
drug design and provide a successful example of academia–industry collaboration.
Keywords: proteins; crystals; ligand binding; drug discovery; academia–industry collaboration
1. Discovering Crystals and Crystallography
My discovery of “crystallography” and “drug discovery” as research themes occurred in 1962.
I was enrolled to do a degree in Natural Sciences with a focus on chemistry but my broad interests
were in research underpinning new developments in medicine. I was also involved in radical politics
and race and gender diversity issues. I had heard that Dorothy Hodgkin had a research team that was
multidisciplinary, multinational and gender balanced in a way that I had not previously seen in Oxford.
A colleague suggested that we apply for a multidisciplinary course that was run for undergraduate
students as an option in the Laboratory of Crystallography where Dorothy was based. We were
accepted and were not disappointed!
The discussions in the breaks and over lunch were about all elements of science, politics and life
in general. The work in Dorothy’s laboratory had medical themes, including vitamin B12, penicillin
and especially insulin, where her research was relevant to long-lasting insulins [1]. However, she
was a crystallographer and progress depended on obtaining crystals in order to define structures and
understand functions. I decided this was a great training whether I ended up in academia, politics,
science research or industry!
Two years later, I had to decide on my fourth-year research—equivalent to a Master’s degree.
At the request of my tutor, Jack Barltrop, I visited Dyson Perrins Laboratory, the home of organic
chemistry in Oxford for most of the past century. However, although I had enjoyed discussion in our
weekly tutorials with Barltrop, I did not fall in love with the experimental organic chemistry in the lab.
Having discovered the Laboratory of Crystallography in a one-term undergraduate project, with its
amazing multidisciplinary and multinational culture, I ran down South Parks Road in Oxford to the
Laboratory and pleaded with them to accept me there.
After a PhD with Tiny Powell learning the techniques, I moved into Dorothy Hodgkin’s laboratory
in 1967 to study crystals of insulin, with the understanding that the knowledge would be useful
in drug discovery. There, I became a member of her group involving Guy Dodson (New Zealand),
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Crystals 2020, 10, 676 2 of 27
Eleanor Dodson (Australia), and Margaret Adams (UK), to be joined by M Vijayan (India) the next year,
Ted and Heather Baker (New Zealand), and Liang Dongcai (China), together with other visitors from
the USA, Argentina, India, Japan, parts of Africa and elsewhere—a truly amazing environment. The
insulin group relocated soon after to Zoology to join Professor David Phillips, later Lord David Phillips,
who had moved from the Royal Institution to take up a new Chair of Molecular Biophysics in 1967.
Dorothy Hodgkin had left-wing views and her husband was a member of the Communist Party
and author of some influential books on Africa that I had read earlier as a student. Nevertheless, she had
very good relationships with pharmaceutical companies, including Novo in Denmark, Wellcome
in the UK and Eli Lilly in the USA—all involved in insulin research for the treatment of diabetics.
The companies provided us not only with valuable pure insulin samples, but also very exciting
discussions about the underlying basic science of insulin crystallisation. The advantage to pharma
companies of using the very small crystals of insulin for treatment of diabetes was that an injection
once a day sufficed, as they had a very slow rate of dissolution in the bloodstream. Company scientists,
in particular Jørgen Schlichtkrull at Novo in Denmark [2], had spent a huge amount of time refining
the conditions for crystallisation and I was amazed by this focus on what seemed to be a very academic
approach to the subject, but it led to new long-lasting insulin forms such as 4Zn-insulin crystals. Thus,
I was introduced to a laboratory in structural biology—the term was not used at that time—using
crystals in my research. The environment was multidisciplinary, having physicists, chemists and
biologists in the team, but also gender balanced and multinational. This became my model for how
science should be. Crystals became the centre of my research life.
2. Solving the Crystal Structure of Insulin
Dorothy Hodgkin began researching on insulin after working with J.D. Bernal in the early 1930s
for her PhD. She had finished her first degree in Chemistry in Oxford and then moved in 1932 to
work in Cambridge, attracted by the research topic of crystallography but also by the charismatic
multidisciplinary approach that J.D. Bernal had to science. In 1934, Bernal received beautiful crystals of
pepsin from Nobel Laureate Theodor Svedberg in Uppsala, Sweden, to see whether they would diffract
X-rays [3]. The first experiments appeared negative but, after a few moments of depression, Bernal,
who was also working on the structure of liquids including water, realised that the crystals should
be kept in an aqueous environment where they would remain stable. He and Dorothy enclosed the
crystals within a thin-walled capillary sitting in a small pool of mother liquor with a reservoir either
side but sealed at each end of the capillary. To their surprise and pleasure, they found an amazing
diffraction pattern, confirming that water was required for retention of protein architecture. Bernal
phoned up the Editor of Nature and the paper was published within a couple of weeks [4]. Dorothy
and J.D. Bernal, known as “Sage”, wrote: “Now that a crystalline protein has been made to give X-ray
photographs, it is clear that we have the means of checking them, by examining the structure of all
crystalline proteins, arriving at far more detailed conclusions about protein structure than previous
physical or chemical methods have been able to give”. The optimism was justified but the time
scales were much longer than they had envisaged. Dorothy returned to Oxford in 1934 but obtained
insulin crystals from Beecham Group plc, a British pharmaceutical company, who were producing the
material for treatment of diabetics. Although Dorothy managed to get diffraction patterns of insulin [5],
the structure proved a challenge.
When I arrived fulltime in the Laboratory of Crystallography to start my PhD in 1964, it was
announced that Dorothy Hodgkin had been awarded the Nobel Prize. This was in fact for the crystal
structures of penicillin and vitamin B12, but there was a general recognition of the progress she had
made with insulin and the expectation that the crystal structure would soon be solved. After Marjorie
Harding, Beryl Rimmer and Sylvia Sheat in the 1950s, Guy Dodson, Eleanor Dodson and Margaret
Adams were continuing to work on it in 1964 and produced some heavy atom derivatives to use in
isomorphous replacement and anomalous scattering.
Crystals 2020, 10, 676 3 of 27
In 1965, there were already crystal structures from Cambridge including haemoglobin from Max
Perutz [6,7] and myoglobin from John Kendrew and colleagues describing the iron-containing haem
group which facilitates oxygen binding [8]. Michael Rossmann, Richard Dickerson and David Davies
from the Cambridge laboratory went on to make major breakthroughs in new labs in the US and Bror
Strandberg in Scandinavia, while David Phillips and Tony North moved to solve the structure of hen
egg-white lysozyme at the Royal Institution in London [9]. This suggested a mechanism of catalytic
activity, the first in three dimensions. Further insights into the mechanism of lysozyme were obtained
from structures of some crystalline lysozyme-inhibitor complexes determined by X-ray analysis at 6 Å
resolution by Louise Johnson and David Phillips [10]. In 1966, I attended a presentation from David
Phillips at the Royal Institution, where he had the sequence of hen egg-white lysozyme hanging from
the ceiling, and the 3D model of the enzyme on the ground in front of him. However, the presentation
by Louise Johnson showing inhibitor binding was equally stunning! It was a day that influenced my
future career strongly!
Dorothy’s structure of vitamin B12 inspired me! My PhD from 1964 to 1967 on crystal structures
of other metal complexes with Tiny Powell ensured that I was trained in the basic techniques. I joined
Dorothy Hodgkin’s group that was next door to work on insulin in 1967. I immediately found myself
thinking about which metal ions might be used to label the insulin more satisfactorily than had been
achieved to that time. Already, lead ions (Pb2+) had been used in an attempt to replace the zinc ions
(Zn2+) in insulin hexamers but this had led to some confusion, due to the fact that Pb2+ acted as an
A-type metal, binding oxygen ligands, whereas Zn2+ was B-type. It was not surprising therefore that
it had high occupancy at a further site near carboxylate sidechains close to the 3-fold axis of the 2
Zn insulin hexamer but half-way between the two zinc ions on the 3-fold axis of the rhombohedral
crystals. I sought to find other metal ions that might bind without displacing the zinc ions. I decided
to try uranyl ions, which I knew would bind to acid groups and see whether I could find a further
useful derivative. This soon led to a low-resolution structure based on the uranyl lead and mercury
derivatives [11].
The low-resolution image excited me and showed how the two zinc ions coordinated three dimers
in the zinc insulin hexamer. It led to an invitation to give a talk at a meeting on protein crystallography
in the US later in 1969. I prepared slides of the low-resolution maps of the crystal structure but left
Oxford soon after 2.8 Å resolution maps were calculated and as the modelling of the zinc-insulin
hexameric structure was ongoing. I gave the talk on the low-resolution insulin crystal structure in
Chicago and then moved to Long Island, where the 1969 International Congress of Crystallography
was taking place at Stony Brook University, New York. I met with Dorothy and discussed the maps
that had already been sent to me with the current molecular interpretation and modelling. As I walked
with Dorothy from our hotel to the International Congress where several thousand participants were
expected, the organiser of the conference came alongside us and started talking to Dorothy. He said
that the plenary keynote lecturer, who was due to talk about the materials retrieved from the moon in
a recent mission, had not been able to contribute due to the need to keep them safe-guarded to avoid
infections that might arise from their release into the laboratory. He had heard that Dorothy’s group
had just solved the crystal structure of insulin and then asked her to give the plenary lecture on insulin
in place of the moon rock. Dorothy was aware that I had prepared slides on the low-resolution crystal
structural information and had just given a talk on the new results. To my surprise she said, “I am
happy to give a talk. I will say a few words and then Tom can talk about the structure in detail”.
This was a day or so before the lecture was due. I reviewed the slides that I had for a traditional
kind of projector. I then very quickly used the material I had on the high-resolution structure to produce
some new slides at the conference centre for a modern projector, and also some overlays of the electron
density map that had been produced at high resolution for insulin crystals. This was an all-night job
with no sleep. I required three kinds of projectors. I went back to the Conference Centre and persuaded
them that, as it was such a huge lecture theatre and a very wide screen, they should allow me to use all
three in parallel. I did not have time to discuss what I was going to say with Dorothy in detail but
Crystals 2020, 10, 676 4 of 27
I confirmed that I was prepared to follow her. Dorothy went up onto the stage and introduced the
topic and said who had been involved and how she had worked on this project since she first obtained
crystals in 1934. She then passed over to me to give the main part of the presentation!
I began with a slide of the crystal electron density on one projector, another of the new insulin
structure prepared overnight on a second projector and on the third overhead projector, I wrote some
music—the tune that Dorothy hummed when she was excited by results. I then began my talk by
announcing that this was the crystal structure of insulin derived from these beautiful electron density
maps, some of the first structures ever defined by X-ray crystallography, and this is the tune that
Dorothy always hummed, probably for the previous 35 years, when she saw the beautiful electron
density maps. My talk was received well, with excitement and appreciation of what the Hodgkin
group had achieved. However, when I met Dorothy afterwards, she said to me, “Tom I really didn’t
know I hummed that tune when I was excited”.
The crystal structure defined the architecture of the insulin hexamer, with three dimers of insulin
coordinated by two zinc ions on the 3-fold axis of the rhombohedral crystals in spacegroup R3 (Figure 1).
The dimers exhibited a pseudo 2-fold axis perpendicular to the 3-fold axis.
