Current Medicinal Chemistry Approaches to the Design of … · 2015. 2. 27. · Current Medicinal...

Current Medicinal Chemistry, 2000, 7, 455-477 455

Approaches to the Design of Effective HIV-1 Protease Inhibitors

F. Lebon* and M. Ledecq

Laboratoire de Chimie Moléculaire Structurale, Facultés Universitaires Notre-Dame de laPaix, 61 rue de Bruxelles, B-5000 Namur, Belgium

Abstract: Recently, western countries have recorded a decrease in thedeath rate imputed to AIDS. This success has been largely attributed to thepresence on the market of chemotherapies that inhibit the infectivity of thepredominant causative agent, the HIV-1 virus, by targeting essential viralenzymes. One of these is the protease (HIV-1 PR) whose activity is aprerequisite for viral replication. Two main sites have been identified as poten-tial targets for the inhibition of HIV-1 PR, the active site and the interface, the latter being largelyresponsible for the stabilization of the enzyme dimeric structure. The compounds that havereached clinical application so far target the active site of HIV-1 PR. These molecules act astransition state analogues and result from modifications of the peptidic scaffold intopeptidomimetics. In order to improve their bioavailability, systematic biological screening and denovo design have been used to suggest new non-peptide inhibitors combining both antiviralpotency and favorable pharmacokinetic properties. In parallel, compounds targeting otherpotential sites of inhibition have been tested. Peptides and peptidomimetics based on theterminal sequence of the enzyme, a site which is proposed to be less susceptible to mutations,have been shown to lead to HIV-1 PR inactivation. Cupric ion was described to bind a sequenceon the protease surface, which includes cysteine and histidine residues, leading to theinhibition of the enzyme. In the future, these non-active site inhibitors could provide analternative in anti-HIV drug combination strategies.

Introduction process catalyzed by a viral integrase (HIV-1 IN). Oncecell activation occurs, the proviral DNA is transcribedinto genomic RNA and mRNA. Translation of viralmRNA gives rise to three precursor polyproteins gag(Pr55gag), gag-pol (Pr160gag-pol) and env (gp160),containing respectively the structural proteins (p17,p24, p7 and p6), the viral enzymes (RT, IN and PR) andthe envelope glycoproteins (gp41, gp120). Thesepolyproteins assemble at the membrane and form newviral particles that become infectious upon cleavage ofthe precursor polyproteins gag and gag-pol by a viralprotease (HIV-1 PR). The resultant understanding ofHIV-1 life cycle at the molecular level has suggestedmany targets for intervention, and in particular the threeviral enzymes HIV-1 RT, IN and PR, essential for virusreplication [5]. As early as 1985, AZT was approved asthe first HIV-1 antiviral drug targeting the viral RT. AZTexerts its pharmacological effect by being incorporatedinto the growing DNA chain and preventing furthergrowth. However, with AZT, as with many nucleosideanalogues, toxicity is still a problem [6]. Non-nucleosides (NN) inhibitors, targeting an allosteric siteof RT have been successfully designed andsynthesized. First generation NNRTIs are rapidlyinactivated through mutations of the enzyme ([7, 8] andreferences therein) however, a potent NNRTI that is

The pandemic spread of the acquiredimmunodeficiency syndrome (AIDS), with more than 30million people infected since the beginning of theinfection, has promoted an unprecedented scientificand clinical effort to understand and combat this lethaldisease. First reported in the beginning of the 80's,AIDS is associated with a depletion of T lymphocytescausing various opportunistic infections among whichis the rare combination among young individuals ofPneumocystis carinii and Kaposi's sarcoma. Followingthe isolation of AIDS causative agent, the humanimmunodeficiency virus (HIV-1) [1, 2], intense researchhas led to an understanding of the processes involvedin the replication machinery of HIV-1. Its viral genome istypical of retroviruses and consists of duplicate copiesof single stranded RNA [3, 4]. Once the cell becomesinfected with the virus, the genetic information flowsfrom RNA to DNA by means of a viral reversetranscriptase (HIV-1 RT). The viral DNA genomebecomes permanently integrated in the host cell by a

*Address correspondence to this author at the Facultés UniversitairesNotre-Dame de la Paix, Laboratoire de Chimie Moleculaire Structurale,Rue de Bruxelles 61, B-5000 Namur, BELGIUM; Tel: +32 81 724569; fax:+32 81 724530; email: [email protected]

0929-8673/00 $19.00+.00 © 2000 Bentham Science Publishers B.V.

