1036 Journal of Medicinal Chemistry,1994,Vol.37,No.8 Perspective chestheicerctions the tolgiotlbiochemitndturalbiohogitiaesential he op ry sites,rest in better poten the above topractic an wo d othe The eif mples oietieg Structural principle method ogy is now deas hi to the rap ign of a ove of very everal itert ons of the chabyrytalliti and structure otionFo cy of carbo . example des ribes the de nou ia to the stru einhibitor theedicenioalstruet des compounds which are currently in clinical trials Sometimes wher rotei a the the pr n ca ycle). bind ing of the Background One of the most active areas of dru rld data orts t s appro ever more appc earch for safe and effec ve the pies for aids ha structure provide ag rch eff on the whi of this dis designs early in drug-design project. sedandproteinmodel-b sed de hatstrategie8noWeitfo nds and biological testing encoded enzyme that cleav ira re 1.bu analysi g and gag-pot mar ily by the ne rest nt de rated that HIV PR per ms an function in the life nermit properties of thes using tion to design ney return it toth e,an emely fast-moving field;there are well 60 If th potent fo Ift est ofa.the ure ca d tes ing.This can be ns for Inhi her ro ds of This ver of cycle ed the y prop sal that these enzyme e relate gde ign and is the The latte are bilobal ngle-chain enzymes1036 Journal of Medicinal Chemistry, 1994, Vol. 37, No. 8 chemist to preserve the critical interactions with the protein, while modifying the ligand or substrate analog to interact more precisely with the receptor or enzyme and to occupy subsidiary sites, resulting in better potency and specificity. With the structure of the target protein-ligand complex, one can better understand the structureactivity relationships of existing compounds, suggest new analogs to synthesize in current series, and develop novel concepts and ideas for completely new ligand moieties. This methodology is now known as structure-based drug design. Obtaining the experimental three-dimensional structure of the protein target frequently requires considerable effort to clone the appropriate DNA, express, purify, and characterize multi-milligram amounts of the protein, followed by crystallization and structure solution.8 For NMR studies, l5N and 13C isotope labeling is frequently crucial to the structure determinati~n.~ Not only is the three-dimensional structure of the protein target desired, but especially complexes with the lead ligand compounds that are of interest to the chemist. Sometimes, when sufficient quantities of the protein are not readily available, the structure of the protein can be derived by homology modeling (Figure 1, green cycle).'OJ The binding of the ligand to the protein can also be examined in this way.12 The large protein structure databasel3 and the exploding protein sequence data￾bases'"'6 are making this approach ever more applicable. While such models are not accurate in the details of their structure, they can provide a good, rapid, albeit ap￾proximate, view of the active site and its interactions with the ligand to the chemist to help generate new ideas and designs early in a drug-design project. Both the ligand-based and protein model-based design strategies can be iterated with chemical synthesis of the designed compounds and biological testing followed by further rounds of ligand or protein model analysis, synthesis, and biological testing (Figure 1, blue and green cycles). However, this process is driven primarily by the experimental data provided by the biological testing. From the structural perspective, one is always extrapolating from the original structural information, and the biological data do not easily permit correcting errors in the structural models. The existence of an experimental structure for the protein-ligand complex, besides being more accurate, allows one to go beyond examining the details of the binding site and using this information to design new analogs. It allows one to take the designed, synthesized, and assayed compound, return it to the crystal or NMR tube, and redetermine the structure of this complex with this new compound (Figure 1, red cycle). Thus, one can determine experimentally whether the design concept was structurally correct. If the molecule was potent, was it potent for the correct reasons built into the design? If the compound was weaker than expected, how and why did the design concept fail? Best of all, the new structure can be used as the basis for another round of analysis, design, synthesis, and compound testing. This process can be iterated with further rounds of design, synthesis, testing, and so on to ultimately produce potent and specific compounds. This version of the design cycle has proven to be the most powerful implementation of structurally based drug design and is the subject of this review. Close collaboration between the medicinal chemist, molecular modeler/theoretical chemist, pharmacologist, molecular Perspective biologist, biochemist, and structural biologist is essential to the optimal utilization of this design cycle. Three examples are presented here to illustrate how the above ideas are being successfully reduced to practice in a number of important cases leading to the design and synthesis of novel, more potent, and specific compounds than would otherwise have been achieved. The examples illustrate the value of structural information at different stages of the drug-design process. Structural principles and ideas led to the rapid design of a novel class of very potent and highly specific HIV protease inhibitors for the treatment of AIDS. Several iterations of the structure￾based drug-design cycle were utilized to optimize the potency of carbonic anhydrase inhibitors for the treatment of glaucoma. The final example describes the de nouo design, followed by iterative cycles of optimization, of a dramatically new series of thymidylate synthase inhibitors which can serve as anticancer agents. The structure-based drug-design efforts in all of these three cases have led to compounds which are currently in clinical trials. Design and Structure of Symmetry-Based Inhibitors of HIV-1 Protease Background. One of the most active areas of drug discovery research in the world today concerns efforts to stem the tide of the AIDS pandemic. The world-wide search for safe and effective therapies for AIDS has prompted an intensive research effort on the structure and biology of the human immunodeficiency virus (HIV- 1) which is the causative agent of this disease. This research had led to the elucidation of a myriad of specific viral targets for drug discovery and design with the result that strategies now exist for targeting virtually every aspect of the viral life cycle.17 HIV-1 protease (HIV PR) is a virally-encoded enzyme that cleaves, or processes, viral gag and gug-pol protein precursors during virus assembly and maturation.18 In 1988, it was observed that deletion mutagenesis of the HIV PR gene resulted in the production of noninfectious, immature virus parti~1es.l~ This experi￾ment demonstrated that HIV PR performs an essential function in the life cycle of HIV and thus makes this enzyme an important target for the design of specific antiviral agents for AIDS. Structure-based drug design'g was used to design potent and specific, CZ symmetry-based inhibitors of HIV PR and to optimize the pharmacologic properties of these compounds. This work ultimately led to the first structure￾based clinical compound for this important antiviral target. Additional details of this work can be found in several recent review^.^^^^ The crystallography of HIV PR is an extremely fast-moving field; there are well over 150 published and unpublished crystal structures of various inhibitor complexes in existence today. Many of these structures as well as complexes with substrate-based and asymmetric peptidomimetic inhibitors have also been revie~ed.2~~~5 Structure-Function Considerations for Inhibitor Design. The initial observations that retroviral proteases contain the amino acid triplet, Asp-Thr(Ser)-Gly, and that their proteolytic activity could be inhibited by pepstatin led to the early proposal that these enzymes were related mechanistically to the aspartic protease family of en￾zyme~.~~~~~ The latter are bilobal, single-chain enzymes in which each lobe, or domain, contributes an aspartic acid residue to the active site.28 The active site itself is formed