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I began with a slide of the crystal electron density on one projector, another of the new insulin 
structure prepared overnight on a second projector and on the third overhead projector, I wrote some 
music—the tune that Dorothy hummed when she was excited by results. I then began my talk by 
announcing that this was the crystal structure of insulin derived from these beautiful electron density 
maps, some of the first structures ever defined by X‐ray crystallography, and this  is the tune that 
Dorothy always hummed, probably for the previous 35 years, when she saw the beautiful electron 
density maps. My  talk was received well, with excitement and appreciation of what  the Hodgkin 
group had achieved. However, when I met Dorothy afterwards, she said to me, “Tom I really didn’t 
know I hummed that tune when I was excited”. 
The crystal structure defined the architecture of the insulin hexamer, with three dimers of insulin 
coordinated by two zinc ions on the 3‐fold axis of the rhombohedral crystals in spacegroup R3 (Figure 
1). The dimers exhibited a pseudo 2‐fold axis perpendicular to the 3‐fold axis. 
   
Figure 1. The structure of 2 Zinc‐insulin hexamers. A. Rhombohedral crystals used in X‐ray analysis 
and treatment of diabetes. B. An X‐ray diffraction pattern from the rhombohedral crystals, illustrating 
the 3‐fold axis of space group R3. C. Structure of the 2 Zinc‐insulin hexamers comprising six insulin 
molecules  and  2  zinc  ions.  The  hexamers  of  approximate  32  symmetry  are  viewed  down  the 
crystallographic 3‐fold axis that relates three dimers, each of which have two protomers related by an 
approximate 2‐fold axis symmetry, perpendicular to the 3‐fold axis [12,13]. D. Storage granules in ß‐
cells of the pancreas, which also contain 2 Zn‐insulin hexamers and resemble the crystals used for 
structure determination’ [14]. ‘ 
Soon after we had  returned, Dorothy phoned  the Editor of Nature  to say  that 35 years after 
starting work on  insulin,  the crystal structure was at  last solved. A couple of weeks  later,  it was 
published in Nature [12]. The insulin structure was further refined and the Hodgkin group produced 
a more  detailed  paper  in  1971  [13]. My  own  interests  then were  very much  to  understand  the 
relationship of structure, chemistry and biological activity of insulin. Guy Dodson, Dan Mercola and 
I worked hard at a long review, where we related the structure to the biology in detail and this was 
published in a Advances in Protein Chemistry in 1972 [14]. We discussed the role of the small crystals 
of  zinc‐insulin  hexamers  in  biology  as  a  storage mechanism  of  insulin  in  the  beta‐granules  in 
evolution  (Figure  1). Related  crystals had been used by pharma  since  the  1930s  to  treat diabetic 
patients. We also discussed  the dissociation of  the hexamer  to allow  the monomer  to bind  to  its 
receptor, using conservation of residues  in evolution  to suggest where  the binding site on  insulin 
A B
C D
Figure 1. The structure of 2 Zinc-insulin hexamers. (A) Rhombohedral crystals used in X-ray analysis
and treatment of diabetes. (B) An X-ray diffraction pattern from the rhombohedral crystals, illustrating
the 3-fold axis of space group R3. (C) Structure of the 2 Zinc-insulin examers comprising six
insulin molecules and 2 zinc ions. The hexam rs of approximate 32 sym etry are viewed down the
crystallographic 3-fold axis that r lat s thr e dimers, each of which have two protom rs related by an
approximate 2-fold axis symmetry, perpendicular to the 3- old axis [12,13]. (D) Storage granules in
ß-cells of the pancre s, which also contain 2 Zn-insulin hexamers and resemble he crystals sed for
structure determination [14].
Soon after we had returned, Dorothy phoned the Editor of Nature to say that 35 years after starting
work on insulin, the crystal structure was at last solved. A c uple of weeks later, it was published
in Nature [12]. The insulin structure was further refined and th Hodgkin gr up produced a more
detailed paper in 1971 [13]. My own interests then we e very much to understand the elationship of
structure, chemistry and biological activity f i sulin. Guy Dodson, Dan Mercola and I wo ked hard
at a long review, where we related the structure to the biol gy in detail and this was published in a
Advances in Protein Chemistry in 1972 [14]. We discussed the role of the small crystals of zinc-in ulin
hexamers in biology as a storag mechani m of insulin in th beta-granules in volution (Figure 1).
Crystals 2020, 10, 676 5 of 27
Related crystals had been used by pharma since the 1930s to treat diabetic patients. We also discussed
the dissociation of the hexamer to allow the monomer to bind to its receptor, using conservation of
residues in evolution to suggest where the binding site on insulin might be. For me, this was a further
exciting development and finally convinced me that I ought to stay in crystallography but relate it
firmly to biology.
3. Politics in the City of Oxford
In parallel to science and music, I had become, while an undergraduate in Oxford in 1963, Chair of
the Joint Action Committee Against Racial Intolerance, and then later, when I had graduated and was
doing my PhD, co-founding a City of Oxford organisation, known as Oxford Committee for Racial
Integration. A few years before, very large numbers of West Indian and Bangladeshi immigrants
were recruited to the Morris Motor and Pressed Steel companies in Oxford. There was an urgent
need to have some activity in the city to reassure the immigrant population that they were welcome.
With Michael Dummett, a very distinguished Professor of Philosophy, and Anne Dummett, his wife,
who was politically active in the Liberal Party, several trade union leaders working in Morris Motors
and Pressed Steel and a socially conscious clergyman, the new Oxford Committee for Racial Integration
was established. This involved me heavily in politics of Oxford. I discovered that Oxford was much
more than the centre where undergraduates lived in their colleges, did rowing on the river and multiple
sports on the playing fields. There were also large working communities in East Oxford, with extensive
housing estates that had grown to accommodate workers in the motor industry.
I was then asked by the Labour Party to stand for the City Council in St. Clements Ward, in
East Oxford, close to the centre, where some immigrants had moved and which had previously been
a Conservative stronghold. I campaigned on an environmental policy of preserving the centre of
Oxford, making it a place where pedestrians and cyclists could be, and making sure that the traffic
was not using it as a through route. In 1970, I was the only Labour councillor elected that year and
I became a member of the City Council Planning Committee and interested in developing a new
policy. This involved not only conserving the traditional centre but also developing communities with
improved local environments. This became part of the Oxford City Labour Party manifesto and, in the
following years, we won an increasing majority, so that we took control of Oxford in 1972. We were
then able to pedestrianise the centre of Oxford and preserve many of the traditional areas. Cars were
banned from going along the High Street, directly through the city centre, and instead routes were
limited to a smaller number of cars directed alongside a road next to Magdalen College wall to go from
East towards the North or West.
We had hoped to stop everything but buses, police and ambulances in terms of motorised vehicles
going across Folly Bridge and Magdalen Bridge, but I was called down to Westminster to meet the
Conservative Minister in charge of planning, appropriately named Keith Speed. He announced, “Blundell
you may be in charge of the centre of Oxford planning, but I am in charge of road planning in the UK,
including routes through city centres! You cannot block cars going over the Folly and Magdalen bridges”.
However, we did manage to maintain the pedestrianisation policy and important conservation areas
have remained restricted from motorised transport ever since. Nobody has changed it!
In general, I found huge support for the environmental changes that we developed in the Oxford
City Centre from the general public and indeed from other parties as well as the Labour Party.
However, politics is much more complicated than science and one can have completely objective
arguments for making different decisions that are contradictory of each other. I found myself supporting
environmental developments but also realising that working people in Oxford needed new housing
near the centre. This demonstrated that, even within some areas of Oxford that I had hoped to preserve
from further large building projects, we had to allow house building to avoid long travelling for
Oxford working people. We did manage, however, to make North Oxford a Conservation Area,
even though it had no listed buildings; rather we justified it on the basis of its impressive townscape.
This policy controlled building in the large back gardens of many of the beautiful homes in North
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Oxford. However, the challenge between environment, industry, housing, travel and other aspects of
people’s lives are real and I realised that politics was much more difficult than science.
4. World Travel for Crystallising My Ideas
Dorothy Hodgkin was very sympathetic to my doing something political but she was certainly
worried that I was spending a lot of time from mid-afternoon onwards in politics and also still running
a modern jazz group in the nights, albeit occasionally. My first wife, and mother of my son Ricky,
was upset about the complexity of my life and our marriage broke up. I decided that I had to have a
thinking period.
I, therefore, used several months in 1972 to leave Oxford, with an initial objective of attending a
meeting in Japan. I first travelled on the train to Moscow in order to join the Trans-Siberian railway.
I thought I would have time to think over the options in my life—to crystallise my ideas during travel to
the Far East. This was quite a challenge, as I found myself in a small carriage with no skills in Russian
language that would allow a conversation during the seven days of travel to Khabarovsk. I could not
reach Vladivostok because it was closed to foreign travellers for military reasons. I eventually caught
a plane travelling to Japan to attend and speak at the Ninth Congress of the International Union of
Crystallography 26 August–7 September, 1972, in Kyoto.
I stayed for a while, making new Japanese friends before travelling on through Hong Kong,
to Bangkok in Thailand to Calcutta in India. There, I was invited to give a talk on crystallography
of insulin at one of the institutes before moving down to Bangalore to meet up with Vijayan,
Siv Ramaseshan and other crystallographers whom I had met when they had worked with Dorothy
Hodgkin in the Laboratory of Crystallography in Oxford over the previous eight years. The main
features of my stay in Bangalore were multiple discussions and a lecture at the Indian Institute of
Science focusing on the future uses of crystallography. I also took lessons on the veena and purchased
one of these very large string instruments. I learnt a lot about Indian music but, after a few weeks,
I decided I had better to return to Oxford. Travel was a challenge, as I had to find a further seat for my
veena next to me, as it would have been clearly smashed if I had checked it in. On my return to Oxford,
I found a veena player amongst the Indian community who continued to give me weekly lessons.
On my journey, I had decided to leave Oxford and to give up my politics and jazz. In discussion
with Dorothy when I returned, she suggested that I should move to Sussex University, close to where I
was brought up. Within one day, a University Lectureship was arranged and laboratory space allocated
in the new building in the School of Biological Sciences just north of Brighton. I quickly applied for
and was awarded a grant to work on insulin and glucagon structure, evolution and function from
the Government Science Research Council. I was really lucky! There was already crystallography in
Ron Mason’s laboratory in Inorganic Chemistry—where he had been appointed in 1971—but none
in biology! I searched for the whereabouts of an ex-undergraduate student I had taught in Oxford,
Ian Tickle, who had been one of my best tutees in Oxford. By then, he had become a well-trained
crystallographer, moving for his PhD to the Netherlands. He agreed to return in 1973. Together with
two very bright PhD students that I had been teaching as undergraduates in Oxford, Rod Pullen and
John Jenkins, we moved to Sussex in 1973. I spent the time in between sorting out various policies and
activities on the City Council. Steve Wood applied to do a PhD with me and joined us in Sussex as I set
up a new group in an almost empty building that had just been constructed.
5. Crystallography in Biological Sciences at Sussex University
With a new focus on science and few other distractions as well as a superb young and enthusiastic
research team, we got to work very quickly. I bought a house in Lewes, a few miles from Sussex
University at Falmer on the outskirts of Brighton. I decided to start new biological crystallographic
projects, the first of which was to look at the crystal structure of glucagon, where crystals were soon
produced (Figure 2).
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Figure 2. The structure of  the glucagon  trimer was defined by X‐ray diffraction of cubic glucagon 
crystals [15]. 