456 Current Medicinal Chemistry, 2000, Vol. 7, No. 4 Lebon and Ledecq

much less liable to select for resistant virus has beenrecently introduced [9-12].

have been identified as potential targets for theinhibition of HIV-1 PR, the active site and the interface,the latter being largely responsible for the stabilizationof the enzyme dimeric structure. Recently, light hasbeen cast on a new potential site of inhibition thatinvolves interaction with metal-binding residues on thesurface of the protease.

As the proteolytic activity of the PR is absolutelyrequired for the production of mature, infectious viralparticles, this enzyme was soon recognized as anattractive target for therapeutic intervention [13]. Since1995, four HIV-1 protease (PR) inhibitors have beenapproved by US Federal Drug Administration (FDA).Currently licensed protease inhibitors include Invirase

(saquinavir mesylate, Hoffmann-LaRoche), Norvir

(ritonavir, Abbott Laboratories), Crixivan (indinavirsulfate, Merck) and more recently Viracept (nelfinavirmesylate, Agouron Pharmaceuticals). A number ofother compounds such as Amprenavir (VX-478, GlaxoWellcome), Tripranavir (PNU-140690, Pharmacia-Upjohn) and DMP-450 (Triangle) are in clinicaldevelopment. The reduction in mortality in AIDSpatients after treatment with combination therapyincluding RT and PR inhibitors has been particularlyimpressive [14]. Although they are no cure, they cansignificantly inhibit viral replication. The long-termclinical efficacy of these compounds is still underinvestigation, but the emergence of resistant virusesappears to be a potential limitation for all of them. Thepeptide-like nature and size of most HIV-1 PR inhibitorslimit their oral bioavailability and half-life in man, makinghigh blood levels difficult to achieve and sustain.However, this first success in the history of AIDSchemotherapy encourages research aimed atproposing new generations of anti-proteases, able tosuppress viral replication and to delay the appearanceof resistance. This review is not intended to provide acomprehensive list of all currently available HIV-1antiviral compounds but to describe the variousmethods that were used to discover anti-HIV-1 PRagents and to indicate promising new strategies.

Active-site Inhibitors

Active site inhibitors have been extensively studied.They present a large structural variety that points to thechemical tolerance of the active site. The structuraldetermination of numerous HIV-1 PR/inhibitorcrystallographic complexes has revealed a similar modeof interaction of most inhibitors with the active site ofthe enzyme, as depicted in Fig. (1 ). A common featureobserved is the presence of a tetracoordinated watermolecule linking the bound inhibitor to the flexible β-strands or "flaps" of the HIV-1 PR dimer. This watermolecule accepts two hydrogen bonds from backboneamide hydrogen atoms of HIV-1 PR residues Ile 50 andIle 50' and donates two hydrogen bonds to carbonyloxygen atoms of the inhibitor, thus inducing the fit ofthe flaps over the inhibitor.

Opposite the flaps region, two catalytic aspartatesAsp 25 and Asp 25’ interact through hydrogen bondswith one (in some cases two) hydroxyl group(s)belonging to the non-cleavable transition-state isostereof the inhibitor (In Schechter & Berger’s nomenclature,residues to the left of the isostere are referred to as theP1, P2, P3, etc. position, and those to the right aregiven the designation P'1, P'2, P'3, etc. Similarly, HIV-1PR binding pockets are designated S1, S2, S3 and S'1,S'2, S'3). This hydroxyl group shifts and replaces thecatalytic water molecule that initiates the hydrolysisprocess. On each side of these interactions that weresoon recognized as essential, hydrogen bonds areformed between the inhibitor and residues Gly 27 (27’),Ala 28 (28’), Asp 29 (29’), Asp 30 (30’) and Gly 48 (48’).The inhibitor is further maintained into the enzymaticcleft by a multitude of van der Waals contacts. It isinteresting to note that fullerene derivatives wereshown to be active against HIV-1 PR (Ki=5 µM),elegantly demonstrating the highly hydrophobiccharacter of the active site [19-22]. Severalmethodologies have been applied for lead generationusing 1) the structure of enzyme substrates as astarting point for drug discovery (substrate-basedapproach) 2) biological screening methodologiesdeveloped to test hundreds/thousands of compounds(screening approach) 3) X-ray crystallography data ofHIV-1 PR-inhibitor complexes (biostructural approachor "rational drug design").

