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tructure-based Drug Designy Veerapandian, Pandi.

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New York Marcel Dekker, Inc., 1997.ISBN: 0824798694eBook ISBN: 0585157448Subject: Drugs--Design. Drugs--Structure-activityrelationships. Drugs--Conformation. Drug Design.Structure-Activity Relationship.Language: English

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Structure-based Drug Design

Table of Contents

Structure-Based Drug Design

Preface

Contents

Contributors

1 Inhibitors of HIV-1 Protease

2 Structural Studies of HIV-1 Reverse

Transcriptase and Implications for

Drug Design

3 Retroviral Integrase: Structure as a

Foundation for Drug Design

4 Bradykinin Receptor Antagonists

5 Design of Purine Nucleoside

Phosphorylase Inhibitors6 Structural Implications in the Design

of Matrix-Metalloproteinase Inhibitors

7 Structure—Function Relationships in

Hydroxysteroid Dehydrogenases

8 Design of ATP Competitive Specific

Inhibitors of Protein Kinases Using

Template Modeling

9 Structural Studies of Aldose

Reductase Inhibition

10 Structure-Based Design of

Thrombin Inhibitors

11 Design of Antithrombotic Agents

Directed at Factor Xa

12 Polypeptide Modulators of Sodium

Channel Function as a Basis for the

Development of Novel Cardiac...

13 Rational Design of Renin Inhibitors

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tructure-based Drug Design

14 Structural Aspects in the Inhibitor

Design of Catechol O-

Methyltransferase

15 Antitrypanosomiasis Drug

Development Based on Structures of

Glycolytic Enzymes

16 Progress in the Design of

Immunomodulators Based on the

Structure of Interleukin-1

17 Structure and Functional Studies of

Interferon: A Solid Foundation for

Rational Drug Design

18 The Design of Anti-Influenza Virus

Drugs from the X-ray Molecular

Structure of Influenza Virus Ne...

19 Rhinoviral Capsid-Binding

Inhibitors: Structural Basis for

Understanding Rhinoviral Biology andf...

20 The Integration of Structure-Based

Design and Directed Combinatorial

Chemistry for New Pharmaceut...

21 Structure-Based Combinatorial

Ligand Design

22 Peptidomimetic and Nonpeptide

Drug Discovery: Impact of Structure-

Based Drug Design

Index

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nhibitors of HIV-1 Protease

rzysztof Appeltgouron Pharmaceuticals, Inc., San Diego, California

Int roduction

ince the discovery of human immunodeficiency virus (HIV) as the causative agent of acquiredmmunodeficiency syndrome (AIDS), perhaps the largest and most powerful consortium of scientistver assembled to tackle a single disease has been brought to bear on the problem of AIDS and itseatment. From an unprecedented wealth of information regarding the molecular biology and virolo

f HIV collected in recent years, it became possible to identify numerous intervention points in the vfe cycle that could be exploited in the development of drugs for AIDS therapy (for reviews seeeference 1, 2, and 3). Among these, the virally-encoded enzymes, in particular reverse transcriptasend protease, have emerged as the most popular targets. A separate chapter of this book is dedicated e description of reverse transcriptase and its inhibitors [4]. For the purpose of introduction only, it

hould be noted that nucleoside inhibitors of reverse transcriptase (AZT, ddI, ddC, d4T, and 3TC) haeen widely used in clinical practice since 1987. Since then it has become apparent that this class ofgents, while slowing progression of disease in HIV-infected patients, is limited in both activity and uration of the clinical responses produced. Therefore in the search for better anti-HIV agents, the fo

f effort was expanded to include the search for clinically useful inhibitors of a second viral enzymeamely the protease. In contrast to reverse transcriptase, for which activity is required prior to thetegration of viral genetic information into the host cell chromosomes, the viral protease plays a keyle late in the virus life cycle and inhibitors of this enzyme display equal anti-viral activity in chron

nd acute infection models in vitro [5].

he HIV protease (HIV PR) is encoded by the 5' portion of the retroviral pol gene, which encodes alplicative enzymes. Viral structural proteins (p24,

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17, p9, and p7) and replicative enzymes (protease, reverse transcriptase/ RnaseH, and integrase) areanslated as either polyprotein P55-GAG, or a larger frameshift product P160-GAG-POL. In therocess of virus assembly these polyproteins are proteolytically cleaved by the protease and thisrocessing step, both in its timing and accuracy, is essential for the formation of infectious particles IV [6]. It was also shown early on that the inactivation of HIV PR, either by chemical inhibition or

ertain mutations, leads to the production of immature, noninfectious viral particles [7,8].tructurally HIV PR is a 99-amino-acid protein translated initially as a central part of the P160-GAGOL polyprotein precursor. The autocatalytic processing from the 160 kDa precursor is poorlynderstood, but most likely occurs during the process of budding of pre-formed viral particles from tost cell [9]. After release from the precursor polyprotein, HIV PR forms a homodimer and acts in tr

correctly process GAG and GAG-POL polyproteins—a process required for formation of the viraapsid and nucleoprotein core.

etroviral proteases such as HIV PR are the latest additions to the wellstudied family of aspartic

roteases. This family of enzymes, which includes, among others, proteases such as pepsin, renin, anathepsins D and E, has been intensely studied in the past, and the knowledge gained from studies ofese enzymes allowed early inferences as to the structure and function of the dimeric HIV PR.

Moreover, the intensive effort over the past two decades to make inhibitors of human renin providedmpetus for the early design of inhibitors of HIV PR. In fact, some of the renin inhibitors have turnedut to be effective inhibitors of retroviral aspartic proteases as well and have served as the starting por drug design. As a result of this many early inhibitors of HIV PR were peptidyl in nature and the b

nown example of such compounds is Ro31-8959, better known as saquinavir, a hydroxyethylamineontaining mimetic of a hexapeptide substrate [10]. This potent inhibitor of HIV PR was discovered

sing a substrate-based rational approach to drug design and displays extremely high in vitro activitygainst clinical isolates and laboratory strains of HIV. Saquinavir has been recently approved by theDA for the treatment of AIDS in combination with nucleoside inhibitors of reverse transcriptase, ane discovery of this compound was the first breakthrough and the starting point for many othernovative designs.

etermination of the crystal structures of HIV PR gave new impetus to the design of novel inhibitorne measure of the intensity with which new inhibitors were designed or discovered is the total numf crystal structures of inhibitory complexes, currently exceeding 250, that have been determined ov

e past 5 years. Very detailed crystallographic analysis combined with extensive biochemicalharacterization and site-specific mutagenesis studies made HIV PR perhaps the best characterizednzyme to date.



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ased on the avalanche of papers describing the structure-based design of various HIV PR inhibitorsould be reasonable to assume that, with the exception of saquinavir, all other HIV PR inhibitors tha

ntered the stage of preclinical or clinical development were discovered using the elements of a strucased approach. From the long list of more than 30 inhibitors considered as clinical candidates [11],urrently there are three compounds (saquinavir, ritonavir, and indinavir) already approved by the FD

anti-HIV drugs. Many factors that are requisite for in vivo activity in AIDS patients can only beredicted a priori in a very general sense. For instance, erratic oral bioavailability in humans, first-paetabolism, binding to plasma proteins or tissue distribution may disqualify a perfect in vitro inhibit

f HIV replication and such properties can be very poorly predicted by any process of drug design. Aotential answer to these problems is the parallel design of several chemically distinct compounds thay have similar in vitro activity but significantly different in vivo properties. The application of proructure-based design offers such possibilities and in this text the discovery and optimization offferent series of potent inhibitors of HIV PR will be discussed. In order to familiarize the reader wie architecture of HIV PR and the properties of its active site, the first paragraphs are devoted to the

etailed description of the x-ray structures of the enzyme followed by several examples of inhibitorsound conformation.