The interest here was that whereas insulin puts down sugar levels in blood circulation, glucagon 
puts  them up. They are  the “yin and yang” of blood  sugar  control, very much exemplifying  the 
ancient Chinese philosophy that opposite and contrary forces can act in complementary ways. Our 
paper was published  in Nature  in 1975  [15]. Glucagon, unlike  insulin, did not have a preformed 
structure in solution but rather assembled at the receptor. However, it formed trimers that assembled 
in crystals with cubic symmetry P213 as shown earlier by Murray Vernon King [16], reflecting the 
stable storage form in the secretory granules of pancreatic α‐cells. Our crystal structure demonstrated 
the  arrangement  of  the  trimers  that  also  occur  in  solution  (Figure  2).  The  glucagon‐receptor 
interaction  inferred was the first structurally defined example of “concerted folding and binding” 
that  I had come across.  It  triggered  research on  the  two complementary ways of peptide–protein 
interactions exhibited by polypeptide hormones and growth factors: the first involving preformed 
structures but often with small conformational changes on binding; the other involving a preformed 
receptor with a disordered flexible polypeptide in solution requiring concerted folding and binding 
to crystallise. It was also very apparent even at that stage something had to compensate for the loss 
of entropy when the peptide bound the protein and if so it likely had a very well‐defined binding 
site, suitable for drug discovery! 
The second line of investigation was to understand the requirements of insulin binding to its 
receptor. We did this in collaboration with the group of Helmut Zahn and Dietrich Brandenburg in 
Aachen Germany, who  synthesised novel  insulins,  and  Jorgen Gliemann and Stefan Gammeltoft 
from Denmark, who did the receptor binding analyses. This multidisciplinary team tested the ideas 
we  had  established  in Dorothy Hodgkin’s  laboratory  based  on  the  crystal  structure  of  insulin, 
bringing  together  chemistry,  biochemistry,  structural  biology  and  biological  assays  to  test  our 
hypotheses. The objective of course was to find more efficient insulin‐receptor binders that could be 
used clinically and may have greater affinity and longer half‐lives. This was also published in Nature 
[17]. 
The third manuscript to be published in Nature was a “think piece” focussing on whether the 
evolution of  insulin was Darwinian or due to selectively neutral mutation [18]. This exploited the 
extensive information about insulin, which was the first protein to be sequenced by Fred Sanger and 
for which there were in the 1970s many sequences from other species available. Our paper combined 
this with information of the 3D structures already defined by crystallography, in order to develop 
ideas about its evolution in terms of natural selection and multiple neutral amino‐acid substitutions, 
building on ideas of Motoo Kimura about evolutionary rates at the molecular level [19,20]. The article 
Glucagon trimer Glucagon crystals
Figure 2. The structure of the glucagon trimer was defined by X-ray diffraction of cubic glucagon
crystals [15].
The interest here was that whereas insulin puts down sugar levels in blood circulation, glucagon
puts them up. They are the “yin and yang” of blood sugar control, very much exemplifying the ancient
Chinese philosophy that opposite and contrary forces can act in complementary ways. Our paper
was published in Nature in 1975 [15]. Glucagon, unlike insulin, did not have a preformed structure
in solution but rather assembled at the receptor. However, it formed trimers that assembled in
crystals with cubic symmetry P213 as shown earlier by Murray Vernon King [16], reflecting the stable
storage form in the secretory granules of pancreatic α-cells. Our crystal structure demonstrated the
arrangement of the trimers that also occur in solution (Figure 2). The glucagon-receptor interaction
inferred was the first structurally defined example of “concerted folding and binding” that I had come
across. It triggered research on the two complementary ways of peptide–protein interactions exhibited
by polypeptide hormones and growth factors: the first involving preformed structures but often with
small conformational changes on binding; the other involving a preformed receptor with a disordered
flexible polypeptide in solution requiring concerted folding and binding to crystallise. It was also very
apparent even at that stage something had to compensate for the loss of entropy when the peptide
bound the protein and if so it likely had a very well-defined binding site, suitable for drug discovery!
The second line of investigation was to understand the requirements of insulin binding to its
receptor. We did this in collaboration with the group of Helmut Zahn and Dietrich Brandenburg in
Aachen Germany, who synthesised novel insulins, and Jorgen Gliemann and Stefan Gammeltoft from
Denmark, who did the receptor binding analyses. This multidisciplinary team tested the ideas we had
established in Dorothy Hodgkin’s laboratory based on the crystal structure of insulin, bringing together
chemistry, biochemistry, structural biology and biological assays to test our hypotheses. The objective
of course was to find more efficient insulin-receptor binders that could be used clinically and may have
greater affinity and longer half-lives. This was also published in Nature [17].
The third manuscript to be published in Nature was a “think piece” focussing on whether the
evolution of insulin was Darwinian or due to selectively neutral mutation [18]. This exploited the
extensive information about insulin, which was the first protein to be sequenced by Fred Sanger and
for which there were in the 1970s many sequences from other species available. Our paper combined
this with information of the 3D structures already defined by crystallography, in order to develop
ideas about its evolution in terms of natural selection and multiple neutral amino-acid substitutions,
building on ideas of Motoo Kimura about evolutionary rates at the molecular level [19,20]. The article
depended very much on our detailed knowledge of insulin storage as crystals of zinc-insulin hexamers
Crystals 2020, 10, 676 8 of 27
after processing of the proinsulin single chain to the A and B chains and the importance of the tertiary
structure of the monomer in receptor binding. It illustrated how residues were selectively conserved
not only to maintain and improve the three-dimensional structure of the protomer and interprotomer
contacts in the dimer and hexamer, but also to facilitate processing of the proinsulin to insulin and
interaction of insulin with its receptor. It demonstrated that much of the sequence change was likely
selectively neutral, but conserved areas indicated evolutionary restraints in storage and receptor
activation. The analysis provided one of the first detailed analyses of evolution of protein function from
primitive fish species through to more recently evolved mammals including human. This paper arose
very much from the discussions with John Maynard-Smith who had exciting ideas about evolutionary
change and Sydney Shall, who was involved in understanding insulin function, at Sussex University,
as well with Norman Lazarus who was involved in insulin research at The Wellcome Foundation
Ltd in Kent. The insulin analyses guided the re-design of insulins in the pharmaceutical industry,
indicating what was essential to activity and therefore what might be modified in drug discovery.
While still in Oxford, I had collaborated with Louise Johnson to write a review on protein
crystallography. Since working with David Phillips on the crystal structure of lysozyme, Louise had
moved to the laboratory of Fred Richards at Yale University for postdoctoral research in 1966. There,
she had worked on the crystal structure of another enzyme, ribonuclease. After her postdoctoral
year at Yale, she returned to the UK in 1967 and took up a post with David Phillips at Oxford in the
new Laboratory of Molecular Biophysics that accommodated both the Phillips and Hodgkin protein
crystallographic groups. Three years later, David Phillips asked Louise and myself to write a review
on protein crystallography, which we did by bringing together our knowledge from the lysozyme and
insulin structural work [21]. In Sussex, being away from the Oxbridge environment, I realised that
the knowledge of the protein crystallographic techniques to define structures was not fully recorded
or widely disseminated. Louise and I decided to write a monograph on protein crystallography.
This resulted in over 500 pages of detailed text, bringing together what we had learnt about the use of
crystals in the groups of David Phillips and Dorothy Hodgkin in the previous decade, as well as from
extensive collaborations not only with the US, but also Russia, China, India and elsewhere, where the
subject was quickly evolving [22].
6. Pepsin, Renin, HIV Protease and Structure-Based Drug Discovery
In the mid-1970s, I became aware that the original crystals of pepsin examined by Crowfoot and
Bernal had not led to a 3D structure of the pepsin family. The sequence of pepsin itself was published
by Tang et al. 1973 [23]. In 1975, I decided to start with endothiapepsin, a pepsin homologue from the
pathogenic fungus Endothia parasitica, classed as an acid or aspartic protease and which we quickly
crystallised and solved the structure [24]. The structure had a deep active site cleft, and a symmetrical
arrangement of the two catalytic aspartates located in the duplicated sequence Asp-Thr-Gly related by
the pseudo 2-fold axis in the structure, later found in pepsin (Figure 3). This led to further comparative
structural analyses of the many pepsin-like proteases including the aspartic protease from Rhizopus
chinensis (solved in the laboratory of David Davies in NIH) and the human chymosin [24,25].
In 1976, I had been appointed Professor and Head of the Crystallography Department in Birkbeck
College. Another aspartic protease, renin, was identified as a target for inhibiting angiotensinogen
cleavage leading to the hormone angiotensin, a peptide hormone that causes vasoconstriction and an
increase in blood pressure [26]. This led to discussions of using crystal structures in drug discovery of
antihypertensives, both in academia and in industry.
However, in parallel, we decided to investigate the pseudo-2-fold axis in the crystal structures
of these enzymes that extended beyond the active site residues shown in Figure 3. This led first to a
paper on their evolution through gene duplication from our group published in Nature in 1978 [27],
a collaboration with Jordan Tang, who had defined the pepsin sequence, and Mike James, another
crystallographer who had worked in the Hodgkin group but also working on the aspartic proteases.
We predicted that there must have been a dimeric evolutionary ancestor that consisted of two protomers,
Crystals 2020, 10, 676 9 of 27
each equivalent to one half of the aspartic proteases/pepsins that had subsequently evolved by gene
duplication and fusion.
Crystals 2020, 10, x FOR PEER REVIEW  9 of 27 
 
 
Figure 3. A. The aspartic protease fold found in pepsin and other aspartic proteases, showing a deep 
active site cleft. B. The symmetrical arrangement of the two catalytic aspartates in the active site of 
pepsins  located  in  the duplicated  sequence Asp‐Thr‐Gly  related by  the pseudo  2‐fold axis  in  the 
structure. 
In  1976,  I  had  been  appointed  Professor  and Head  of  the  Crystallography  Department  in 
Birkbeck  College.  Another  aspartic  protease,  renin,  was  identified  as  a  target  for  inhibiting 
angiotensinogen  cleavage  leading  to  the  hormone  angiotensin,  a  peptide  hormone  that  causes 
vasoconstriction  and  an  increase  in  blood  pressure  [26]. This  led  to discussions  of using  crystal 
structures in drug discovery of antihypertensives, both in academia and in industry.   
However, in parallel, we decided to investigate the pseudo‐2‐fold axis in the crystal structures 
of these enzymes that extended beyond the active site residues shown in Figure 3. This led first to a 
paper on their evolution through gene duplication from our group published in Nature in 1978 [27], 
a collaboration with Jordan Tang, who had defined the pepsin sequence, and Mike James, another 
crystallographer who had worked in the Hodgkin group but also working on the aspartic proteases. 
We  predicted  that  there must  have  been  a  dimeric  evolutionary  ancestor  that  consisted  of  two 
protomers,  each  equivalent  to  one  half  of  the  aspartic  proteases/pepsins  that  had  subsequently 
evolved by gene duplication and fusion. 
In  general,  these  developments  led  to  an  antagonistic  response  from  some  distinguished 
members of the academic community, who advised me not to focus on these “speculative” ideas but 
to  think more  about  crystallography. Although  I  respected  them  and  focused  on  experiment,  I 
continued to look for the ancestral dimeric protease of the pepsins/aspartic proteases predicted in the 
1978 Nature paper. It took six years before I realised in 1984 that it existed in the genome of HIV when 
the infectious agent was sequenced and AIDS became a pandemic.   