Design of HIV-1 Protease Inhibitors

Since the first crystallographic structure of HIV-1 PRwas released [15-17], more than 80 crystallographicstructures of native HIV-1 PR or in complex with variousinhibitors are now presently available in the PDB. Thismine of structural information makes HIV-1 PR one ofthe most well known enzyme to date. HIV-1 PRbelongs to the aspartic protease group of enzymes,like renin and endothiopepsin. HIV-1 PR is unique withrespect to other retroviral proteases as it is active as anobligatory homodimer stabilized by non covalentinteractions [15, 16]. Each subunit contributes one ofthe two conserved triads (Asp-Thr-Gly) containing thecatalytically active aspartate residues [18]. Theseaspartates are located at the center of a hydrophobiccleft of sufficient length to accept six amino acids of thesubstrate in an extended conformation. Two main sites

Design of Effective HIV-1 Protease Inhibitors Current Medicinal Chemistry, 2000, Vol. 7, No. 4 457

Fig. (1). Model of the interaction between HIV-1 PR active site and the substrate (or peptidic/peptidomimetic inhibitors). HIV-1PR residues have been represented in green. In peptidic/peptidomimetic inhibitors the hydrolysis unit (in blue) in the peptidechain has been replaced with non-cleavable isosteric fragments. Two essential interactions (in red) involve flaps residues Ile50/50’ and catalytic residues Asp 25/25’ of the protease.

Substrate-based approach molecule is delivered by two aspartic acids to thesubstrate cleavage site, creating a tetrahedraltransition-state intermediate, which rapidly breaks apartto give a C-terminal acid and a N-terminal amine.

The substrate clearly represents a molecule thatinteracts with the enzyme, and as such forms a leadstructure. The similarities between HIV protease andrenin allowed medicinal chemists to make rapid use ofthe information gained from renin inhibitor projects, inwhich transition state mimetics were used to achieveisolated enzyme activity. Similarly, when HIV-1 proteasecleaves a protein substrate as shown in Fig. (2 ), a water

It was recognized at an early stage that mimics of thetetrahedral intermediate of the reaction catalyzed bythe enzyme might be excellent inhibitors. The firstinhibitors that were designed were based on thestructure of the proteolysis products of polyproteins


NH

O

HN

O

NH

HN

O

OHHO N

H

OH

O

H2N

O

OH

O

O

OO

O

O

HO

OH

O

O

O_

__

HO-Hcatalytic water

molecule

catalytic aspartates

peptidic substratetetrahedral transition-state

intermediate hydrolysis products

Fig. (2). Creation of a tetrahedral transition-state intermediate during hydrolysis of a substrate by HIV-1 PR.

Pr55gag and Pr160gagpol, substrates of HIV-1 PR, and inparticular on the hydrolysis site Phe*Pro, specific ofretroviral proteases (see Fig. (3 )).

Viracept (nelfinavir mesylate, AgouronPharmaceuticals) as well as Amprenavir (VX-478, GlaxoWellcome, currently in clinical trials). Those compoundspresent sub-nanomolar activities versus the enzyme[27] and demonstrate high selectivity for HIV proteaseover other aspartyl proteases.