. Thr ee-Dimensional Structure of H IV PR

etroviral proteases such as HIV PR were tentatively assigned to the aspartic protease family on theasis of putative active-site sequence homology [12]. Mammalian aspartic proteases are bilobal, singhain enzymes in which each lobe (or domain) contributes an aspartic acid residue to the active site he active site itself is formed at the interface on the N- and C-terminal domains and exhibitspproximate two-fold symmetry. Since the retroviral proteases are only about one-third the size of th

wo-domain eukaryotic enzymes, they were hypothesized to function as dimers in which each monomontributes a single aspartic acid to the active site [14]. Obligate homodimeric proteases, in additionroviding a regulatory mechanism to control activation of the enzyme, represent the most efficient uf genetic information which, in retroviruses, is naturally parsimonious.

he crystal structures of HIV PR confirmed the predicted dimeric character of the enzyme [15,16]Figure 1). In all published crystallographic investigations of the unliganded form of the enzyme, theonomers are related to each other by crystallographic two-fold symmetry and are necessarily identhe general topology of the HIV PR monomer is similar to that of a single-domain



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Figure 1Stereo view of the α-carbon backbone of HIV PR dimer. (a) The apoenzyme

with flaps in the “open” conformation. (b) Inhibited form of HIV PR with flaps in a“closed” conformation. For clarity, the inhibitor is removed from the active site.

epsin-like aspartic protease and consists of antiparallel β-strands and a short, two-turn α-helixonnected by loops of varying length. The dimer interface is formed by an antiparallel β-sheetomprising two strands from each monomer. The hydrophobic residues from those β-strands and tw

ymmetry-related α-helices form the core of the dimer. The dimer is further stabilized by a net ofydrogen bonds involving the residues around the catalytic aspartic acids. The active site is formed be dimer interface and is composed of equivalent contributions of residues from each monomer. Th

ubstrate-binding cleft is bound on one side by the active site aspartic acid (Asp25/25') and on the otde by a pair of two-fold related, antiparallel β-hairpin structures, commonly referred to as “flaps.”



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he conserved active-site residues (Asp25, Thr26, and Gly27 from both monomers) form a symmetrnd highly hydrogen-bonded arrangement virtually identical to that described for pepsin [17]. The twpartates are nearly coplanar with the “inner” carboxylate oxygens hydrogen bonded to the amide

ydrogens of Gly27/27'. This designation (e.g. Gly 27/27') will be used throughout this text to indicaquivalent residues of the dimer. The two threonines are inaccessible to solvent and are hydrogen-

onded to the main-chain amide groups of the other monomer, forming a rigid network called afireman's grip” [17]. As in the case of the structures of eukaryotic pepsins, there is electron density water molecule bound between the two carboxylates of the active-site aspartates.

n the structure of the apo-form of HIV PR, the flaps from both monomers are related byystallographic two-fold symmetry and can be considered as being in an open conformation. In theructures of related proteases from Rous Sarcoma Virus and HIV-2, the flaps are eitherystallographically disordered or in a partly closed conformation [18]. This suggests that, in solutioe absence of ligands, the flaps are rather flexible and that the stable conformation of the flaps obsethe crystal structure of the apo-enzyme of HIV PR could be considered to result from kinetic trapp

uring the crystallization process.

n the apo-form of HIV PR, the active site residues are located at the bottom of a rather shallow groopon binding an inhibitor, the protease undergoes significant structural changes, particularly apparee flap region. As a result, a tunnel-like site is formed, which runs diagonally across the dimerterface. The tunnel has a volume of approximately 1140 Å3 and is 23 Å long. Because of the dimer

ature of HIV PR, the active site has approximate two-fold symmetry with the dyad axis intersectingane of the catalytic aspartates. Along the active site tunnel, starting from the central aspartates, thee distinct subsites S1, S2, S3, and S4, and corresponding symmetry related subsites S1', S2' S3', an

4' (Figure 2). It should be noted that in this chapter, the convention of Schechter and Burger [19] we used to describe enzyme specificity subsites (S1, S1', etc.) and the corresponding side chains ofhibitors (P1, P1', etc.). The boundaries of the subsites are formed by residues from both monomersIV PR. All subsites, with the exception of S4/S4', which are exposed to solvent, are bounded by moiphatic side chains and have hydrophobic character. The borders of the S1/S1' subsites are formed e side chains of Ile23/23', Ile50/50', Ile84/84', Pro81/81', the γ carbon of Thr80/80', carboxylates of

ctive site Asp25/25', and the carbonyl oxygens of Gly27/27'. The S2/S2' subsites are bounded byal32/32', Ile50/50', Ile47/47', Leu76/76', Ala28/28', and the carboxylates of Asp30/30'. The S3/S3',

ubsites are partly exposed to solvent and are bordered by the side chains of Leu23/23', Val82/82',

ro81/81', and the guanidinium groups of Arg8/8', which form a salt bridge with the carboxylates ofsp29/29'. Most of



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Figure 2Schematic representation of the specificity subsites of the HIV PR active site with bound

peptidic inhibitor JG-365. Amino acids forming the boundaries of the particular subsites are shown.

e hydrogen bond donor and acceptor functional groups of the active site are located in an approximane that lies along the long axis of the tunnel and is somewhat perpendicular to the plane of the

ubsites. The hydrogen-bonding functionalities include the carboxylates of the catalytic aspartates, tharbonyl oxygens of Gly27 and Gly48, the amide nitrogens of Asp29' and Gly48, the carboxylate ofsp29', and the dimer symmetry-related groups on the other side of the active site. Additional group

apable of forming hydrogen bonds with ligands are located in the outer part of the S2/S2' subsite anclude the amide nitrogens and the carboxylates of Asp30/30'. There are five conserved waterolecules in the active site of HIV PR. Four of the waters are symmetrically distributed in the S3/S3

ubsites and one, hereafter called Wat301, is located near the two-fold axis of the dimer and, in theresence of most inhibitors, is approximately tetrahedrally coordinated by the hydrogen bonds formeetween carbonyl oxygens of the ligand(s) and the amide nitrogens of Ile50/50' of the flaps. In thegand-bound form of HIV PR, Wat301 is completely inaccessible to solvent, and it has been speculaat its functional substitution could be energetically favorable [18] or at least may lead to discovery

ovel nonpeptidic inhibitors [20]. Thus, there are 18 hydrogen bond donors or acceptors in



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e active site of HIV PR-16 that could form hydrogen bonds directly and two in which the interactionediated by the conserved Wat301. The total solvent accessible surface area of the eight subsites of thIV PR active site is approximately 1150 Å2. Because of the large number of groups with hydrogen-brming potential, 450 Å2 of the surface has a polar character, and the nonpolar area of the subsites isightly larger, approximately 700 Å2.

Structural F lexibili ty of HIV PR

the process of viral assembly, HIV PR specifically cleaves nine cleavage sites on GAG and GAG-Polypeptides [21]. Examination of the amino acid composition of the recognized substrate sites (Tabledicates their hydrophobic character and significant sequence variability. The loose specificity of HIVost likely reflects its functions in a world of reduced complexity within the confines of the buddingrion. The length of the viral protein precursors (approximately 1500 amino acids) reduces the numbe

otential sequences the protease must discriminate from in selecting its nine cleavage sites. Therefore,IV PR and other retroviral proteases are not enzymes that have evolved to carry out a single reaction

pid rate, but rather enzymes with minimum specificity required to cleave the viral precursors in apecific and orderly manner.

he loose specificity requirements demonstrated by effective binding and catalytic processing of all niquences, albeit at different rates [22], was the

Table 1 The Sequences of the Proteolytic Processing Sites of HIV-1

HIV-1 PRCleavage sites

Scissile bond

P17/P24 V S Q N Y P I V Q

P24/P2 K A R V L A E A M

P2/P7 S A T I M M Q R G

P7/P1 E R Q A N F L G K

P1/P6 R P G N F L Q S R

TF/PR V S F S F P Q I T

PR/RT C T L N F P I S P

RT/RN G A E T F Y V D G

RN/IN I R K V L F L D G

Schechter-Bergernotation

P5 P4 P3 P2 P1 P1' P2' P3' P4' P

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TF—transframe, PR—protease, RT—reverse transcriptase, RH—RNAse H, IN—integrase. The location of theprocessing sites in HIV-1 were determined by protein sequencing of HIV-1 virion proteins.