Lynn Sibanda and I built models of HIV protease in the lab but I was reluctant to try to publish 
them  in view of  the criticism  from colleagues. Rather, we started an experimental programme by 
producing  crystals  that  came  to  fruition only  in 1989  (Figure 4). Between 1984 and 1989,  several 
groups embarked on defining the crystal structures of the retroviral proteases with a focus on HIV 
protease as a drug target. A computational model was produced by colleagues Laurence Pearl and 
Taylor from our laboratory in Birkbeck [28] based on the aspartic protease evolutionary relationship. 
Later, X‐ray crystal structures were defined by Alex Wlodawer and coworkers for Rous sarcoma virus 
protease  [29] and  for HIV protease by Navia et al., 1989  [30], Wlodawer et al., 1989  [31] and  the 
Blundell laboratory [32].   
A B
Figure 3. (A) The aspartic protease fold found in epsi and other spartic proteases, showing a
deep active site cleft. (B) The symmetrical arrangement of the two catalytic aspartates in the active
site of pepsins located in the duplicated sequence Asp-Thr-Gly related by the pseudo 2-fold axis in
the structure.
In general, these developments led to an antagonistic response from some distinguished members
of the academic community, who advised me not to focus on these “speculative” ideas but to think
more about crystallography. Although I respected them and focused on experiment, I continued to
look for the ancestral dimeric protease of the pepsins/aspartic proteases predicted in the 1978 Nature
paper. It took six years before I realised in 1984 that it existed in the genome of HIV when the infectious
agent was sequenced and AIDS became a pandemic.
Lynn Sibanda and I built models of HIV protease in the lab but I was reluctant to try to publish
them in view of the criticism from colleagues. Rather, we started an experimental programme by
producing crystals that came to fruition only in 1989 (Figure 4). Between 1984 and 1989, several groups
embarked on defining the crystal structures of the retroviral proteases with a focus on HIV protease as
a drug target. A computational model was produced by colleagues Laurence Pearl and Taylor from our
laboratory in Birkbeck [28] based on the aspartic protease evolutionary relationship. Later, X-ray crystal
structures were defined by Alex Wlodawer and coworkers for Rous sarcoma virus protease [29] and
for HIV protease by Navia et al., 1989 [30], Wlodawer et al., 1989 [31] and the Blundell laboratory [32].
The resemblance of these putative ancestral dimers to renin suggested that inhibitors similar
to those of renins and other pepsin-like enzymes might be effective [34]. Further research in the
Blundell laboratory was developed collaboratively with Pfizer [35], with their work on the expression
and characterisation of HIV protease and ours on the structure; this collaboration was an exercise in
knowledge exchange and sharing between academia and industry and widely adopted by different
companies! By 1997, four successful AIDS antivirals (saquinavir from Roche Pharmaceuticals,
ritonavir from Abbot, indinavir from Merck and nelfinavir from Agouron) were on the market.
They demonstrated the importance of understanding the genome not only in terms of the functions of
gene products, but also of their crystal structures for use in structure-guided drug discovery, as recorded
recently in an excellent history of macromolecular crystallography and its fruits [36].
Crystals 2020, 10, 676 10 of 27
Figure 4. Evolution of pepsin family of aspartic proteases from an ancestral dimeric protease. (A)
Pepsin and other aspartic proteases are monomers with a pseudo 2-fold axis, shown by the red arrow
above, relating the two halves of the enzyme, each with the Asp-Thr-Gly motif, and suggesting a
dimeric ancestor [27]. (B) A modern day retroviral protease such as HIV protease has a 2-fold axis,
shown by the red arrow above, resembles the predicted dimeric enzyme, as first suggested by Toh et
al., 1985 [33], and confirmed experimentally by several groups experimentally to exist to the present
day in viral proteases [34–39].
Our efforts at structure-guided drug discovery in academia in the 1980s included applications
of protein crystallography and interactive computer graphics [37]. We continued to focus on crystal
structures of the inhibitor complexes of aspartic proteases using transition-state substrate analogues
(Figure 5) [38,39].
Crystals 2020, 10, x FOR PEER REVIEW  11 of 27 
 
 
Figure 5. The structural basis of design of antihypertensives targeting renin guided by knowledge of 
H bonding of substrate analogues (left‐hand side) and pocket occupation (right‐hand side). 
7. Four Decades of Crystals and Drug Discovery in Large Pharma   
Our work on crystallography of the aspartic protease inhibitors had already attracted attention 
in the pharmaceutical industry in the late 1970s. I was invited by Pfizer in Groton USA to discuss the 
renin–angiotensinogen structural work in relation to drug discovery, and was offered a contract as a 
scientific  advisor  in  1980.  I  soon  discovered  that  there was  also  a  very  exciting  drug  discovery 
research  programme  in  Pfizer  at  Sandwich  in  the UK.  Simon  Campbell  had  developed  a  very 
impressive, multidisciplinary and interactive group of scientists. I became a regular visitor to Pfizer 
at  Sandwich  and  to  the  laboratories  in Groton, Connecticut, which  in  the  1980s were  the major 
research centres. 
Simon  Campbell’s  radical  approach  to  drug  discovery  in  Sandwich  led  to  very  successful 
outcomes as recorded in an interview by Joanna Owens for Nature Reviews Drug Discovery much 
later in 2006 [44]). Amlodipine, initially approved by the FDA in 1987, continues to be a widely used 
drug for the treatment of high blood pressure and angina; it sells for $6 billion a year! Fluconazole, a 
broad‐spectrum antifungal agent, active by both the oral and the intravenous routes, was developed 
for the treatment of superficial and systemic infections. Viagra and Revatio contain the same active 
ingredient, sildenafil, which was designed to treat pulmonary arterial hypertension. Simon Campbell 
noted what appeared at first to be a challenge to the use of the drug, when  it was found to cause 
erections in male patients. He realised the opportunity and it became a major medication for use to 
treat erectile dysfunction, selling $2.3 billion a year! Simon was one of the first industrial researchers 
to be elected to the Royal Society and he was knighted in 2015. 
In parallel to my visits to Pfizer, I was invited to advise other companies including ICI in the 
UK, which later became Zeneca, and then merged with Sweden’s Astra to become AstraZeneca. I was 
also  heavily  involved  in  Celltech  Group  plc, which was  a  leading  British‐based  biotechnology 
business based in Slough. It was founded by Gerard Fairtlough in 1980 with finance from the National 
Enterprise Board—a Labour Party Initiative of which I was very supportive. I was a scientific advisor 
in the 1980s, while it was focused mainly on antibody production in Slough, with a smaller effort in 
small‐molecule drug discovery. Visits were always exciting, interactive and productive.   
I then had a break while I ran research councils between 1991 and 1996, first the Director General 
of the Agricultural and Food Research Council (AFRC) with the plan to merge with the biological 
sciences  funded within  the Science and Engineering Research Council  (SERC)  to become  the  first 
Chief  Executive  of  the  BBSRC  in  1994.  The  objective  was  to  bring  more  basic  bioscience  into 
agricultural,  food and biotechnology  industries,  in much  the  same way as MRC did  for medical 
research.   
Figure 5. The structural basis of design of antihypertensives targeting renin guided by knowledge of H
bonding of substrate nalogues (left-hand side) and pock t occupation (r ght-han side).
The approach found its most successful development with the renin inhibitor–aspartic protease
complex s defined b high resolution crystallography in our laboratory and in a collaboration with
Michael Szelke and published in Nature in 1987 [40]. However, we continued to use endothiapepsin
as an easier basis for crystal structural basis for drug discovery of renin inhibitors, for example in
Sali A. et al. [41,42]. In parallel, we developed discussions of the antiviral agents for the treatment of
AIDS using similar approaches [43], which described the crystal structure of HIV protease and various
antiviral agents for the treatment of AIDS.
Crystals 2020, 10, 676 11 of 27
7. Four Decades of Crystals and Drug Discovery in Large Pharma
Our work on crystallography of the aspartic protease inhibitors had already attracted attention in
the pharmaceutical industry in the late 1970s. I was invited by Pfizer in Groton USA to discuss the
renin–angiotensinogen structural work in relation to drug discovery, and was offered a contract as a
scientific advisor in 1980. I soon discovered that there was also a very exciting drug discovery research
programme in Pfizer at Sandwich in the UK. Simon Campbell had developed a very impressive,
multidisciplinary and interactive group of scientists. I became a regular visitor to Pfizer at Sandwich
and to the laboratories in Groton, Connecticut, which in the 1980s were the major research centres.
Simon Campbell’s radical approach to drug discovery in Sandwich led to very successful outcomes
as recorded in an interview by Joanna Owens for Nature Reviews Drug Discovery much later in
2006 [44]). Amlodipine, initially approved by the FDA in 1987, continues to be a widely used drug
for the treatment of high blood pressure and angina; it sells for $6 billion a year! Fluconazole,
a broad-spectrum antifungal agent, active by both the oral and the intravenous routes, was developed
for the treatment of superficial and systemic infections. Viagra and Revatio contain the same active
ingredient, sildenafil, which was designed to treat pulmonary arterial hypertension. Simon Campbell
noted what appeared at first to be a challenge to the use of the drug, when it was found to cause
erections in male patients. He realised the opportunity and it became a major medication for use to
treat erectile dysfunction, selling $2.3 billion a year! Simon was one of the first industrial researchers to
be elected to the Royal Society and he was knighted in 2015.
In parallel to my visits to Pfizer, I was invited to advise other companies including ICI in the UK,
which later became Zeneca, and then merged with Sweden’s Astra to become AstraZeneca. I was also
heavily involved in Celltech Group plc, which was a leading British-based biotechnology business
based in Slough. It was founded by Gerard Fairtlough in 1980 with finance from the National Enterprise
Board—a Labour Party Initiative of which I was very supportive. I was a scientific advisor in the 1980s,
while it was focused mainly on antibody production in Slough, with a smaller effort in small-molecule
drug discovery. Visits were always exciting, interactive and productive.
I then had a break while I ran research councils between 1991 and 1996, first the Director General
of the Agricultural and Food Research Council (AFRC) with the plan to merge with the biological
sciences funded within the Science and Engineering Research Council (SERC) to become the first Chief
Executive of the BBSRC in 1994. The objective was to bring more basic bioscience into agricultural,
food and biotechnology industries, in much the same way as MRC did for medical research.
In 1996, I returned to be a member of the main Board of CellTech. My time on the Board seemed
to be completely focused on mergers, first with Chiroscience plc, to become Celltech Chiroscience
before buying Medeva plc. In 2000, we bought other companies in Europe and eventually Oxford
Glycosciences in July 2003. However, in 2004, Celltech was acquired by UCB, a Belgian small pharma,
which I had not visited before and we become UCB Celltech. It was an interesting expansion but
nothing quite equalled the science and innovation of the early days of Celltech, when it had an
interactive and multidisciplinary culture not unlike the early days of Pfizer in Sandwich in the 1980s.