However, peptidic inhibitors lack appropriatephysical chemical properties and metabolic stability tobe ideally suited as therapeutics. They display poor

PR/RT Cys Thr Leu Asn Phe * Pro Ile Ser Pro Ile

P3 P2 P1 P' 1 P' 2 P' 3

Fig. (3). Amino acid residues of the PR/RT cleavage site, highly specific of retroviral proteases and their associated positionP1, P2, P3 / P'1, P'2, P'3 in the enzyme active site.

bioavailability and are rapidly metabolized in vivo.Systematic modification of the substrate-basedpeptides using rational design techniques (see below)led to peptidomimetic compounds which abandon asmuch as possible the peptidic backbone while retainingthe essential functionalities for HIV-1 PR active siteinhibition (for reviews on peptidomimeticmethodologies see [23-26]). Among them themarketed drugs Invirase (saquinavir mesylate,Hoffmann-LaRoche), Norvir (ritonavir, AbottLaboratories), Crixivan (indinavir sulfate, Merck) and

The first step towards HIV-1 PR peptidomimeticinhibitor design is to modify the substrate backbone byreplacing the hydrolysis unit in the peptide chain withnon-cleavable isosteric fragments and to modifypeptidic side chains into non-peptide groups.

Modification of Substrate Backbone

The transition-state isosteres most frequentlyemployed for the synthesis of HIV-1 PR inhibitors arelisted in table 1. They comprise statine, hydroxyethyl-

Table1. Most Frequently Used Transition-state Isosteres in the Design of HIV-1 PR Substrate-based Inhibitors

HN

OOH

R

Statine

HN

HN

OH

R

O

R'

Hydroxyethylamine

HN

O

OH

R

R'

Hydroxymethylene

HN

NH

R

R'

O

Reduced amide

HN

OH

R

R'

O

Hydroxyethylene (Dihydroxy)

HN

O

R

R'

F F

α,α-difluoroketone

....etc


amine (-CHOH-CH2-NH-), hydroxymethylene (-CHOH-),reduced amide (-CH2NH-), hydroxyethylene (-CHOH-CH2-), dihydroxyethylene (-CHOH-CHOH-), α,α-difluoroketone (-CO-CF2-), …etc.

In order to overcome resistance and to enhance thepharmacokinetic profile of KNI-272, JE-2147 3 wasdesigned [35], which is a potent HIV-1 PR inhibitor.The compound shows good pharmacokineticproperties attributed to the cyclized P2 moiety, whichseems to confer a good balance betweenhydrophobicity and lipophilicity [36].

To illustrate these concepts, we have selectedsome representative compounds of each bioisostericclass that were designed using the substrate-basedapproach. Hydroxyethylamine

Statine The first HIV-1 PR inhibitor to reach the market wassaquinavir 4 . It is characteristic of peptide-based HIV-1PR inhibitors in which a transition-state analog mimicsthe tetrahedral intermediate formed during cleavage ofthe scissile Phe*Pro amide bond of the naturalsubstrate. A vast number of compounds weresynthesized in which steric and electronic properties ofeach side chain and terminal substituent were

Pepstatin A and acetyl-pepstatin 1 are generalinhibitors of the family of aspartic proteases [28], and inparticular of HIV-1 PR. The absence of the P’1 sidechain reduces hydrophobic contacts with the viral PRactive site and leads to relatively low potency (µM) formost statine-containing inhibitors [29].

H3CHN

NH

HN

NH

HN

OH

O

O

O

O

O

O

Val Leu

OHAlaOHVal

Leu acetyl-pepstatin K i=20 nM

1

To increase potency, norstatine bioisosteres wereconsidered. The norstatine unit is one methylenegroup shorter than statine and allows the reintroductionof the normal P’1 residue. KNI-272 2 , a conformationallyconstrained compound was shown to be a selectiveand super potent inhibitor of HIV-1 PR, with low toxicity.The high degree of potency of this compound wasattributed to conformational preorganization as theoverall conformation of KNI-272 in the HIV-1 PRcomplex is remarkably similar to that observed in singlecrystal structures of KNI-272 in the free form [30, 31].However, the pharmacokinetics of KNI-272 arecharacterized by limited bioavailability, limiteddistribution and rapid elimination [32-34]. Moreover,HIV-1 develops in vitro a high level of resistance to KNI-272.

individually modified. Incorporating combinations ofpreferred side chains into individual molecules resultedin the generation of Ro31-8959 4 [27].

Nelfinavir 5 results from the optimization ofsaquinavir and LY289612 9 , which both suffer frompoor oral bioavailability, presumably due in part to theirretained peptide character [37-39]. The first cycle ofimprovement was the discovery of a novel S-phenylcysteine P1-P3 spanning group. The sulfur provedimportant for good hydrophobic interaction with theenzyme wall. The P2 group was then optimized with theelongated P1-P3 group. The best combination ofactivity and pharmacology was observed for themesylate salt AG1343 5 [40].