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rst indication that the recognition subsites of the HIV PR can display flexibility upon binding ofubstrates or inhibitors. Early crystal structures of the HIV PR apo-enzyme and complexes with pepthibitors showed several conformations of the active site forming flaps, which include the residues

Met46/46' to Ile54/54' [15,16]. Increased availability of coordinates of HIV PR complexed with varihibitors and crystallized in different crystallographic space groups allowed for more rigorous

xamination of domain movements and structural changes in the active site.he alignment of several crystal structures of HIV PR in a common frame of reference, which mostommonly includes the region around the symmetryrelated active site triad Asp25/25'-Thr26/26'-ly27/27', will highlight those regions of the backbone where significant displacement occurs upon

ccommodating the individual inhibitors. Examination of the aligned structures, which includedxamples of all classes of inhibitors, indicated only small variation of the backbone and limitedovements in the two binding loops, comprising residues Leu76-Ile84 from both monomers. The lorm the outer walls of subsites S1/S1' and S3/S3' with inward-facing hydrophobic side chains ofoleucines and valines. The flexibility of these loops, which in some cases can move outward by asuch as 2.5 Å, has a significant impact on the volume of the specificity subsites, which in turn can

ccommodate corresponding P1/P1' and P3/P3' moieties of various sizes. Interestingly, the predominsistance-causing mutations are located on the same loops and involve changes in residues Val82/8

nd Ile84/84' (see below). It should be noted, that while the alignment of several crystal structuresrovides information about the flexible regions, the extent of flexibility of the residues around the HR active site can be limited by crystal packing forces and may represent a crystallographic artifact. l characterized crystal forms of HIV PR [23] the loops 76–84 and 46–56 participate in crystal latticrmation and the particular conformation of these loops can be driven by crystallization conditions teractions with other molecules related by the crystallographic symmetry.

. I nhibitors of HIV PR

n general, inhibitors of HIV PR can be divided into three distinct groups. The first group includeseptidic inhibitors that utilize various transition-state dipeptide analogs such as statine, hydroxyethynd hydroxyethylamine incorporated into peptidic frameworks of differing lengths. Several crystalructures of this type of inhibitor complexed with HIV PR were solved and the structural informatiorovided a wealth of information as to the minimum size of inhibitors, geometry of hydrogen bondsrmed within the active site, and the structural flexibility of the subsites (for reviews see Reference

nd 23). The second and perhaps largest group of HIV PR inhibitors includes peptidomimetic



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ompounds that utilize similar transition-state analogs and retain at least one peptide bond with a sidhain corresponding to a naturally occurring amino acid. Several compounds from this group havexcellent pharmacokinetic and antiviral properties and, in fact, all three HIV PR inhibitors approvedinical use (saquinavir, ritonavir, and indinavir) belong to this class of compounds. The last and the

mallest group of HIV PR inhibitors has a distinct nonpeptidic character. Compounds from this class

ere discovered either by screening libraries of existing compounds or by structure-based de novo esign. Illustrative examples of inhibitors belonging to all three classes and a brief description of thescovery of selected compounds are presented below.

. Peptidic I nhibitors of HIV PR

he concept of peptidic inhibitors of HIV PR can be exemplified by the crystal structure of the statinontaining peptidic compound AG1002 (Figure 3) [23]. In AG1002, the statine moiety replaces theissile dipeptide while the flanking amino acids were derived from the natural substrate cleaved by

R. The inhibitor binds to the active site in an extended conformation with the central hydroxyl grou

e statine moiety forming hydrogen bonds with the active-site aspartic acids 25/25'. The peptidicackbone and the side chains of the

Figure 3Stereo view of the peptidic inhibitor AG1002 bound to the active site of HIV PR.

The distribution of the specificity subsites S and S' is similar to that shown inFigure 2. The boundaries of the HIV PR active site are indicated by the dotted

surface.



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hibitor form 16 hydrogen bonds and occupy subsites from S4 to S1, S2', and S3'. The carbonylxygens of P2 and P1' accept two hydrogen bonds from the flap water Wat301, which in effect is netrahedrally coordinated. Due to the structural nature of statine, which lacks the P1' side chain, the Socket remains unoccupied. The S1 subsite is only partially filled by the P1 side chain of leucine. Th2 and P2' side chains of asparagine and glutamine form hydrogen bonds with Asp30' and 30, while

iphatic carbons of both side chains make several hydrophobic contacts in the S2 and S2' pocketsspectively. Despite the large number of hydrogen bonds formed within the HIV PR active site,G1002 has rather low inhibitory potency with a binding constant of 0.55 µM The low binding consost likely reflects the absence of the P1' group, the free energy required for desolvation of the

ydrophilic side chains, and the charged N- and C-termini as well as entropic effects caused by theexible nature of the heptapeptide.

ther interesting examples of peptidic inhibitors are compounds utilizing other transition-state analog. reduced amide-containing hexapeptide MVT-101 [24], hydroxyethylene-containing octapeptide 5548e [25], and hydroxyethylamine-containing heptapeptide JG-365 [26]. All these compounds bine active site of HIV PR in a similar extended conformation and the small differences in the geomet

f hydrogen bonds formed with HIV PR can be attributed to the different character and length of theansition-state analogs. The chemical structures and inhibition constants of these inhibitors areummarized in Table 2. Note that the inhibition constants cited throughout this chapter and in Tables

and 6 were determined in different laboratories—often using significantly different assayonditions—and therefore might not be meaningfully comparable.

ue to their substantial size and peptidic nature, inhibitors from this class were not suitable for clinicpplication. Nevertheless, the structural information derived from many crystal structures of peptidic

hibitors bound to the HIV PR active site was critical for subsequent modeling and design of the neeneration of peptidomimetic and nonpeptidic inhibitors of HIV PR.

. Peptidomimetic I nhi bitors of H IV PR

esign and Structure of Ro-31-8959 (Saquinavir)

he strategy of designing saquinavir was based on the transition-state mimetic concept, an approachas been used successfully in the design of potent inhibitors of renin and other aspartic proteases [10rom the variety of nonscissile transition-state analogs of a dipeptide, the hydroxyethylamine mimetas selected because it most readily accommodates the amino acid moiety characteristic of the Phe-

nd Tyr-Pro cleavage sequence of the



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troviral substrates. In the first step of design, the dipeptide analog consisting ofhe[CH(OH)CH2 N]Pro was used to determine the minimum sequence required for potent inhibitionrom this study a compound was selected that included benzyloxycarbonyl at the N-terminal side ofhibitor followed by the P2 asparagine, the hydroxyethylamine isostere with side chains of

henylalanine and proline in the P1 and P1' positions respectively and the NH-t- butyl group at the Crminal part. In the following design, the side chain of proline was consequently modified to aperidine and finally to a decahydroisoquino-



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ne moiety, and the N-terminal benzyloxycarbonyl group was replaced by the quinoline-2-carbonyl.sulting compound, Ro-31-8959, was one of the first peptidomimetic inhibitors with very high antiv

otency and became a benchmark for further design of HIV PR inhibitors [10].

he high-resolution crystal structure of saquinavir bound to the active site of HIV PR was solved inany laboratories [23,27]. The incorporation of decahydroisoquinoline moiety, which can be consid a conformationally restrained mimic of cyclohexylalanine, has some important consequences. Firse length of the C-terminal part of the inhibitor has been restricted to the P2' residue which, in

aquinavir, consists of a NH-t- butyl group. Second, it restrained the conformational freedom of theherwise peptidic backbone, minimizing the entropic penalty to the free energy of binding. In theystal structure of saquinavir with HIV PR (Figure 4), the decahydroisoquinoline in the preferred ch

hair conformation, makes extended hydrophobic contacts in the S1' subsite. The bond between theethylene carbon and the nitrogen of decahydroisoquinoline is in the low-energy equatorial

onformation and the nitrogen, even if protonated, is not in a position to form a hydrogen bond with ctive-site residues. The central hydroxyl group is in the R(syn) conformation and is within theydrogen-bond-forming distance with both carboxylates

12640-0012a.gif

Figure 4Stereo view of the peptidomimetic inhibitor Ro 31-8959 (saquinavir) bound

to the active site of HIV PR. The distribution of the specificity subsites S andS' is identical to that shown in Figure 2. Note the stacking interaction

between the quinoline moiety and the P1 side chain of phenylalanine.