8. Software Underpinning Drug Discovery
In the 1980s, there was often no crystal structure available for new drug targets, on which to base an
understanding of function and develop structure-guided drug development. Our academic laboratory,
therefore, decided to develop modelling software for this purpose. The early papers focused on the
use of computer graphics for interactive modelling of the human and mouse renins [45], for which
energy calculations were used to optimise protein–ligand interactions. These studies led to a highly
cited review on prediction of protein structures and the design of novel molecules published in Nature
by Lynn Sibanda and myself, along with Mike Sternberg and Janet Thornton who had been recruited
to the Department in Birkbeck [46]. This focused on knowledge-based approaches, depending on
identification of analogies in secondary structures, motifs, domains or ligand interactions between a
protein to be modelled and those of known three-dimensional structure, often described as comparative
Crystals 2020, 10, 676 12 of 27
or homology modelling. Our approach underlined the importance of using multiple structures of
close homologues with good crystallographic resolution to model the tertiary framework and then
using rule based approaches to model insertions and deletions in loop regions, followed by energy
minimisation and molecular dynamics approaches. Our new software, COMPOSER, was described in
two papers. the first on the three-dimensional frameworks derived from a simultaneous superposition
of multiple structures and knowledge-based building of insertions and deletions [47], and the second
on the rules for replacement of side chains [48]. However, the development of these methods made us
all aware of the need for protein sequence/structure information Peter Murray-Rust coordinated as a
database published in Nature [49].
A major challenge with respect to comparative/homology modelling is to select the templates to
model the proteins, a process often called fold recognition. Andrej Sali had joined the laboratory to do
experimental work for drug discovery, as described above, but his skills in computer programming made
him keen to develop software as he became aware of the importance of good modelling procedures.
In 1989, we published an approach to the definition of topological equivalence in homologous
protein structures [50]. In parallel, others developed software addressing this challenge, including
David Eisenberg with a similar methodology involving inverse folding and local environments [51],
and software, using a new approach to protein fold recognition, known as THREADER by David Jones
in the laboratories of Janet Thornton and Willie Taylor [52].
In parallel, John Overington together with Andrej Sali in our laboratory investigated environment-specific
substitution tables, based on analysis of crystal structures, to evaluate sequence and structure
compatibility in evolution [53]. We also devised methods using environment-specific amino acid
substitution tables to assess tertiary templates for the prediction of tertiary folds [54]. This was followed
QSLAVE, software developed by Mark Johnson and John Overington for alignment and searching
for common protein folds using a data bank of structural templates [55]. However, the advance that
has had the greatest impact from our laboratory was a further approach to comparative modelling.
I had originally suggested we should exploit spatial restraints in a way that was similar to programs
developed in our lab for use in the refinement of crystal structures. However, Andrej Sali developed
ideas way beyond my first suggestion in his software MODELLER, which exploited spatial restraints
derived from the structures of homologues [56]. The original paper has at the time of writing been
cited more than 12,000 times!
In the following decade, we focused on software and databases that were useful for modelling,
for example JoY, a method for sequence-structure representation and analysis that facilitates the
alignment of proteins and understanding the structural homology. It depends on annotating local
environments of amino acids in the three-dimensional crystal structures and ensuring alignments are
compatible with amino acids occurring at the equivalent positions [57]. In parallel, we developed a
further database, HOMSTRAD, comprising alignments of protein sequences and crystal structures for
homologous families [58]. Ji-Ye Shi in the group, an amazing PhD student from China, wrote FUGUE,
which developed ideas, first exploited in QSLAVE, for sequence-structure homology recognition using
environment-specific substitution tables and structure-dependent gap penalties [59]. We also used the
local environment substitution tables developed earlier to devise software to predict the impacts of
amino acid substitutions.
Of course software is difficult to maintain and its success depends on having excellent collaborators
and students, but also on encouraging them to take away the software so as to develop and maintain it
as a centre of their research. The most convincing aspect of this has been in the work of Andrej Sali who
has maintained the Modeller software for nearly 30 years, ensuring its wide use and frequent citation.
9. Using Crystals to Explore Chemical Space for Drug Discovery
The multiple structures defined crystallographically allowed exploration of the chemical space of
the small molecules that will bind to a potential drug target, both computationally and experimentally.
In 1984, Peter Goodford founded a software company Molecular Discovery Ltd, working in the area of
Crystals 2020, 10, 676 13 of 27
drug discovery. Its aim was to develop software, initially GRID, which uses a probe to explore the
interaction of a small molecule with a protein of known structure [60]. The energy values are computed
at grid positions throughout and around the molecule. Various probes can be used including small
molecules with aliphatic, nitrogen, aiming donors and carboxyl and hydroxyl acceptors. Molecular
Discovery Ltd. developed a range of other software, which is widely used. This influenced a range of
software such as LIGSITE that provides automatic detection of potential small-molecule-binding Sites
in Proteins [61], Fpocket which uses Voronoi tessellation [62] and Pocket Depth [63], a depth-based
algorithm for identification of ligand binding sites in proteins. These and related approaches can
recognise up to 95% of binding sites in known protein–ligand structures and have facilitated use of
protein structures in drug discovery.
Another incredibly influential development in drug discovery was the work at Pfizer, Groton,
Connecticut, USA, by Chris Lipinski in 1997, who defined criteria such as solubility and permeability
for drug molecules in his Rule of 5 [64]. This was influenced by analyses of successful drugs, then on the
market, with respect to pharmacokinetics in the human body including ADME (Absorption Distribution
Metabolism and Excretion). The Rule of 5 requires the drugs have no more than 5 hydrogen-bond
donors or acceptors, no more than 5 rotatable bonds and a molecular mass less than 500 daltons as
well as an octanol water partition co-efficient log P not greater than 5. These restraints together with a
maximum of four rings led to a decrease from 1080 possible molecules down to 1063 compounds [65].
They emphasised the huge challenges in exploring chemical space by searching for molecules with
drug-like size of molecular weight 500 daltons. Awareness of the Rule of 5 in the industry has led to
very efficient drug discovery, particularly for human targets.
10. Crystallography and Fragment-Based Drug Discovery
In the 1990s, drug developers became increasingly aware that, even after adopting the Rule of 5,
a huge number of molecules of drug-like size remained in order to explore chemical space. This led to
the idea that smaller molecules of around 300 molecular weight, known as fragments, might be used,
as this would lead to increased promiscuity and require smaller libraries.
A fragment is around the minimum size required to bind to a so-called “hotspot”. A hotspot is
usually in a deep cavity on the protein surface, where there is a juxtaposition of polar and non-polar
regions, making water molecules “unhappy”. This arises from the fact that bound water molecules in
the apo-form will have lost rotational and translational entropy compared with their being in a water
environment. If a fragment binds at the hotspot the loss of entropy in binding in one orientation is
compensated by the gain of rotational and translational entropy, usually of several liberated waters.
We, together with collaborators from the Crystallographic Data Centre in Cambridge, have developed
more recently computational approaches to defining hot spots [66,67]. Once the fragment is bound in
a way that it has a clear orientation, it can be elaborated to establish further interactions within the
pocket or closely surrounding region without increasing the loss of translation and rotational entropy.
Fragments usually have millimolar binding and it is possible to maximise interactions of each step
in the elaboration of the fragment in order to gain nanomolar binding with a drug-like molecule of
approximately 500 daltons molecular weight.
This approach, now known as fragment-based drug discovery, was originally developed using
NMR approaches to monitor the fragment binding [68]. However, structure-guided methods using
crystallography are most efficient as they define the position of the fragment before growing it or
linking to other fragments. The use of X-ray crystallography in fragment screening (Figure 6) has
been pioneered in Astex [69,70]. Astex was founded by Harren Jhoti, Chris Abell and myself in 1999
and funded by Abingworth Investments, which I had advised for many years before. The laboratory
was established with three postdocs in the Blundell and Abell laboratories in the Departments of
Biochemistry and Chemistry in Cambridge University, where it was demonstrated that X-ray analysis
could define the fragment positions accurately with high-resolution X-ray methods. In parallel,
Harren Jhoti, who had been at GSK, established himself initially in the Grafton Centre, in the centre of
Crystals 2020, 10, 676 14 of 27
Cambridge to recruit his team, before moving to the Cambridge Science Park to establish the early
stage drug discovery effectively. Tim Haines, an entrepreneur funded by Abingworth, joined as CEO
and Harren initially was Chief Scientific Officer, but after five years became CEO.
Crystals 2020, 10, x FOR PEER REVIEW  14 of 27 
 
We,  together  with  collaborators  from  the  Crystallographic  Data  Centre  in  Cambridge,  have 
developed more recently computational approaches to defining hot spots [66,67]. Once the fragment 
is bound in a way that it has a clear orientation, it can be elaborated to establish further interactions 
within  the  pocket  or  closely  surrounding  region without  increasing  the  loss  of  translation  and 
rotational  entropy.  Fragments  usually  have  millimolar  binding  and  it  is  possible  to  maximise 
interactions of each step in the elaboration of the fragment in order to gain nanomolar binding with 
a drug‐like molecule of approximately 500 daltons molecular weight.   
This approach, now known as fragment‐based drug discovery, was originally developed using 
NMR approaches to monitor the fragment binding [68]. However, structure‐guided methods using 
crystallography are most efficient as they define the position of the fragment before growing  it or 
linking  to other  fragments. The use of X‐ray crystallography  in fragment screening  (Figure 6) has 
been pioneered in Astex [69,70]. Astex was founded by Harren Jhoti, Chris Abell and myself in 1999 
and funded by Abingworth Investments, which I had advised for many years before. The laboratory 
was established with  three postdocs  in  the Blundell and Abell  laboratories  in  the Departments of 
Biochemistry  and  Chemistry  in  Cambridge  University,  where  it  was  demonstrated  that  X‐ray 
analysis  could  define  the  fragment  positions  accurately with  high‐resolution  X‐ray methods.  In 
parallel, Harren Jhoti, who had been at GSK, established himself initially in the Grafton Centre, in 
the centre of Cambridge to recruit his team, before moving to the Cambridge Science Park to establish 
the  ea ly  stage dr g d covery  ef ectively. Tim Haines,  an  entrepr eur  funded  by Abingworth, 
joined as CEO and Harr n  nitially was Chief Sci ific Officer, but after five years became CEO. 
 
Figure  6.  The  evolution  of  structure‐guided  drug  discovery.  A  schematic  comparison  of  the 
conventional drug discovery process with very large libraries of drug‐like compounds of molecular 
weight ~500 daltons, compared with the Astex fragment‐based structure‐guided approach in which 
libraries < 1000 compounds of molecular weight < 300 daltons are screened using biophysical methods 
defining structures and affinities, before being elaborated to drug‐like compounds with nano‐molar 
affinities [69,70]. 
Fragment‐based drug discovery usually now involves an initial screen using fluorescence‐based 
thermal‐shift measurements, ligand‐based NMR, surface plasmon resonance and X‐ray screening of 
crystals [71]. The fragment is first shown to bind the protein, and the structure of the protein‐chemical 
fragment complex defined by X‐ray or NMR. The kinetics are  investigated using surface plasmon 
resonance  and  the  thermodynamics  using  isothermal  calorimetry.  Fragment‐based  screening  is 
widely used and many of the X‐ray screening has been established at synchrotrons, in the UK by Dr 
Target       Screening    Hit           Lead compound
Astex Drug discovery process
Conventional Drug Discovery Process
Figure 6. The evolution of structure-guided drug discovery. A schematic comparison of the
conventional drug discovery process with very large libraries of drug-like compounds of molecular
weight ~500 daltons, compared with the Astex fragment-based structure-guided approach in which
libraries < 1000 compounds of molecular weight < 300 daltons are screened using biophysical methods
defining structures and affinities, before being elaborated to drug-like compounds with nano-molar
affinities [69,70].