O

HN

NH

N O

O

N

O

S

OHS

NH

O

Ph

2

HONH

O O

N

S

NH

O

OH

Ph

KNI-272 Ki=5.5 pM JE-2147 Ki=0.33 nM

3


N

O

HN

O

NH

N

CONHtBu

H

H

OHCONH2

4

N

CONHtBu

H

H

HN

O

HO

OH

S

Ph

5

Saquinavir : Ro31-8959 Ki=0.12 nM Nelfinavir : AG1343 Ki=2.0 nM

Following publication of a series of potenthydroxyethylamine HIV-1 PR inhibitors, exemplified bysaquinavir 4 , it was hypothesized that incorporation of abasic amine into the backbone of the hydroxyethyleneL-685,434 series (1 0 ) might improve the bioavailabilityand aqueous solubility of this series of compounds [41,42]. Indinavir 6 results from the optimization of a seriesof hydroxyaminopentane amide compounds, thatincorporate a basic amine into the hydroxyethyleneinhibitor backbone [43-45]. L735,524 6 is a potentinhibitor and exhibits good oral bioavailability andpharmacokinetic profiles.

extending out to molecular weights of over 1000. In thecase of VX-478 7 [46], the aim was to reduce themolecular weight and increase solubility of thecompound while maintaining potency, by usinginformation from the crystal structure to minimize thestrain energy in the binding conformation [47-49].

Reduced Amide

The first crystallographic structure of an inhibitor incomplex with HIV-1 PR was that of compound MVT-1018 , based on a reduced amide isostere [50].

The absence of the central hydroxyl group, meantto interact with the catalytic aspartates Asp 25 and Asp25’ partially explains the low affinity of this class ofcompounds. Only a few potent inhibitors are based onthis bioisostere.

The problem with many large HIV-1 PR inhibitors isthat the liver tends to clear them quickly fromcirculation. The molecular weight distribution of allmarketed oral and systemic drugs shows a maximum inthe bell-shaped curve around 350 daltons and a tail

N

N

N

OHHN

CONHtBu

Ph

O

OH

Indinavir : L735,524 K i=0.38 nM

6

O

OHN

SN

OPh

OH

NH2

O

7

Amprenavir : VX-478 Ki=0.6 nM


HN

NH

HN

NH

HN

NH

O

O

OThr

Ile

Nle O

ONle

Gln

Arg

O

NH2

8

MVT-101 Ki=780 nM*

Hydroxyethylene-dihydroxyethylene 74,695, 1 1 ) [56-58] bioisosteres were proposed, inwhich hydration of the central fluorinated ketonemoiety generates mimics of the tetrahedralintermediate.

Numerous HIV-1 PR hydroxyethylene inhibitors arebased on the Phe*Pro cleavage site, as illustrated byLY289612 9 and L-685,434 1 0 , the hydroxyethyleneprecursors of marketed inhibitors nelfinavir andindinavir [37, 51, 52].

The clinical progression of MDL 74,695 series mayhowever be hampered by its poor bioavailability and its

N

HN

O

NH

O

NHt-BuO

OH

H2N

O

9

L-685,434 IC50=0.35 nM

10

BocHN

O

HN

OHOHPh

LY289612 IC50=1.5 nM

The poor oral bioavailability and aqueous solubilityof these molecules led to the addition of a basic amineinto the hydroxyethylene inhibitor backbone. Theresulting compounds, nelfinavir and indinavir, revealedpotent inhibitors and exhibited good pharmacokineticprofiles.

ability to select rapidly for drug resistant viruses [59,60].