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f the active-site aspartates. Similar to Ag1002 and other peptidic inhibitors, the carbonyl oxygens o2 and P1' amides are within hydrogen-bonding distance of the flap water molecule; however, theeometry of the second hydrogen bond is distorted due to the additional spacing between both carboroups. The nitrogen of the t- butylamide is displaced from the normal P2' position by approximatelyand, as a result, cannot form a direct hydrogen bond with the carbonyl oxygen of Gly27. Instead th

utylamide nitrogen interacts via highly ordered water molecules with the amide nitrogen of Asp29 e carbonyl oxygen of Gly27. The aliphatic t- butyl moiety occupies the S2' subsite and the positione backbone in this region prohibits any further extension into the S3' pocket. The P1 and P2 side

hains of phenylalanine and asparagine, respectively, occupy the corresponding subsites and have amilar conformation to the equivalent groups observed in peptidic inhibitors. In the crystal structure-terminal quinoline-2-carboxylate is moved to the side and, as a result, the carbonyl oxygen formsydrogen bonds with the ordered water molecule and with the amide nitrogen of Asp29'. The quinolng is in a low-energy conformation with respect to the preceding carbonyl oxygen, which places thomatic nitrogen in unfavorable close contact (3.3 Å) to the carbonyl oxygen of the flap Gly48.

ecause of the absence of any further contacts with the HIV PR active site residues, the contributione quinoline moiety to the free energy of binding remains unclear. Perhaps in solution, a stackingteraction of the P1 phenyl ring and the aromatic quinoline restricts the conformational freedom of R

1-8959, in effect diminishing the free-energy loss due to the entropic and desolvation effects.

aquinavir, despite its distinct peptidomimetic character is a very potent inhibitor of HIV PR with anhibition constant of 0.9 nM and an antiviral IC50 in vitro of 0.020 µM [10]. Although it suffers frow oral bioavailability (5–10% in humans), it became an important starting point for the design ofcond generation, less-or nonpeptidic inhibitors. Saquinavir became the first HIV PR inhibitor

pproved by the FDA for treatment of AIDS.

esign and Structure of ABT-538 (Ritonavir)

n interesting concept for designing specific HIV PR peptidomimetic inhibitors with internal two-foymmetry was first formulated by John Erickson and his colleagues from Abbott Laboratories [28].hey reasoned that if HIV PR incorporates symmetry into its active site structure, compounds thatimic this symmetry might be novel, more specific, and potent inhibitors and, furthermore, due to thdirectionality of peptide bonds, might be sufficiently less peptidic in character and pharmacologica

uperior to the classical peptide-based compounds. The crystal structure of one of the first compound

om this series (A74704) verified the assumption of symmetrical binding conformation in the

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ctive site of HIV PR. The inhibitor consists of the central diamino alcohol moiety with symmetricalstributed phenylalanine side chains and two flanking, Cbz-blocked, valine residues. Except for theymmetric hydroxyl group, A74704 binds to the active site in a symmetric mode and the inconsistestribution of the terminal Cbz groups is most likely caused by crystal lattice contacts and may notflect the binding mode in solution [28].

he design of symmetrical inhibitors was further extended to include a series of diamino, diol core uwhich the C2 axis bisects the bond connecting the two hydroxy-bearing carbon atoms [29]. Suchhibitors consistently showed greater potency than A74704, but the relative potencies of the diolsffered for different diastereomers, and they did not exhibit a uniform dependence on theereochemistry at the hydroxymethyl position. Surprisingly, high-resolution crystal structures of HIR with all possible diol diastereomers, (S,S, R,R and R,S) revealed that most of the inhibitors bind early asymmetric fashion placing only one of the diol hydroxyl groups on the C2 axis dissecting th

ctive site of HIV PR and the catalytic carboxylates of Asp25/25'. The asymmetric placement of dioauses translation of inhibitors along the long axis of the active site and, as a result, the midpoint of tompounds is displaced by up to 0.9 Å from the two-fold axis of the HIV PR. Nevertheless, the dihengles of the symmetry-related bonds are in most cases within 10°, and the inhibitors maintain overaymmetry in the bound conformation [23,29].

he ABT-538 design was a direct consequence of the pioneering work with peptidomimetic compouith the internal C2 symmetry [30]. Since the high-resolution crystal structures of a family of diol-

ontaining compounds indicated that in most cases only one of the diol hydroxyls interacts with theatalytic aspartic acids 25/25', in subsequent designs the noninteracting hydroxyl group was replacedhydrogen. This substitution reduced the free energy penalty required for desolvation of the hydroxy

roup and increased the inhibitory potency without perturbing the binding mode of the compounds [n the further search for related inhibitors with improved oral bioavailability, the focus of effortoncentrated on the effect that molecular size, aqueous solubility, and hydrogen-bonding capability hn pharmacokinetic behavior. This study resulted in a smaller compound, A-80987, in which the P2'de chain of valine was eliminated and the terminal 2-pyridinyl group was replaced by 3-pyridinyloiety that makes van der Waals contacts in the S2' subsite and forms a hydrogen bond with the amtrogen of Asp30 [31]. The pharmacokinetic properties of A-80987 were significantly improved ovrger, symmetrical compounds from this series and, at the same time, the high antiviral activity typir these inhibitors was largely unaffected. In subsequent optimization, which focused on the metabo

ability of these inhibitors in vivo, the electron-rich and oxidation-prone pyridinyl groups were reply thiazoles.



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hiazoles are less electron-rich isosteres of pyridines and therefore it was speculated that compoundith such substitution may have improved metabolic stability [30]. The modeling of A-82200 in whie N-terminal pyridinyl group was substituted by a 4-thiazolyl moiety indicated that the 5-membereng binds in the S3 subsite and can be further derivatized at the 2 position by an isopropyl group. Thopropyl functionality makes van der Waals contacts with Val82 and fills the hydrophobic part of th3 subsite in nearly optimal fashion.

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he resulting compound, ABT-538 (Table 3), binds to the active site of HIV PR in an extendedonformation. The central, asymmetric hydroxyl group is within hydrogen-bonding distance of theatalytic aspartates 25/25', and the P1/P1' phenylalanine side chains are symmetrically distributed inorresponding subsites. The nitrogens of the symmetric amide bonds on both sides of the centralminoalcohol are barely within the hydrogen-bonding distance of the carbonyl oxygens of Gly27/27

.4 Å) and the carbonyl oxygens of these amide bonds participate in the tetrahydral coordination ofap water molecule Wat301. On the N-terminal side of the compound, the P2 side chain of valine file S2 subsite and the terminal 2-isopropyl-4-thiazolyl makes hydrohobic contacts with the residues e S3 pocket and has a stacking interaction with the P1 phenylalanine. On the C-terminal side, the 5iazole is positioned to interact within the S2' subsite, and the nitrogen on the 5-membered ring isithin hydrogen-bonding distance of the amide of Asp30'.

espite two peptide bonds present in ABT-538, this compound has substantial oral availability inumans and a very high antiviral activity in vivo [30]. Recently, ABT-538, better known as ritonaviras been approved by the FDA for treatment of AIDS in combination with inhibitors of the reverseanscriptase.