Fragment-based drug discovery usually now involves an initial screen using fluorescence-based
thermal-shift measurements, ligand-based NMR, surface plasmon resonance and X-ray screening of
crystals [71]. The fragment is first shown to bind the protein, and the structure of the protein-chemical
frag ent complex defined by X-ray or NMR. The kinetics are investigated using surface plasmon
resonance and the thermodynamics using isothermal calorimetry. Fragment-based screening is widely
used and many of the X-ray screening has been established at synchrotrons, in the UK by Dr Frank von
Delft and his colleagues at the Diamond Light Source. Frank had been in our laboratory in Cambridge
but not involved in fragment-based approaches. His method combines a high-throughput synchrotron
data collection from crystals with some very nice software called PanDDA [72], in which the electron
density for the fragment bound state is calculated taking into account the occupancy of the fragment
and weighting in the apo- and bound-states properly.
Astex established funding not only from venture capital but also through collaborations initially
with each of AstraZeneca, Novartis, Janssen, and GSK. This allowed drugs to be moved through
clinical trials. By 2012 Astex had eight candidate drugs in clinical trials, some internally developed and
others in the collaborations. The cost of the in-house clinical trials is huge and it was clear we needed a
major investment. In 2013, Astex was sold to a Japanese Pharma giant, Otsuka, in a $886 million deal,
but the Cambridge laboratories have been maintained and expanded.
In 2016, Astex achieved a milestone with a US FDA filing of a new drug known as Ribociclib,
in a collaboration with Novartis, finally gaining FDA approval in March, 2017. The drug is used in
combination therapy for advanced breast cancer with Letrozole as first-line treatment. Ribociclib was
followed in April, 2019 by FDA approval of a further drug, Erdafitinib, with Janssen for treatment of
metastatic urothelial carcinoma. Astex remains very much as it was on the Science Park as part of
Otsuka, but now focusing on fragment-based drug discovery not only in cancer but also in diseases of
the central nervous system. Harren Jhoti continues to play a role in Astex and in the larger Otsuka
Crystals 2020, 10, 676 15 of 27
family of companies in Japan and the United States, as well as UK. I continue to be on the Board of
Astex Pharma and also to chair the Science Advisory Board.
11. Crystals and Antimycobacterial Discovery for Infectious Disease
In 2006, Ken Duncan of the Gates Foundation contacted Astex to see if there was an interest
in collaboration with the Bill and Melinda Gates Foundation as part of the Integrated Methods for
Tuberculosis Drug Discovery (IM TB) to use fragment-based discovery for the design of new treatments
for tuberculosis. At that time Astex was working on oncology targets and was aware that it needed to
maintain this focus in order to establish its position in drug discovery and get further investment for
its early development and clinical trials. Furthermore, the market for therapeutics for tuberculosis was
likely to be in developing countries, where the population cannot afford the costs of drugs that are used
in economically developed countries. It seemed sensible therefore that this was done in a university
context. Therefore, Chris Abell and I volunteered to develop a parallel approach using crystal structures
and fragment-based drug discovery in our laboratories in the University of Cambridge.
In Africa, the position was that over 50% of tuberculosis cases had often not been diagnosed,
the BCG vaccine provided only marginal protection for a few years and drug therapy was long and
complicated, taking six to nine months for drug sensitive tuberculosis and as long as two years for
drug-resistant tuberculosis. The first line drugs were Rifampicin, Ethambutol, Pyrazinamide and
Isoniazid [73], but multiple drug-resistant tuberculosis was on the rise with only two new tuberculosis
drugs in 30 years.
We had already developed some early work in the University on targeting protein–protein
interactions, which was another area of greater challenge than targeting the active sites of protein
kinases and other druggable targets favoured by big pharma (see below). We were therefore able to
establish collaboration with the Gates Foundation fairly quickly. There were two initial objectives—one
was to substantially improve the ability to discover, identify and validate targets linked with TB
persistence and secondly to find and optimise small-molecule inhibitors of validated targets.
The focus on tuberculosis, nevertheless, required new thinking, as the cell wall of Mycobacterium
tuberculosis provides a challenge that differs from that of human cells, there are many efflux pumps
in M. tuberculosis and enzymes that can degrade candidate molecules that do reach the intracellular
region. These challenges, however, are well suited to fragment-based discovery, which allows the
design of molecules that meet new requirements [74]. We followed the fragment-based approach
taken for the cancer targets in Astex (see Figure 7), but introducing further selection criteria as the
fragments were elaborated. In order to understand the challenges and to select suitable targets
Bernardo Ochoa-Montano in our group set out to develop a database of crystallographic and modelled
structures of the M. tuberculosis proteome, called CHOPIN [75]. We considered this to be a suitable
name, given the fact that CHOPIN probably died of tuberculosis. The database has models of around
80% of the gene products; it first accepts any crystal structures and then exploits those of homologues
using the software developed in the Blundell over the past three decades in particular FUGUE [59] and
MODELLER [56]. The higher oligomeric states were modelled as well as the individual protomers.
In addition, the binding sites were defined and hotspots used to identify those that were likely to
be druggable.
The team set off to select targets for which there were crystal structures and to apply the methods
developed for fragment-based discovery. We considered some well-established targets in tuberculosis
including the enoyl-acyl carrier protein reductase, which is involved in the synthesis of mycolic
acids and is the target of the first and second-line tuberculosis drugs isoniazid and ethionamide.
We also looked at KatG, which is a heme enzyme catalase peroxidase, which is involved in activation
of isoniazid. Although we have defined crystal structures for all proteins that we have targeted,
most recently we have used cryo-EM, which has undergone a resolution revolution. This combined
approach has been applied to follow the drug discovery against KatG, cryo-EM providing images at
3 Å resolution, work developed by Asma Munir and Amanda Chaplin in our group [76]. Our work
Crystals 2020, 10, 676 16 of 27
has led to low-micromolar and nanomolar candidate molecules. The real challenge with tuberculosis,
however, is persuading companies to take any candidate drug through clinical trials. Even for wealthy
charitable foundations like the Bill and Melinda Gates Foundation it is a challenge.
Crystals 2020, 10, x FOR PEER REVIEW  16 of 27 
Bernardo  Ochoa‐Montano  in  our  group  set  out  to  develop  a  database  of  crystallographic  and  
modelled structures of the M. tuberculosis proteome, called CHOPIN [75]. We considered this to be a 
suitable name, given the fact that CHOPIN probably died of tuberculosis. The database has models 
of around 80% of the gene products; it first accepts any crystal structures and then exploits those of 
omologues using the software devel ped i  the Blund ll over t  past t re  decades in particular 
FUGUE  [59]  and  MODELLER  [56].  The  higher  oligomeric  states  were  mode led  as  well  as  e  
individual protomers. In addition, the bindi g sites were defined and hotspots used to identify those 
Figure 7. Using  structural  information  for designing better  compounds  targeting a Mycobacterium 
tuberculosis target. Overlay of five fragment hits to a protein target, the ethionamide (ETH) receptor, 
a transcriptional regulator, defined by X‐ray analysis. These can then be used to guide the design of 
a drug‐like molecule, by either fragment linking or fragment growth, for example see review by Vitor 
Mendes and Tom Blundell, 2017 [74]. 
The team set off to select targets for which there were crystal structures and to apply the methods 
developed  for  fragment‐based  discovery.  We  considered  some  well‐established  targets  in 
tuberculosis including the enoyl‐acyl carrier protein reductase, which is involved in the synthesis of 
mycolic  acids  and  is  the  target  of  the  first  and  second‐line  tuberculosis  drugs  isoniazid  and 
ethionamide. We also looked at KatG, which is a heme enzyme catalase peroxidase, which is involved 
in activation of isoniazid. Although we have defined crystal structures for all proteins that we have 
targeted, most recently we have used cryo‐EM, which has undergone a resolution revolution. This 
combined approach has been applied to follow the drug discovery against KatG, cryo‐EM providing 
images at 3 Å resolution, work developed by Asma Munir and Amanda Chaplin in our group [76]. 
Our work has led to low‐micromolar and nanomolar candidate molecules. The real challenge with 
tuberculosis, however, is persuading companies to take any candidate drug through clinical trials. 
Even for wealthy charitable foundations like the Bill and Melinda Gates Foundation it is a challenge.   
Bill Gates himself visited the group in Cambridge a few years later when the work was ongoing. 
He requested that his visit was not advertised and he arrived alone to be greeted by the combined 
Chemistry  and  Biochemistry  teams. He  spoke with  each member  of  our  structural  biology  and 
chemistry teams, who introduced themselves by name and country/language—members were truly 
international, with over thirty languages between them. Bill Gates recounted how Melinda had seen 
a programme about tuberculosis in Africa and had encouraged him to be involved in this area, one 
of the major steps in establishing the Gates Foundation. I discussed with him how tuberculosis was 
a real challenge to my own family on my wife’s side, who live in Matabeleland Zimbabwe and where 
Site 3 Site 2 Site 1
7. t ycobacte i
l sis target. erla of five fragment hits to a protein target, the thionamide (ETH) receptor, a
transcriptional regulator, defined by X-ray analysis. Thes can then be used to guide the design of a
drug-like molecule, by either fragment linking or fragment growth, for exa ple see r i it
o Blundel , 2017 [74].
Bill Gates himself visited the group in Cambridge a few years later when the work was ongoing.
He requested that his visit was not advertised and he arrived alone to be greeted by the combined
Chemistry and Biochemistry teams. He spoke with each member of our structural biology and
chemistry teams, who introduced themselves by name and country/language—members were truly
international, with over thirty languages between them. Bill Gates recounted how Melinda had seen a
programme about tuberculosis in Africa and had encouraged him to be involved in this area, one of
the major steps in establishing the Gates Foundation. I discussed with him how tuberculosis was a
real challenge to my own family on my wife’s side, who live in Matabeleland Zimbabwe and where
a number of family members had contracted the disease, mainly as they moved down to work in
South Africa.
In parallel to these developments, we have also targeted other mycobacteria. One of these is
Mycobacerium abscessus, which is an infectious agent that infects many Cystic Fibrosis patients. Professor
Andres Floto from the Clinical School leads the research programme in Cambridge for the Cystic
Fibrosis Trust. With targeting M. abscessus for cystic fibrosis, the challenge is that patients are few
in number making it financially it less attractive for pharmaceutical companies [77]. Nevertheless,
successful development of drugs for the market has been achieved by Vertex, a company that is in
both the United States and the UK. Our work has focused always on obtaining crystal structures
but as there are many fewer structures for the M. abscessus proteome, we have again developed a
database, in this case called Mabellini, to extend the structural knowledge experimentally defined
by crystallography [78]. With Dr Sherine Thomas in the lead in our laboratory we have targeted
several enzymes, including M. abscessus TrmD (tRNA-(N(1)G37) 4 methyltransferase). TrmD is an
essential tRNA modification enzyme in bacteria that prevents errors in the reading frame during
protein translation and represents an attractive potential target for the development of new antibiotics.
We reproduced the crystal structure and used fragment-based drug discovery to the design a new class
Crystals 2020, 10, 676 17 of 27
of inhibitors with low-micromolar in vitro TrmD inhibitory activity [79]. Several of these compounds
exhibit activity against planktonic M. abscessus and M. tuberculosis as well as against intracellular
M. abscessus and M. leprae, indicating their potential as the basis for a novel class of broad-spectrum
mycobacterial drugs.
After a lecture in 2016 at the International Mycobacterial Congress in Fort Collins, Colorado, USA,
I was approached by the American Leprosy Mission to see whether we would collaborate with them,
using the techniques we had developed for M. tuberculosis and abscessus, but focusing targets from
the closely related M. leprae. The stigma of leprosy has meant that, even in countries such as India or
Brazil, where the disease is rampant, it is not widely known that it is contracted by~200,000 people
each year. Leprosy, which causes chronic infections of the skin and peripheral nerves, is treated using
the same multidrug therapy as tuberculosis including Dapsone, Rifampicin and Clofazimine.
