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Drug design: HIV Molecules made to measure HIV protease inhibitors have been one of the big successes of rational drug design. Clare Sansom looks at the impact of structural biology on drug discovery PROTEIN DATA BANK DATA PROTEIN 50 | Chemistry World | November 2009 www.chemistryworld.org There are fashions in drug discovery, on this new virus. enzymes are over 300 residues In short as in many fields of technical Oroszlan had proved that a similar long and contain two copies of a development, and these even follow virus, the murine leukaemia virus, HIV protease characteristic tripeptide sequence common enough patterns to be matured by proteolytic cleavage – inhibitors – now a key necessary for enzyme activity: mapped. Gartner’s ‘hype cycle’ that is, as new virus capsules bud component of multi- aspartic acid, threonine (or serine), describes the path of a technology from an infected host cell, proteases drug HIV treatments and glycine – a trio generally referred from initial adoption, over the slice up its payload of polyproteins – are a prime example to as DTG. In contrast, the retroviral peak of inflated expectation and into individual, functional proteins of structure-based drug protease is a much smaller enzyme through the consequent trough of and enzymes. Oroszlan had also design – only 99 residues in HIV – and disillusion, until reaching its ‘plateau found that the protease cleavage HIV’s protease contains only one DTG sequence. It of productivity’, by which time it sites were resistant to all known enzymes were validated was clear from the beginning that has evolved to fit its particular niche mammalian proteases, suggesting as a potential drug target this protein could not be active as a in the market and become stable. the presence of an essential retroviral in 1985, sparking a race monomer. And structure-based drug discovery protease. to unravel the enzyme’s Mammalian aspartic proteases is a technique that has followed The sequence of HIV revealed structure such as pepsin have a bilobal the cycle exactly. It was extremely that it contained a similar protease X-ray crystal structure, with a large cleft in which fashionable during the 1980s, after to murine leukaemia virus. In 1985, structures of the enzyme the substrate binds and a flap that the structures of the first important Oroszlan published a paper showing, began appearing in 1989 folds down over it when it is bound. drug targets, including DNA and the through site-directed mutagenesis, –and the first drugs The two characteristic tripeptides enzyme dihydrofolate reductase, that protease was essential for HIV designed to inhibit it are symmetrically related and found were published. Disillusionment maturation and was hence a valid were licensed for use just at the bottom of the cleft, and the set in during the following decade target for antiviral therapy. At a six years later active site is supported by a tight with the downgrading of these time when the medical community Structure-based drug network of hydrogen bonds that ‘rational’ approaches in favour of was struggling to cope with rapidly design has since become Tom Blundell and Laurence Pearl high throughput screening and developing HIV epidemics, that was an established drug formally from Birkbeck College, combinatorial chemistry. The extremely welcome news. discovery tool University of London, UK, later pendulum has since swung back, and termed a ‘fireman’s grip’. One structural biology is now a mature A question of clan scientist who did much of the early science that has found its niche. Enzymologists have divided the work in this field was David Davies at Yet it was during the years when proteases into broad ‘clans’, divided the US National Institutes of Health, this technique was so resolutely out by their mechanism of action and who remembers: ‘some time in the of fashion that it scored perhaps named according to the residues or late 70s I took wire models of the its most stunning success to date: chemical groups involved in that pepsin structure – all the models we rationally designed HIV protease mechanism. The HIV protease had then – to a meeting where I was inhibitors. The first such drug, sequence seemed most closely able to discuss it with Jordan Tang Roche’s saquinavir, entered the clinic related to proteins in the aspartic (who first sequenced the protein), in 1995 and, almost immediately, protease clan, a group that includes Blundell, and others. We came up life expectancy for people with the well-studied mammalian with the idea that there might have HIV and Aids began to improve. digestive enzyme pepsin (see Gartner’s hype cycle been an ancestral aspartic protease Only two years after saquinavir was box p52). However, there were describes the evolution with a single DTG triplet that licensed, Roy Gulick, a prominent also significant differences. The of new technologies, was active as a dimer, and that the physician at Cornell University, New sequences of pepsin and related including drug design mammalian proteases might have York, US, involved