esign and Structure of L-735,524 (Indinavir)

ndinavir is another example of very potent peptidomimetic compound discovered using the elemente crystal structure-based design [32] and SAR (structure activity relationship). The starting point foe design was a series of compounds containing the hydroxyethylene isostere of a scissile dipeptide3]. An example of compounds from these series is L-685,434, which consists of a tert-butylcarbam

roup forming the P2 moiety, symmetrically distributed phenylalanine side chains in the P1/P1', and

danol group in the P2' portion of the inhibitor. Although very potent, the optimized molecules fromis series lacked aqueous solubility and an acceptable pharmacokinetic profile [32]. The Merck grouypothesized that incorporation of a basic amine-containing functionality, such as theecahydroisoquinoline group of saquinavir, into the backbone of L-685,434 series might improve theolubility and bioavailability of this type of compound. Also the replacement of the P2/P1unctionalities, the tert-butylamide and phenylalanine side chain by the decahydroisoquinoline tert-utylamide, would generate a novel class of hydroxylaminepentanamide isostere with potentiallymproved metabolic stability in vivo. An additional strong argument for using decahydroisoquinolinn isostere of P1/P2 moieties was the restricted conformational freedom of the enclosed-into-a-ring

asic amine, which should decrease the entropy change upon binding to HIV PR in a similar fashionat observed in saquinavir. In



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e resulting chimeric inhibitor the central hydroxyl group forms hydrogen bonds with the catalyticpartic acids 25/25' and the hydrophobic side chains of the P1/P1' decahydroisoquinoline and

henylalanine respectively are separated from the central hydroxyl-bearing carbon by the methylenenkers forming a pseudosymmetrical arrangement. In the subsequent optimization of inhibitors fromis novel series, a smaller piperazine group was substituted for the decahydroisoquinoline group, wh

ffered a possibility to expand from the N4 position to the partially lipophilic S3 subsite. One of the ompounds from the piperazine series possessed a benzyloxycarbonyl moiety attached to the piperazng and the additional hydrophobic interaction in the S3 subsite was reflected by substantial increasoth intrinsic potency and in the ability to inhibit viral spread in infected cells in vitro. Finally, theplacement of the benzyloxycarbonyl group by the 3-pyridylmethyl moiety (Table 3) provided bothpohilicity for binding to the HIV PR active site and a weakly basic nitrogen that increased aqueousolubility and oral bioavailability. The crystal structure of L-735,524 (indinavir) bound to the active f HIV PR [34] indicates that the 3-pyridylmethyl group attached to the N4 position of the piperazinng makes hydrophobic contacts with the residues in the S3 and S1 pockets and the tert-butyl moiety

lls the S2 subsite in the fashion previously observed in the structure of saquinavir. The positions of2 and P1' carbonyls maintain the proper alignment to form hydrogen bonds with the flap waterWat301. The terminal indanol group of indinavir occupies the S2' subsite with the hydroxyl group

ithin hydrogen-bonding distance of the amide nitrogen of Asp29.

he high aqueous solubility and largely nonpeptidic character of indinavir may be responsible for thood oral bioavailability, respectable pharmacokinetic profile, and high antiviral activity observed wis compound. Similar to saquinavir and ritonavir, indinavir has been recently approved by the FDAeatment of AIDS.

. Nonpeptidic I nhibitors of H IV PR

he nonpeptidic inhibitors of HIV PR can be divided into two subclasses. Compounds that belong torst group maintain the general binding mode of the peptidomimetic inhibitors including formation e key hydrogen bonds with the active site residues. An example of such nonpeptidic inhibitors of HR is AG1343 (nelfinavir). The second group of nonpeptidic HIV PR inhibitors includes compoundsith a binding mode significantly different from that described for the peptidomimetic compounds.

Most inhibitors in this latter class were initially discovered by screening natural-products libraries orructurebased de novo design. The most interesting examples of the nonpeptidic inhibitors from this

roup are the independently discovered but structurally



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lated 4-hydroxypyrans and 4-hydroxycoumarins, the cyclic urea-based DMP323 series, and AG12

esign and Structure of AG1343 (Nelfinavir)

nalysis of the crystal structure of saquinavir with HIV PR indicated that while the nonpeptidicomponents of the ligand, namely the decahydroisoquinoline and the t-butylamide moieties fill the S

nd S2' subsites nearly optimally, the N-terminal portion offered the possibility for remodeling, aimee elimination of the peptidic character. Also, the contribution of the quinoline to the binding affinitIV PR was difficult to rationalize. Since the removal of quinoline resulted in a nearly 1000-fold losnding constant, it was concluded that the stacking interactions of the P1 phenyl ring and the P3omatic moiety of quinoline are necessary for the conformational stability of Ro 31-8959. In an atteredesign the N-terminal part of the ligand, the nonpeptidic portions of the P1' and P2' were mainta

ut for reasons of synthetic accessibility, the decahydroisoquinoline moiety was replaced by an orthoubstituted benzylamide [35]. Crystallographic analysis of both compounds showed that saquinovir ae modified LY289612 bind essentially identically to the active site of HIV PR and their inhibition

onstants and antiviral activity were very similar (Table 4 and Table 3).

n the first attempt to functionally substitute the P2 side chain of asparagine, the isophthalic-acid-ontaining compound was modeled and the low-energy conformation of the aromatic ring, required fnding in the S2 subsite, was stabilized by a tertiary carboxamide in the P3 region of the inhibitor [3he analysis of the binding mode and interactions of the isophthalic ring in the S2 subsite indicated apophilic pocket deep on the border between the S2 and S1' subsites, which could be conveniently fith a methyl group extending from the 2 position of the ring. The resulting compound II in Table 4ost of the peptidomimetic character of LY289612 but retained its inhibitory potency.

n an independent line of design, the relationship between the P1 phenylalanine side chain and the P3uinoline was investigated. In the crystal structure of saquinavir bound to the active site of HIV PRFigure 4), the aromatic ring of the P1 phenylalanine makes several van der Waals contacts withsidues forming the S1 subsite. Computer modeling indicated that an extension of the phenylalaninede chain to phenethyl (homophenylalanine) would lead to prohibitive close contacts of the phenyl rith the aliphatic side chains of HIV PR. On the other hand, replacement of the γ -carbon of theomophenylalanine by sulphur, which has a more acute C-S-C bond angle, would direct the aromating into the neighbouring S3 subsite without changing the desired lipophilic nature of the P1 side ch

he increased area of hydrophobic interactions in the S1 and S3 subsites by compounds with the S-henylcysteine and



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naphthylcysteine derived side chains in P1 resulted in a substantial increase in the inhibition const7]. The increase in the binding affinity to the low picomolar range in enzyme inhibition assay, allor subsequent truncation of the P3 quinoline moiety. The final compound from this miniseriesompound III in Table 4) consisted of the ortho-substituted benzamide in the P1' and P2', S-

aphthylcysteine in P1 and asparagine in P2. Despite reduced molecular weight, the inhibition const

f this compound for HIV PR was comparable to LY289612.he observation that a larger, nonpeptidic moiety in the P1 could eliminate the need for the P3 sidehain led to hybrid molecules that incorporated ring structures as the P2 component and maintained 1 S-naphthylcysteine side chain of compound III. In this miniseries several bicyclic functionalitiesere modeled as the P2 substituents and one example, compound IV utilizing a tetrahydroquinolineroup, is shown in Table 4 [38]. In subsequent modeling, it was noticed that the P2 bicyclic functionight be replaced by 2,3-disubstituted phenyl rings. In particular, a methyl substitution in position 2ould increase the area of hydrophobic interaction in a manner previously observed in the isophthalries. Addition of a hydrophilic functionality attached at position 3 could increase the solubility of t

ompound and contribute to the binding constant by forming a hydrogen bond with the carboxylatexygen of Asp30. A compound with a 2-methyl-3-hydroxy substitution pattern was synthesized andhowed an improved inhibition constant of 3 nM in the HIV PR enzyme assay (Table 4). The crystalructure of compound V with HIV PR was solved and indicated the predicted binding mode with thossibility of a stacking interaction between the P2 phenyl and the P1 thio-naphthyl groups and thexpected hydrophobic and hydrogen-bonding interactions of the P2 moiety with the protein side cha

the corresponding specificity pocket [38].