In 2016, Dr Sundeep Chaitanya Vedithi, Director of Drug Discovery for the American Leprosy
Mission, joined our laboratory in Cambridge and began educate us about the challenges of leprosy [80].
We had many new things to learn including the fact that the genome of M. leprae has undergone
reductive evolution and has only 1615 genes, although there are 1293 pseudo genes. This means that the
mycobacterium is very host dependent and difficult to culture. Sundeep has developed the programme
both by developing the database known as HANSEN in which around 70% of the models are good
quality and most of the rest have some reasonable structures built in the usual way [81]. We have
also used computational saturation mutagenesis to predict structural consequences of systematic
mutations in the beta subunit of RNA polymerase in M. leprae [82]. In parallel, the American Leprosy
Mission is funding fragment-based discovery in the laboratory, with Dr. Marta Acebron focusing on
the experimental crystallographic and drug discovery programme.
12. Multicomponent Systems as Drug Targets in Cancer
Most biological systems involve multicomponent systems in order to introduce sensitive regulation
into the cell and whole organism. Fifty years ago most researchers assumed that regulatory and
signalling processes involved binary interactions, for example through a single receptor and its ligand.
In the 1960s and 1970 when we defined the structures of insulin and glycogen, we were active at
inferring which regions of the structures were important for activity but little was known about the
sequence or structure of the receptors, or indeed about other components of the signalling systems.
Our first attempts to define structures of complexes came much later in our work on nerve growth
factor in 1991 when our 2.3 Å resolution structure demonstrated a new protein fold comprising three
antiparallel β-strands forming a flat surface facilitating dimerisation [83]. We later described the mouse
7S NGF, a complex of nerve growth factor with four binding proteins (two α-NGF and two γ-NGF),
which assembles as a symmetrical hetero-hexamer organised around the β-NGF dimer [84]. The crystal
structure of nerve growth factor in complex with the ligand-binding domain of the TrkA receptor was
defined in 1999 demonstrating a similar assembly to that of the 7S NGF complex [85]. A further six
years later led to the characterisation of a symmetric dimer binding the extracellular domains of two
receptor molecules in a similar way, so dimerising the receptor [86].
In parallel to these studies, we examined the structure of FGF paralogues interacting with the FGF
receptors. In 2000, we defined the structure of fibroblast growth receptor extracellular domain bound
to ligand FGF and heparin sulphate glycosaminoglycan polysaccharide (Figure 8) [87]. The molecular
association of heparin with FGF and its receptor was known to be essential for biological activity, but the
interactions defined in our crystal structure indicated a multicomponent complex. Our contention that
this complex structure was biologically relevant caused extensive controversy!
However, another group had suggested that a 2FGF: 2FGFR: 2heparin ternary complex existed on
the basis of a 3 Å resolution crystal structure by colleagues [88]. This was inconsistent with the results
obtained in collaboration with Luca Pellegrini and Ashok Venkitaraman [89], so we proceeded in the
following years to check the stoichiometry by other biophysical methods including size-exclusion
chromatography [90] and isothermal titration calorimetry [91]. These and other experiments led
Crystals 2020, 10, 676 18 of 27
to the conclusion that the earlier crystal structure that appeared to indicate a 2:2:2 complex of the
FGF-FGFR-heparin probably arose from two sites are partially occupied in a way that would be
generated by disorder of two 2:2:1 complexes around the crystallographic 2-fold axis
Crystals 2020, 10, x FOR PEER REVIEW  18 of 27 
 
NGF dimer  [84]. The crystal structure of nerve growth  factor  in complex with  the  ligand‐binding 
domain of the TrkA receptor was defined in 1999 demonstrating a similar assembly to that of the 7S 
NGF complex [85]. A further six years later led to the characterisation of a symmetric dimer binding 
the extracellular domains of two receptor molecules in a similar way, so dimerising the receptor [86].   
In parallel to these studies, we examined the structure of FGF paralogues interacting with the 
FGF receptors. In 2000, we defined the structure of fibroblast growth receptor extracellular domain 
bound to ligand FGF and heparin sulphate glycosaminoglycan polysaccharide (Figure 8) [87]. The 
molecular association of heparin with FGF and its receptor was known to be essential for biological 
activity, but the interactions defined in our crystal structure indicated a multicomponent complex. 
Our contention that this complex structure was biologically relevant caused extensive controversy! 
 
 
Figure 8. A. The crystal structure of  the basic repeat unit of  the extracellular domain of  the 2FGF: 
2FGFR:1heparin complex. With extended heparin it forms a 2D repeat higher‐order complex on the 
cell surface. B. A schematic of the higher‐order signalling complex, in which the heparan sulphate 
(blue and black lines), links the FGFR receptors (green) and FGF (red) [87]. 
A
B
Figure 8. (A) The crystal structure of the basic rep at unit of the extracellular domain of the 2FG :
2FG R:1heparin complex. With extend d h parin it forms a 2D repeat higher-order complex on the cell
surface. (B) A schematic of the higher-order signallin complex, in which the heparan sulphate (blue
and black lines), links the FGFR r ceptors (green) and FGF (red) [87].
Most efforts at drug discovery on FGF receptors were focused on the intracellular kinase domain,
giving rise to useful molecules. Multiple small-molecule inhibitors targeting this family of kinases have
been developed, and some of them are in clinical trials. The pan-FGFR inhibitor erdafitinib, originally
discovered by Astex Pharmaceuticals and licensed to Janssen Pharmaceuticals as JNJ-42756493 for
Crystals 2020, 10, 676 19 of 27
further development, has recently been approved by the U.S. Food and Drug Administration (FDA)
for the treatment of metastatic or unresectable urothelial carcinoma [92].
An attempt to improve selectivity by targeting the protein–protein interactions in the extracellular
region of FGF receptors was developed in a collaborative exercise involving sixteen different groups
coordinated by Marc Herbert from Sanofi-Aventis, from Montpellier, France. [93]. The new FGFR
inhibitor, SSR128129E (SSR), binds to the extracellular part of the receptor. It does not compete
directly with FGF for binding to FGFR but inhibits in an allosteric manner. SSR was the first reported
small-molecule allosteric inhibitor of FGF/FGFR signalling, acting via binding to the extracellular
part of the FGFR. This orally deliverable, small-molecule multiFGFR inhibitor showed promising
therapeutic anti-cancer efficacy
These early experiments emphasised the challenge for targeting drugs at protein–protein
interactions. In general, the protein–protein interfaces were relatively flat and the number of druggable
sites very few. However, it was evident from other studies in our laboratory of protein–protein
interactions that when one component was involved in concerted folding and binding onto a pre-defined
site then the pockets were generally deeper. Our initial studies, involving Luca Pellegrini, of such
a system involved the interaction of Rad51 with BRCA2. The flexible region of BRCA2 involved in
concerted folding and binding with the Rad51 involved a conserved sequence of FxxA, in which the
phenylalanine (F) initially docks into a deep pocket (Figure 9). Pellegrini et al. (2002) demonstrated
that the intervening residues, which are variable in evolution, then fold to allow the conserved alanine
to dock into a second pocket ([94]).
Crystals 2020, 10, x FOR PEER REVIEW  19 of 27 
 
However, another group had suggested that a 2FGF: 2FGFR: 2heparin ternary complex existed 
on the basis of a 3 Å resolution crystal structure by colleagues [88]. This was inconsistent with the 
results obtained in collaboration with Luca Pellegrini and Ashok Venkitaraman [89], so we proceeded 
in  the  following  years  to  check  the  stoichiometry  by  other  biophysical methods  including  size‐
exclusion  chromatography  [90]  and  isothermal  titration  calorimetry  [91].  These  and  other 
experiments led to the conclusion that the earlier crystal structure that appeared to indicate a 2:2:2 
complex of the FGF‐FGFR‐heparin probably arose from two sites are partially occupied in a way that 
would be generated by disorder of two 2:2:1 complexes around the crystallographic 2‐fold axis   
Most  efforts  at  drug  discovery  on  FGF  receptors were  focused  on  the  intracellular  kinase 
domain, giving rise to useful molecules. Multiple small‐molecule inhibitors targeting this family of 
kinases  have  been  developed,  and  some  of  them  are  in  clinical  trials.  The  pan‐FGFR  inhibitor 
erdafitinib, originally discovered by Astex Pharmaceuticals and licensed to Janssen Pharmaceuticals 
as JNJ‐42756493 for  further development, has recently been approved by  the U.S. Food and Drug 
Administration (FDA) for the treatment of metastatic or unresectable urothelial carcinoma [92]. 
An  attempt  to  improve  selectivity  by  targeting  the  protein–protein  interactions  in  the 
extracellular  region of FGF  receptors was developed  in a  collaborative exercise  involving  sixteen 
different groups coordinated by Marc Herbert from Sanofi‐Aventis, from Montpellier, France. [93]. 
The new FGFR inhibitor, SSR128129E (SSR), binds to the extracellular part of the receptor. It does not 
compete directly with FGF for binding to FGFR but inhibits in an allosteric manner. SSR was the first 
reported  small‐molecule  allosteric  inhibitor  of  FGF/FGFR  signalling,  acting  via  binding  to  the 
extracellular part of the FGFR. This orally deliverable, small‐molecule multiFGFR inhibitor showed 
promising therapeutic anti‐cancer efficacy 
These  early  experiments  emphasised  the  challenge  for  targeting  drugs  at  protein–protein 
interactions.  In  general,  the  protein–protein  interfaces  were  relatively  flat  and  the  number  of 
druggable sites very few. However, it was evident from other studies in our laboratory of protein–
protein interactions that when one component was involved in concerted folding and binding onto a 
pre‐defined site then the pockets were generally deeper. Our initial studies, involving Luca Pellegrini, 
of  such  a  system  involved  the  interaction  of Rad51 with  BRCA2.  The  flexible  region  of  BRCA2 
involved in concerted folding and binding with the Rad51 involved a conserved sequence of FxxA, 
in which the phenylalanine (F) initially docks into a deep pocket (Figure 9). Pellegrini et al. (2002) 
demonstrated that the intervening residues, which are variable in evolution, then fold to allow the 
conserved alanine to dock into a second pocket ([94]. 
 
Figure 9. A. The crystal structure of the RAD51–BRCA2 complex, in which during DNA homologous 
recombination a FxxA motif from BRCA2 folds onto Rad51, with the phenylalanine (F) occupies a 
BRC1 HSFGGSFRTASNKEI
BRC2 EVGFRGFYSAHGTKL
BRC3 ETSDTFFQTASGKNI
BRC4 EPTLLGFHTASGKKV
BRC6 EVGPPAFRIASGKIV
BRC7 ANTCGIFSTASGKSV
BRC8 SSAFSGFSTASGKQV
A. RAD51-BRC4 interaction B RAD51-RAD51 interaction
KLVPMGFTTATEFHQ HsRAD51
RLVPMGFVTAADFHM    ScRAD51
KLVPMGFTTATEFHQ    GgRAD51
KLVPLGFLSARTFYQ    DmRAD51
AANLGTFMRADEYLK   PfRadA
Figure 9. (A) The crystal structure of the RAD51–BRCA2 complex, in which during DNA homologous
recombination a FxxA motif from BRCA2 folds onto Rad51, with the phenylalanine (F) occupies a deep
pocket, before the C-terminal region folds as a helix on to the surface. (B) A similar interface involving
an FxxA motif is involved in the assembly of the RAD51–RAD51 polymer on DNA [94].