in clinical trials of protease inhibitors, predicted that ‘we may finally have the tools to turn HIV infection into a long- term, manageable and treatable disease, much like hypertension and diabetes’. Today, there are ten drugs in this class in clinical use – the latest, Tibotec’s darunavir, was licensed in 2006 – and they are key components of multi-drug HAART [highly active anti-retroviral therapy] treatments. Rapid response The global HIV pandemic is still less than 30 years old. The first cases of the disease that became Aids were reported to the US Centers for Disease Control and Prevention in 1981; four years later, not only had the disease agent been elucidated, but the complete genome sequence of that agent, the retrovirus later named HIV, had been determined. Scientists such as Steve Oroszlan, a researcher in viral oncology at the US National Cancer Institute, rapidly set to work www.chemistryworld.org Chemistry World | November 2009 | 51 Drug design: HIV evolved through gene duplication. would be similar enough to that of We didn’t realise then that enzymes pepsin to be inhibited by known with this structure could still be generic aspartic protease inhibitors, found in nature.’ and that some of these might be By the late 80s, there were three good enough inhibitors to be leads groups working on crystal structures for drug design.’ Blundell, who was of retroviral proteases: Blundell’s, at independently thinking along the Birkbeck College; Alex Wlodawer’s, same lines, had started talking to DR TIM EVANS / SCIENCE PHOTO LIBRARY LIBRARY PHOTO SCIENCE / EVANS TIM DR at the US National Cancer Institute, collaborators at Pfizer in Sandwich, Frederick, Maryland; and a group UK, about testing known aspartic led by Manuel Navia at Merck, Sharp protease inhibitors against and Dohme in New Jersey, US. ‘It HIV. ‘Pfizer in the US had was very difficult to get enough pure a series of compounds HIV protease to make crystals,’ that inhibited aspartic remembers Wlodawer. ‘Our proteases that are known first structural studies were targets for drugs against on the related Rous sarcoma hypertension,’ he says. ‘We virus [RSV] protease, as that were intrigued by the idea that protein was more readily an antihypertensive might available. Our first crystal be a lead compound for drugs structure of the HIV enzyme against Aids.’ used protein that Steve Kent, from California Institute of Structure racing Technology, had produced by By 1989, the three groups were chemical synthesis.’ working against the clock, Pearl was a postdoctoral and against each other, to solve researcher at the Institute and publish their structures. The of Cancer Research (ICR) at Merck group was the first to submit Sutton in Surrey, UK, when the a manuscript on the HIV protease HIV sequence was published. structure; they sent it to Nature, ‘After obtaining my PhD with who sent it to Blundell to review. Tom Blundell, I found myself in the He realised that although the bulk fortunate position of knowing a of the structure was correct, the great deal about aspartic protease C-terminal part was inconsistent structure and mechanism, and with the models that he had been being in a lab doing state-of-the- building, and that his models seemed art work in retrovirology – a group more plausible. ‘However, knowing at the ICR had just discovered that well how medically important this CD4 was the receptor for HIV,’ he structure was, and believing it to remembers. He studied alignments be largely right, I recommended of the HIV sequence with other that Blundell, Tang and Davies had The mammalian digestive it for publication.’ He and Pearl retroviral proteases, and noticed that speculated about, and he worked hormone pepsin is a close laid out the experimental and ‘everything needed for the aspartic with Willie Taylor at Birkbeck to relative of HIV protease predicted structures side by side in protease mechanism was completely build a structural model of a cut- an accompanying News and Views conserved in these sequences, but down half protease. ‘Our model could article. The paper came out a week it was only half there’. He started form a dimer with perfect two-fold after the Wlodawer group had to wonder whether these proteins symmetry,’ he says. ‘Furthermore, published the structure of the Rous could be the ‘ancestral half-enzyme’ we concluded that its mechanism sarcoma virus enzyme, and in the same week Irene Weber, working Clan connections with Wlodawer, published a model of the HIV enzyme based on the RSV The structural biology of the aspartic proteases protease structure. When Wlodawer, has a very long history and pepsin in particular and three months later, but to higher has a unique place in this. The first ever protein resolution, Blundell, published their x-ray diffraction pattern was obtained by John x-ray structures, they proved that Desmond Bernal and his student Dorothy the Merck group had indeed traced Crowfoot [later Hodgkin] at the University of the C-terminal end of the chain Cambridge from crystalline pepsin.

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