s with the optimized compounds from other series, compound V suffered from low aqueous solubi

he replacement of the P1' aryl group by the basic amine-containing decahydroisoquinolineramatically increased the solubility and allowed for truncation of the P1 S-naphthylcysteine side ch

S-phenylcysteine without any loss of inhibitory activity. The resulting compound VI, AG1343 orelfinavir, has an inhibition constant of 1.9 nM in the HIV PR enzyme assay and respectable antiviractivity with an IC90 of 60 nM [39]. The nonpeptidic character, pH-dependent solubility profile, and mall molecular weight of nelfinavir may contribute to its good pharmacokinetic profile in humans0,41]. Currently, this compound is being tested and is in the advanced phase of clinical trials.

he crystal structure of nelfinavir bound to the active site of HIV PR is shown in Figure 5. The gene

nding mode of this compound, in particular the path of the backbone, is similar to the binding modeptidyl inhibitors. Nevertheless, the lack of any peptide bonds utilizing naturally occurring amino



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Figure 5Stereo view AG1343 (nelfinavir) bound to the active site of HIV PR.

cids qualifies nelfinavir to be a member of the group of nonpeptidic inhibitors of HIV PR. The uniqnd perhaps crucial hydrogen-bonding interaction of the P2 hydroxyl group with the carboxylate oxyf Asp30, combined with the smaller area of hydrophobic contacts in the S1 and S3 specificity subsi

e the principal differences from other clinically active HIV PR inhibitors and may contribute to astinct resistance pattern and point to additional utility of nelfinavir in the treatment of AIDS.

esign and Structure of DMP323

cyclic urea-containing HIV PR inhibitor, DMP323, was discovered using de novo structure-basedesign principles. Similar to the concept of Erickson and his co-workers from Abbott Laboratories, troup from DuPont-Merck attempted to take advantage of the two-fold symmetry of HIV PR inesigning compounds that maintained the interaction of the diol with the catalytic aspartic acids 25/2nd at the same time were able to functionally displace the ubiquitous flap water molecule Wat301.

hey hypothesized that incorporation of the binding features of this structural water molecule into anhibitor would be beneficial because of the entropic gain due to its displacement and because the

onversion of a flexible linear inhibitor into a rigid, cyclic structure with restricted conformation shorovide an additional, positive entropic effect. In the initial design, a cyclohexanone with the ketonexygen as the structural



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ater mimic was used and in subsequent synthetic targets the cyclohexanone ring was enlarged to a embered ring to incorporate a diol functionality. This target was further modified to a cyclic urea,hich can be symmetrically substituted from both nitrogens without creating unnecessary stereocenthe crystal structures of about 10 cyclic-urea-based inhibitors with HIV PR were solved [42]. In allases, the C2 symmetric inhibitors bind to the HIV PR active site with the diad symmetry axes of the

rotease and the compounds being nearly coincident. The 7-membered ring of the inhibitors is rougherpendicular to the plane of the catalytic aspartates 25/25' and both hydroxyl groups of the diol areositioned to interact with their carboxylates. The carbonyl oxygen of the inhibitors accepts hydrogeonds from backbone amides of symmetrically distributed residues Ile50/50' of the flap. In the structf DMP323, symmetrically substituted moieties of hydroxymethylbenzyls and phenylalanines extendwards the S2/S2' and S1/S1' subsites respectively and are involved in van der Waals interactions we hydrophobic residues of these pockets [42].

he interaction of DMP323 with the residues of HIV PR are restricted to the central four specificityubsites of the active site. Despite this limited area of hydrophobic interaction and hydrogen bondingstricted to the central cyclic urea functionality, DMP323 is a very potent inhibitor of HIV PR with

ood antiviral activity in vitro (Table 5). The limited solubility of this compound was perhapssponsible for erratic oral availability in humans, and after short trials, DMP323 was withdrawn froe clinical investigation. Nevertheless, the discovery of this class of compounds represents a veryteresting and, by now, classical example of de novo structure-based drug design.

esign and Structure of AG1284

nother compound discovered by the application of de novo structure-based design is AG1284 [43]

e initial design of a lead compound, the nonpeptidic hydroxyethyl-t-butylbenzylamide portion ofY289612 occupying the S1' and S2' subsites was retained as a “starting module.” In attempting to fe pockets related by the dimer two-fold symmetry it was discovered that, by extending a two-carboagment from the central hydroxyl carbon, the S1 subsite could be accessed by an aromatic ring. Thng was oriented orthogonal to the observed P1 phenyl group of the classical inhibitors and this allo

urther extension off the ortho position towards the S2 subsite. In order to maintain the critical hydroond to the flap water Wat301, in the initial compounds an acylated amino group was used, replacedubsequent designs by a benzamide functionality. In this model, the geometry of the hydrogen bondse flap water was somewhat perturbed, and the nitrogens of the t-butyl amides on both sides of the

ompound were in a position to interact favorably with solvent,



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nding constant and also in reduced aqueous solubility. Also, due to the very tight fit of both naphthoieties in the S1 and S1' subsites, subsequent design targeting the S3/S3' subsites proved to be diff

nd synthetically challenging [44]. In the search for a simpler solution, the di-tertiary amides wereesigned using the crystal structure of compound II (Table 6) as a starting model. Branching from thmide nitrogens provided an interesting possibility to access S2–S3/S2'–S3' subsites while

multaneously increasing the solubility and stability of the compounds. In the first design, theydroxyethyl moieties were fused to the amide nitrogens and the hydroxyl groups were intended to fydrogen bonds with the amide nitrogens of Asp29/29' (compound III in Table 6). The addition of bydroxyethyl groups resulted in a rather significant increase in the binding constant, and the racemicixture had the K i of 1.1µM. When the crystal structure of compound III complexed with HIV PR w

olved at 2.2 Å resolution, it was observed that the inhibitor had undergone an inversion in bindingode relative to the secondary amide series. The phenyl groups of compound III occupied the S2/S2

ubsites, switching positions with the t- butyl groups, which were in turn occupying the S1/S1' pockeFigure 6). Due to this change in binding mode, the R enantiomer would be expected to be preferred

lative to S. The final position of the hydroxyethyl moieties was less effected by the change, and boydroxyls were within hydrogen-bonding distance from the amide nitrogens of Asp 29/29'. In the S2ockets, the phenyl groups occupied only a fraction of subsites, but the interaction was strengthenedghly ordered water molecules involved in electrostatic interaction with the aromatic rings and byrming hydrogen bonds to Asp30/30'. Interestingly the position of the hydrogen bonds with respecte flap water was significantly disturbed in the new binding mode, and the conserved Wat301 was nnger tetrahydrally coordinated [43,45].

his unanticipated change in binding mode presented a potential for new avenues of design differentom those of the secondary amides. The ability to design into neighboring subsites depends to a largxtent on the positions of bond vectors suitable for substitution in the bound conformation of a givenhibitor. These vectors in the crystallographically discovered new binding mode of compound III w

ositioned ideally to access unfilled space in the S3/S3' pockets. The discovery of this new conformaf compound III highlighted the power of crystallographic feedback in the process of inhibitor designd, without this structural information, further design in this series would have been severely imped

nspection of the crystal structure of compound III bound to the active site of HIV PR revealedpophilic cavities extending off the S1/S1' subsites adjacent to the t-butyl groups of the benzamidineoiety. The cavities are bordered by flexible loops around Pro81/81' and previous crystallographic

udies indicated that both loops can move back by up to 2.5 Å, extending the size and



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Figure 6Change of the binding mode of compound III observed during

iterative design of AG1284. (a) Crystallographically determined binding mode of compound II. Pseudosymmetrically distributed aryl groups

are bound in the S1 and S1' specificity subsites. (b) Crystallographicallydetermined binding mode of compound III. Note the inversion of the binding

mode. The ortho-substituted benzyl groups bind in a pseudosymmetricfashion in the S2 and S2' subsites.