In a collaboration with Ashok Venkitaraman from the Clinical School in Cambridge and Marko
Hyvonen and May Marsh in our group in the Department of Biochemistry, we screened using the
fragment based approach producing multiple micromolar fragment binding (Figure 10) including
merging of fragment and peptide data to eventually give inhibitors with nanomolar binding [95,96].
However, even when we obtained nanomolar compounds targeting the Rad51 BRCA2 system and
obtained good nanomolar binding, it proved impossible to persuade any companies to join us.
Crystals 2020, 10, 676 20 of 27
Crystals 2020, 10, x FOR PEER REVIEW  20 of 27 
 
deep pocket, before  the C‐terminal region  folds as a helix on  to  the surface. B. A similar  interface 
involving an FxxA motif is involved in the assembly of the RAD51–RAD51 polymer on DNA [94]. 
In a collaboration with Ashok Venkitaraman from the Clinical School in Cambridge and Marko 
Hyvonen and May Marsh in our group in the Department of Biochemistry, we screened using the 
fragment based approach producing multiple micromolar fragment binding  (Figure 10)  including 
merging of fragment and peptide data to eventually give inhibitors with nanomolar binding [95,96]. 
However, even when we obtained nanomolar compounds targeting the Rad51 BRCA2 system and 
obtained good nanomolar binding, it proved impossible to persuade any companies to join us. 
 
Figure 10. Crystal structures of  fragments binding  to  the phenylalanine  (F) pocket of RAD51  that 
mediates the RAD51–BRCA2 interaction during DNA homologous recombination. The micromolar 
affinities of the individual fragments are given. Figure devised by Marko Hyvonen [95]. 
We have also targeted protein–protein interactions in the alternative DNA repair system, Non‐
Homologous End  Joining  (NHEJ). This  involves  the recognition of DNA double‐strand breaks by 
Ku70/80,  the  recruitment of a huge DNA protein kinase  (DNA‐PKcs)  that  interacts with Ku, and 
multiple components that form scaffolds and strings that regulate the assembly as the DNA ligase is 
recruited to repair the assembled ends [97]. Individual components, even the largest molecules such 
as DNA‐PKcs with 4128 amino acids have been defined by X‐ray analysis at 4.3 Å [98]. Furthermore, 
such systems often change over  time, providing major challenges  to crystallography, and  require 
dissection  using  novel  single‐molecule  forceps  and  related  techniques  [99].  These  structures  are 
proving useful in the design of new cancer therapeutics, not only through targeting the DNA‐PKcs 
kinase active site [100], but also at protein–protein interfaces, especially where polypeptide with an 
intrinsically disorder region folds and binds, such as the binding of the Artemis tail to a site on the 
DNA‐Ligase IV in NHEJ [101,102]. 
13. Crystals and Drug Discovery: The International Scene 
This personal history has shown that for more than five decades, the use of crystals and X‐ray 
analysis to define structures has contributed to the discovery of new medicines in a very significant 
way. As  it  is a personal history,  I have mainly described developments  in  the UK, particularly  in 
Oxford, London and Cambridge, where Max Perutz,  John Kendrew, David Phillips and Dorothy 
Hodgkin made very impressive contributions to haemoglobin, myoglobin, lysozyme and insulin. 
However,  these developments  in  structural  biology  and  drug  discovery were  international, 
having strong parallels not only in the United States, in particular in Yale, Harvard, UCLA and UCSF, 
but also in Russia, China, India, Brazil, South Africa and elsewhere. For example, when I first visited 
KD = 540 M KD = 600 MKD = 1000 M
KD = 730  M KD = 430 M
Figure 10. Crystal structures of fragments binding to the phenylalanine (F) pocket of RAD51 that
mediates the RAD51–BRCA2 interaction during DNA homologous recombination. The micromolar
affinities of the individual fragments are given. Figure devised by Marko Hyvonen [95].
We have also targeted protein–protein interactions in the alternative DNA repair system,
Non-Homologous End Joining (NHEJ). This involves the recognition of DNA double-strand breaks
by Ku70/80, the recruitment of a huge DNA protein kinase (DNA-PKcs) that interacts with Ku,
and multiple components that form scaffolds and strings that regulate the assembly as the DNA ligase
is recruited to repair the asse bled ends [97]. Individual components, even the largest molecules such
as DNA-PKcs with 4128 a ino acids have been defined by X-ray analysis at 4.3 Å [98]. Furthermore,
such systems often change over time, providing major challenges to crystallography, and require
dissection using ovel single-molecule forceps and related techniques [99]. These structures are
proving useful in the design of new cancer therapeutics, not only through targeting the DNA-PKcs
kinase active site [100], but also at protein–protein interfaces, especially where polypeptide with an
intrinsically disorder region folds and binds, such as the binding of the Artemis tail to a site on the
DNA-Ligase IV in NHEJ [101,102].
13. Crystals and Drug Discovery: The International Scene
This personal history has shown that for more than five decades, the use of crystals and X-ray
analysis to defi e structures has c ntributed to the discovery of n w medicines in a very significant way.
As it is a perso al history, I ve mainly described developments in th UK, particula ly n Oxford,
London and Cambridge, where Max Perutz, John Kendrew, David Phill ps and Dorothy Hodgkin
made very impressive contributions to haemoglobin, myoglobi , lysozyme and insulin.
However, these developm nts in structural biology and drug discovery were international, having
strong parallels not only in the U ited States, in part cular in Yale, Harvard, UCLA and UCSF, but also
in Russia, Chin , India, Brazil, Sou Africa and elsewhere. For example, when I first visited Russia for
the Inter ational Cryst llography Meeting in 1966, Boris Vainsht in, who was Head of the Inst ute
of Crystallography, was already strongly supporting the work on biological systems. At this time,
Natalia Andreeva work on pepsin, the structure of which was eventually published in 1976 at high
resolution [103,104].
Developments in China were more complex. A key factor in establishing protein crystallography
in China was the impact of Fred Sanger on Chinese scientists who had been in Cambridge in the
early 1950s, while Sanger produced the first sequence of a protein, insulin. When back in China and
challenged to do something for China during the Great Leap Forward in 1958, they decided to follow
Crystals 2020, 10, 676 21 of 27
the advice of Frederick Engels a century before: “if you know the formula of something, then synthesise
it.” They realised that they knew the formula of insulin, so why not follow the radical Frederick Engels,
who was much respected in China, and synthesise it. Soon after, encouraged by the visit of Dorothy
Hodgkin to Shanghai in 1960, they were inspired to crystallise synthetic insulin and check that it was
identical to bovine insulin. Liang Dong Cai was subsequently sent to Oxford in 1966 to learn protein
crystallography. He returned in 1967, apparently for the Cultural Revolution, but worked on the crystal
structure of insulin in the Institute of Biophysics in Beijing, where he eventually became Director [105].
The incorporation of crystallographic structural work into drug discovery in US large pharma
including Pfizer occurred soon after in the late 1970s and early eighties, in Belgium in Janssen
Pharmaceutica, in Denmark in Novo, and in many other countries throughout the world, including
China. This has been a truly international translation of basic science with an impressive impact on an
applied area.
14. Recent Developments and Perspectives in Crystals, Crystallography and Drug Discovery
Over recent years, new technologies have improved our knowledge of crystals and crystallography
to contribute new insights. Amongst these have been a new generation of light sources [106].
First-generation rings were built for high-energy physics research and only the second generation
were designed from the start as light sources. The third-generation rings came online in 1992 with
straight sections for insertion devices and lower electron beam emittance. Undulators gave further
impressive gains in brightness. Since then, fourth-generation light sources exceed the performance of
previous sources by an order of magnitude or more, not only in brightness but also coherence and
pulse duration with an impressive wavelength range from the vacuum UV to hard X-rays. Linac-based
Free-Electron Lasers [107] offer subpicosecond pulses and huge technological opportunities for the
future analysis of molecules and crystals.
Other developments have arisen from computer analysis, often using molecular dynamics
or normal mode analysis based on time averaged structures derived from X-ray crystallography.
These have had major impacts on understanding cryptic pocket formation in protein targets [108].
Cryptic pockets are often exposed on protein targets when drugs bind and so provide alternatives to
classical binding sites for drug development. Simulation-based approaches demonstrate that cryptic
sites do not correspond to local minima in the computed conformational free-energy landscape of the
unliganded proteins. Temperature-based enhanced sampling approaches also do not help, although
simulations with fragments can stabilise cryptic sites and help in defining them in a way than can be
used for drug discovery [108].
However, the major impact in structural biology over recent years has been electron microscopy,
where the resolution revolution depended on the development of new cryo-EM machines, new detectors
and new software [109,110]. These developments were recognised in the award of the 2017 Nobel
Prize for Chemistry to Richard Henderson, Jacques Dubochet and Joachim Frank “for developing
cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution”.
Our own laboratory has been impacted by this revolution. As described in the previous section, the
structure of the very large enzyme DNA-PKcs was solved by the X-ray analysis at 4.3 Å [98], but
recently, structures have been obtained of DNA-PKcs by cryo-EM at 2.8 Å and its complex with Ku70/80
and DNA at 3.8 Å resolution (previously 6.6 Å) [111], examples of how the resolution revolution has
changed structural biology over the past decade. It has even allowed Harren Jhoti and Pamela Williams
and the team at Astex along with collaborators at Isohelio Ltd, to develop fragment-based drug
discovery using cryo-EM, demonstrating that fragment-sized molecules can be accurately described
when bound to large proteins, allowing cryo-EM to contribute even further to drug discovery [112]).
The next decade will be a very exciting time for many academics and pharma companies as they
follow the trend to use cryo-EM for structural biology and drug discovery. Nevertheless, crystals and
crystallography will continue to play a major role in understanding protein function and drug discovery.
Crystals 2020, 10, 676 22 of 27
Funding: This research received no external funding.
Acknowledgments: T.L.B. thanks his amazing research team in the University of Cambridge for maintaining a
productive research environment, including Amanda Chaplin, Shikang Liang, Vitor Mendes, Sherine Thomas,
Sheikh Arif, Pedro Torres, Ales Hnizda, Bridget Bannerman, Sundeep Chaitanya Vedithi, postgraduates Arian
Jamasb, Ali Alsulami, Chris Beaudoin, Liviu Copoiu, Asma Munir, Antonia Kelafa-Stravidi, Robert Appleby and
So Yeon Kim. T.L.B. is particularly thankful for continuing support in the office from Niki Miller. He thanks the
many funding agencies that have supported his research described here over more than five decades, including
current funding from the Wellcome Trust Programme Grant (O93167/Z/10/Z; 2011–2016) and Investigator Award
(200814/Z/16/Z; 2016 -), The Bill and Melinda Gates Foundation (HIT-TB (OPP 627 OPP1024021) and Shorten-TB
(OPP1158806), The Cystic Fibrosis Trust (RG 70975), Fondation Botnar (RG91317) and The American Leprosy
Missions (G88726). He thanks Harren Jhoti and David Rees (Astex Pharma) for sharing advances and many helpful
discussions. T.L.B. thanks Gerard Evan, Department of Biochemistry, University of Cambridge, for allowing T.L.B.
access to laboratory space and facilities for the 11 years since T.L.B.’s “retirement”.
Conflicts of Interest: I have no conflict of interest to declare.
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