Modification of Peptidic Side Chains intoNon-peptide Groups

Non-peptide groups will further be preferred overpeptidic side chains. A non natural D amino acid canreplace a natural L amino acid [61]. For example, inorder to keep the affinity of compound LY289612 9(IC50=1.5 nM) towards HIV-1 PR while enhancing its

Difluoroketones

Several nanomolar inhibitors containingdifluoroketone [53-55] or difluorostatone (MDL

O NH

HN

NH

O

O

O

O

O O

F F

N

OMDL 74,695 IC50=5 nM

11


N

O

S

NH

NH

O

SO2Me

O NHt-Bu

OH

*

O

ValNH

BocPhe

OH

OMe

IC50=3.5 nM

*=D

IC50=8 nM

1213

Boc=butoxycarbonyl

pharmacokinetic profile, a distant analogue of D-cysteine 1 2 was inserted that was meant to interactwith the S2 subsite of the enzyme active site [52, 62].

imagined and synthesized (as in 1 5 [68-70]) withvarious bioisosteres [71, 72] and in pepsin inhibition[73]).

To obtain pharmacologically useful analogs, theconformation of the peptide can be stabilized into the

The observation that peptides interact exclusively inan extended conformation with HIV-1 PR active site has

IC50=0.22 µM

NH

O

HN

HO

N HN

O

Ph

OH

Cbz

Ph

Cbz=carboxybenzyl14

Ph O

O

HN

NH

O

O

HN

NH

OO

O

NH

Ph

OH

Ki=7.9 nM

15

biologically active conformation by the introduction ofbridges between different parts of the molecule. Thelink can involve a single unit (as in compounds 1 3 [63]and 1 4 [64-67]) or connect two side chains. A motifshown to stabilize enzyme-bound, extended β-structures is cyclisation between P1 and P3 side chains.Several original molecules linking subsites P1 and P3(or P’1 and P’3) in a macrocyclic structure have been

led to the concept of a new type of peptidomimeticsystem in which a greatly modified backbone isintegrated into cyclic structures to produceoligopyrrolinones 1 6 [74].

These compounds are more resistant towardsmetabolism than their peptidic analogues and benefitfrom a high transport into infected cells [75-77].

O

OHN

OPh

OH

O

HN

HN O

NH2

OPh Ph

16

oligopyrrolinone IC50=1.3 nM


NN

O

HN

O

NH

HN

O

NH

N

O

N

OH

OH

Ph

Ph17

A77003 Ki=84 pM

S

N

O

HN

O

NH

HN

O

O N

SOH

Ph

Ph

18

Ritonavir : ABT-538 Ki=15 pM

An extension of the substrate-based approachutilized an unusual feature of HIV-1 protease. Thehomodimeric nature of the enzyme led to thesuggestion that symmetrical inhibitors might beeffective [78]. One of the postulated advantages of thisapproach was selectivity, as the symmetrydemonstrated by the viral enzyme was unique. Theresulting inhibitors did indeed demonstrate high levelsof selectivity. Beginning with the symmetric inhibitorA77003 1 7 (Ki=84 pM), which displayed low oralbioavailability but adequate anti-HIV activity andaqueous solubility, systematic investigation ofperipheral (P3 and P’2) heterocyclic groups designed todecrease the rate of hepatic metabolism providedanalogues with improved pharmacokinetic properties.Replacement of pyridyl groups with thiazoles, a lesselectron-rich bioisostere, provided increased chemicalstability towards oxidation while maintaining sufficientaqueous solubility for oral absorption and led toRitonavir 1 8 [79, 80].

Screening Approach

The second general strategy for obtaining leadcompounds is by screening libraries of compounds forbiological activity against HIV-1 PR. The aim is todiscover inhibitors of reduced peptidic nature whichgenerally exhibit improved pharmaco-dynamic andkinetic properties than peptide-based compoundstypical of the substrate-based approach. The moststriking example is probably the discovery ofphenprocoumon 1 9 as a lead non-peptide inhibitor ofHIV-1 PR, which was identified via broad screening ofthe Pharmacia & Upjohn compound library [81]. Thegoal was to design more potent compounds throughSAR studies, and retain the desirable pharmacokineticcharacteristics of the 4-hydroxycoumarin class ofcompounds. Crystallographic studies providedinformation on the binding mode of these novel nonsubstrate-based inhibitors, and revealed that the 4-hydroxycoumarin derivatives interact directly with the