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olume of the active site. With this in mind, the dimethylbenzyl group was attached to compound IIIe additional phenyl ring was accommodated well in the lipophilic pocket of the S1'/S3' sides. As th1 pocket was not fully occupied, a Monte Carlo-based De Novo Ligand-Generating programMCDNLG) [46] was used to identify other amide substituents that might fill this subsite moreffectively. From several moieties identified by the MCDNLG program, a larger cyclopentylethyl gr

howing very good shape complementarity to the S1/S3 subsite was selected for synthesis. In additioue to the asymmetrical nature of this compound, additional space was identified at the bottom of th2' pocket that was conveniently filled with either a methyl or a chlorine group on the 5 position of tenzamidine ring. The inhibition constant of the resulting compound (compound IV in the Table 6) w008 µM, which represents approximately a 2500-fold improvement over the first compound from tries.

he crystal structure of compound IV or AG1284 complexed with HIV-1 PR was solved, revealingxcellent complementarity between the ligand and protein. The ligand forms only 4 hydrogen bondsith either protein functional groups or ordered water molecules, in contrast to the nine hydrogen bormed by peptidomimetic LY289612, despite their similar binding affinities. The nonpeptidic chara

f AG1284 may have contributed to good oral bioavailability and pharmacokinetics in three animalpecies [43].

espite very good inhibitory potency on the enzyme level, AG1284 has rather modest antiviral activvitro (Table 6). The reason for this discrepancy is unclear but could be related to the low water

olubility and higher affinity for membranes, which may effect cell partitioning. A similar lack oforrelation between the potency of enzyme inhibition and antiviral activity has been previously obseith other HIV PR inhibitors [11].

ydroxypyrans and Hydroxycoumarins

he lead compounds for the 4-hydroxypyran and 4-hydroxycoumarin series were discovered inological screens as low potency inhibitors of HIV PR [47–49]. Successful structure solution of botad compounds with HIV PR enabled rapid optimization of their enzyme inhibitory potencies and aIV activities, and one of these compounds, U96988, has already entered Phase I clinical testing9,50]. The binding mode of this type of inhibitor differs substantially from the classical

eptidomimetic compounds and is somewhat similar to de novo-designed compounds from the cycli

rea series. In the case of 4-hydroxycoumarin, the two oxygen atoms of the lactone functionality areositioned within hydrogen-bonding distance of the two NH amides of Ile50/50' on the flap, replacine ubiquitous water molecule Wat301. The 4-hydroxyl group (Table 5) is located within hydrogen-

onding distance of the two catalytic



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partic acid residues Asp25/25' and this hydrogen-bonding network of the 4-hydroxycoumarin define essential pharmacophore of this new class of inhibitors. In the structure of U96988, this

harmacophore is pseudosymmetrically subsituted by an ethyl and a phenyl group at the C-3a and anhyl and a benzyl group at the C-6a positions. These four substituents extend into the central core of2/S2' subsites, where they make van der Waals contacts with the hydrophobic residues of the active

9]. With a molecular weight of 362 U96988 is the smallest inhibitor of HIV PR in clinical testing. uffers from rather low antiviral activity (ED90 of ~ 10 µM)but can be considered as the first in a serf this promising class of nonpeptidic HIV PR inhibitors.

. Str uctur al Basis of Resistance of HIV PR Inhibitors

he dimeric character and the two-fold symmetry of the active site, in which the monomers contribuquivalent residues to symmetrically distributed specificity subsites, led to early speculations that HIR may be less susceptible to resistance than, for example, reverse transcriptase. In the case of retrov

roteases, a single base mutation in the viral genome corresponds to two changes in the three-mensional structure and two structurally identical changes in the active site could result in an enzyith a drastically modified specificity profile and impaired catalytic activity. Identification of HIV Pariants in cell-culture experiments clearly indicated, however, that this class of drugs is not immunee challenge of viral resistance. It should be stressed that HIV, unlike other human viruses, is

haracterized by a dynamic viral turnover in the steady state [51,52]. The rapid replication rate coupith the lengthy duration of infection will favor the emergence of resistant mutants to targeted antiv

gents [53].

he accumulated data from cell-culture sequential-passage experiments with several structurally

fferent inhibitors and from the resistant variants identified during clinical exposure to four HIV PRrgeting drugs indicate a very complex pattern of mutations in the structure of HIV PR. In contrast tutations in the reverse transcriptase, which frequently cause multihundredfold resistance [54], sing

ase changes in the HIV PR gene (i.e., two identical substitutions per protease dimer) lead in most c5–10-fold decrease in the antiviral potency of a given drug [11]. It has been shown for the most

inically studied HIV PR inhibitors, such as indinavir and ritonavir, that the clinical manifestation osistance (increase in the viral load and decrease in the CD4 count) requires the simultaneous

ppearance of several mutations [55,56]. For example the resistant HIV strain isolated from patientsxposed for 40 weeks to indinavir carried mutations at residues 10/10'L > R, 46/46'M > I, 63/63'L >

2/82'V > T, and 84/84'I > V [59,60]. However, the combination of these five



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Figure 7Cartoon representation of the HIV PR dimer. The sites of primary

resistance-causing mutations in the active site are indicated. For clarity, the names of theresidues are shown for one monomer only.

utations (ten assuming the dimeric nature of HIV PR) changed the susceptibility of the resistant strindinavir by only eight-fold if compared to the wild type HIV.

he resistance-causing mutations are localized in a few “hot spots” in the structure of HIV PR and ce divided into two groups. The first group consists of the primary mutations located directly in thective site and includes changes at residues Val82/82', Ile84/84', somewhat less frequently at Gly48/nd, in the case of nelfinavir, Asp30/30' (Figure 7). Residues 82/82' and 84/84' are located on theexible loops that form the outer walls of the S3/S3' and S1/S1' subsites, respectively. In the resistanariants, valine 82/82' is most frequently substituted by the smaller side chain of alanine or the largede chains of phenylalanine or isoleucine [57,58]. The change in position 82/82' is usually accompay a substitution of Ile84/84', most commonly to the smaller amino acids alanine or valine [57]. From

e clinically tested compounds, ritonavir and indinavir, which were optimized to form strongydrophobic interactions with the side chain of Val82/82' in the S3 subsite, suffer most significantlyom any change at this position. On the other hand, the antiviral activities of saquinavir, and nelfinahich do not form any interaction in the S3/S3' subsite are not affected by mutations at Val82/82' ane only marginally cross resistant to changes involving Ile84/84' [57,58,62]. The resistance-causing



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utation of Asp30/30' to asparagine seems to be specific for nelfinavir and was initially observed inulture sequential passage experiments [62]. Recently, the same phenotypic change was confirmed ae predominant mutation in the resistant variants appearing in AIDS patients exposed to low doses ois HIV PR inhibitor [64]. The molecular basis of resistance involving this mutation can be rational follows: in the crystal structure of nelfinavir with the wild type HIV PR, the 3-hydroxyl group of

3-substituted phenyl group is within hydrogen-bonding distance of the carboxylate oxygen of Asp3e S2 subsite. Due to the expected coulombic character of this interaction, the hydrogen bond formeith the negatively charged carboxylate of Asp30 would be expected to be a relatively strong one. T

hange of the negatively charged carboxylate of Asp30/30' to the amide oxygen of the asparagine sidhain should reduced the strength of this interaction. Apparently the loss in the enthalpic contributioe free energy of binding is only partially balanced by the entropic gain caused by the difference in

esolvation of a charged vs. neutral side chain of the receptor, leading to decreased binding affinity oelfinavir and eventually to viral resistance.

n additional resistance-causing mutation that qualifies as a primary mutation involves the change oly48/48' to valine. This particular mutation seems to be specific for saquinavir and was observed bcell-culture sequential passage experiments and in AIDS patients exposed to this inhibitor [61,65]

ocated on the lower strands of the active-site forming flaps, Gly48/48' can be considered a part of t4/S4' subsites. The replacement of the glycine hydrogen by the rigid side chain of valine has mostkely a dual effect: first it has a direct impact on the interaction of the quinoline moiety of saquinaviith the active site of HIV PR, and second it may change the mobility of the flaps, which in turn wil

ffect the binding kinetics of the natural substrates or inhibitors. Although none of the other clinicallsted inhibitors form any interaction with this part of the flap, the HIV variants with mutation ofly48/48' seem to be cross-resistant to all compounds, which is reflected by a 3–5-fold reduction ofeir antiviral activity [55,62].

While the effect of primary mutations on reduced binding affinities of inhibitors can be at least partixplained in view of the accumulated structural data, the function of secondary, or compensatoryutations in the resistant HIV PR is difficult to rationalize as yet. The predominant compensatoryutations observed in the resistant variants involve residues Leu63/63', Ala71/71', Met46/46',sn88/88', Leu10/10', and Leu90/90' (Figure 8) [60,63]. Changes of these residues alone do not confral resistance, but their appearance increases the viability of the virus carrying the primary mutatiothe active site of protease. All these residues are located far away from the active site of HIV PR d

ot participate in any apparent way in the inhibitor binding and it seems unlikely that they form a lonnge interaction with the natural



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Figure 8Cartoon representation of the HIV PR dimer. The sites of compensatory

mutations are indicated.

ubstrate. Also, the reported compensatory mutations are conservative in nature and have no effect oe overall distribution of atomic charges on the surface of HIV PR.

equence polymorphism at the Leu63/63' position, located on the surface at the base of HIV PR, haseen observed in clinical isolates of the virus not exposed to any HIV PR inhibitors. Variations ofla71/71', where the side chains are buried very close to Leu63/63', are less commonly found in clinolates. After a prolonged challenge by HIV PR inhibitors, Leu63/63' changes to proline and Ala71/valine.

he side chains of Met46/46' are fully exposed to solvent and these residues are located on the β-airpins that form the active side flaps. It has been speculated that the compensatory change of

Met46/46' to isoleucine or phenylalanine may affect the dynamics of the flap movement, which in tu

ould influence the rates of catalytic activity of HIV PR impeded by the primary mutations in the actte [58].

ny changes to Asn88/88' and Leu90/90', buried in the body of HIV PR, most likely affect the structability of the enzyme. The side chains of Asn88/88' form buried hydrogen bonds and replacement ois residue by aspartic acid or serine not only eliminates some of these bonds but also introduces

nfavorable interactions in the core of HIV PR. Similarly, Leu90/90' is buried in a tight hydrophobicpace close to the “fireman's grip” motif, which



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volves the catalytic asparates 25/25'. The structural effect of a mutation of Leu90/90' to the largerethionine is rather difficult to predict since it can either rigidify or destabilize the HIV PR dimer oray have an effect on the catalytic efficiency of the “resistant” enzyme.

he complicated pattern of HIV resistance to protease inhibitors, in particular the appearance ofompensatory mutations that alone do not confer any resistance, suggests that the key to understandie basis of decreased susceptibility of the virus to a given drug is the kinetics of specific processinge GAG and GAG-POL polyproteins. The reduction in sensitivity of a mutant HIV PR towards anyhibitor can be conveniently reflected by the ratio of K i mutant/K i wild type. However, this reducedhibitor sensitivity is only one component that distinguishes mutant-form from wild-type proteases.rus encoding of a mutant HIV PR to be viable, the mutant protease must be capable of a minimallthough not yet quantified) level of enzymatic activity towards all substrates required for maturatioe virions. This proteolytic efficiency is reflected in the specificity constants (K cat/K m) as determinedutant and wild type HIV PRs. In order to rationalise these potentially conflicting relationships betw

nzymes, substrates, and inhibitors, Gulnik and his colleagues [66] introduced the term “Vitalityactor,” in which

n order for the “Vitality Factor” to be predictive for the level of resistance expected for a particular r combination of drugs for a given resistant strain of HIV, the determination of the specificity constK cat/K m for mutants) must be repeated for all nine known substrates processed by HIV PR. Thehibition constants of a given compound should not depend on the substrate, but the K cat/K m ratios d

nd therefore vitality values will differ for different substrates. It will be expected that the mean for ane “vitality” values will be predictive for the change in antiviral activity for a particular compoundlthough those data will be derived from in vitro experiments and are clearly not without somemitations, they may help in understanding the molecular basis of resistance and may contribute somalue to possible multidrug strategies for the clinical management of AIDS.

I. Perspective

IV PR inhibitors with acceptable oral availability and pharmacokinetic properties offer great promi

r the treatment of HIV infection and AIDS. Efficacy studies of indinavir, ritonavir, or nelfinavir usasma viral RNA as a marker have demonstrated up to three log reductions in RNA copy numbers te



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ustained in many patients [67–69]. In contrast, nucleoside antiretroviral therapy that targets reverseanscriptase rarely results in more than one log reduction of viral RNA, indicating fairly poor inhibif viral replication by this class of compounds. One of the reasons for the apparent greater in vivontiviral activity of HIV PR inhibitors could be the mechanistic difference of the two enzymes and thspective activities in the viral life cycle. However, growing evidence of retroviral resistance to

rotease inhibitors remains a concern. The availability of several chemically distinct HIV PR inhibitcluding the second generation of compounds currently under preclinical development, offers aossibility of combining two or more drugs that share little cross-resistance. Also, it seems reasonabvaluate these compounds in combination with various nucleoside and nonnucleoside reverseanscriptase inhibitors. Early clinical data from such combination therapy indicates reduction oftroviral RNA in plasma to levels lower than the currently available limit of detection [70]. This is trst indication that the application of well-chosen combination therapy can place AIDS patients inrolonged virologic and clinical remission.

ndoubtedly, protein crystallography and other elements of structure-based drug design were widelypplied in the discovery of HIV PR inhibitors. It will be prudent to assume that, in the absence ofructural feedback, rapid discovery of several chemically different and potent inhibitors of HIV PRould have been severely impeded if not even impossible. However, structure-based drug design stimains a new and developing technology. Further success of this drug discovery technique largely

epends on the development of methods of computational chemistry. Several computational approacuch as ALADDIN [71], DOCK [72], and MCDNLG [46] have been applied with a limited degree ouccess in a search for novel inhibitors of HIV PR and these methods will be developed further. Theost difficult and challenging computational task required for full implementation of structure-based

rug design involves assigning a priority to designed compounds before their synthesis, by computatf the absolute free energy of binding or by prediction of the relative difference in the binding constaf chemically related compounds. While the former approach is technically very difficult, due to thef configurational space that must be sampled and the limited accuracy of the force field that describomic interactions in the molecular system [73], the latter approach has had some successes [74,75]evertheless, owing to the various assumptions and approximations that underlie these techniques, sethods are useful only as order-of-magnitude estimates [74]. Further improvements of these metho

eavily depend on the availability of structural and thermodynamic data for several closely relatedompounds that could be used to calculate parameters required for the implementation of suchermodynamic-integration cycles. The large number of high-resolution crystal structures of HIV PR



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omplexed with various inhibitors offers a unique opportunity for the development of suchomputational methods if the structural data can be coupled with thermodynamic measurements ofhibitor-protein binding. These include direct measurements by microcalorimetry of the association

onstant, K, and in addition the enthalpy, entropy, heat capacity, and stoichiometry of binding. Theombination of such thermodynamic and structural data will lead to a more precise understanding of

ctors that influence binding and, ultimately, will lead to new general design principles that can bepplied to drug discovery in the area of AIDS as well as other challenging diseases.

cknowledgments

wish to thank all my co-workers from Agouron who contributed to these studies, in particular J.avies, S. Reich, M. Melnick, V. Kalish, A. Patick, L. Musick, and B-W. Wu. Steven Kaldor from Eilly is acknowledged for his contribution in designing AG1343 (nelfinavir). I would like to thankichard Ogden for critical reading of the manuscript and D. Olson for expert assistance in preparinganuscript.

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