O O

O

OH

O O

S N

CF3

OO

OH

PD099560 Ki=1 µM

1920

Tipranavir : PNU-140690 Ki<1 nM


21

GR137615 K i=3.8 nM

HN

NH

O

O HN

S

O

N

HN

O

NHN

OH

H

OCOB10H11 C2

C2H11B10OCO

N

N

COOH

N

N

COOH

OCOB10H11 C2

OCOB10H11 C2

Cu(II)

Porphyrine IC50=0.975 µM

22

two isoleucine residues on the flaps of the enzyme, Ile50 and 50’, replacing the tetracoordinated watermolecule observed in the binding of substrate-basedinhibitors (Fig. (4 )]. Iterative cycles of structure-baseddesign led to Tipranavir 2 0 (PNU-140690, clinicaltrials), a potent and orally bioavailable nonpeptidicprotease inhibitor [82-88]. (further reading: Parke-Davis[89-97]).

An analysis of nonpeptide compounds with usefulpharmacological properties led to the identification ofnatural porphyrins as HIV-1 PR inhibitors (as compound2 2 ). However, the potential usefulness in clinicaltherapy of these compounds is questioned as thepresence of albumin prevents inhibition of HIV-1 PR bythis class of compounds [103].

Screening of traditional medicines and diverse plantextracts, a largely untapped source for structurallynovel chemicals, has led to the identification ofcompounds with anti HIV-1 PR activity (as compound2 3 ) [104-112].

Screening programs have highlighted how poorlyselective the HIV-1 PR active site is (or how little weunderstand of structural similarity!) since it canaccommodate structurally very diverse molecules. Hereare some examples of original attempts to find non-peptide HIV-1 PR inhibitors. Novel series of penicillin-derived C2-symmetric inhibitors of HIV-1 PR wereidentified (as exemplified by compound 2 1 ) [98-102].Although the compounds were selective and exhibitedno cytotoxicity, they suffered from rapid elimination.The disappointing pharmacokinetics prevented thedevelopment of these compounds as potentialantivirals in the treatment of HIV infections.

Biostructural Approach or "Rational DrugDesign"

The third strategy for generating lead compound isthe biostructural approach. Antiviral research hasbenefited enormously from advances in molecularbiology and protein chemistry, which have providedpure proteins in sufficient quantities to allow structural

O

N

OOO

NO

OH

OOH

23

Spirodihydrobenzofuranlactame IC50=11µM


HN NH

OH H

O O

OHHO

HN NH

Y

OH OH

HN NH

Y

OHH

O

O

O

HO

O

O

O

HO

O

O

O

HO

_ _ _

Asp 25/25' Asp 25/25' Asp 25/25'

A.B. C.

lle 50/50' lle 50/50' lle 50/50'

(Original Citation) Reproduced by permission of The Royal Society of Chemistry

5.5Å

Fig. (4). Pharmacophores for HIV-1 PR inhibitors: A. Peptidic and peptidomimetic inhibitors. The tetracoordinated watermolecule is simultaneously bound to residues Ile50/150 of the protease and to the carbonyl groups of peptidomimetic inhibitors.Hydroxyl groups interact with the catalytic residues Asp25/125. B. Nonpeptide inhibitors. The proton acceptor Y mimics thetetracoordinated water molecule. C. Coordination complexes. The pharmacophore includes the water molecule necessary tothe protease’s hydrolysis process.

studies to be carried out. These developments,coupled with advances in X-ray crystallography, NMRspectroscopy, and computing technology, have greatlyaccelerated the determination of 3D structures ofproteins. Visualization of these structures bysophisticated computer graphics has made rationaldrug design feasible. Structural data of the enzymebinding site is now frequently used during theoptimization process of compounds that originate fromsubstrate-based design or screening methodologies.An important key design element in many approacheshas been to use the C2 symmetrical nature of theenzyme and the presence of the structural watermolecule in the active site to build in specificity andpotency (Fig. (4A )). The design strategy that led tononpeptide cyclic ureas as potent HIV-1 PR inhibitorswas to incorporate the structural water into the inhibitorframework (Fig. (4B )).

The favorable pharmacokinetics of cyclic ureas ledto the identification of the first cyclic urea candidateshortly after its initial design and synthesis [113].Unfortunately, poor solubility, first-pass metabolism andlimited formulation possibilities resulted in poorperformances in Phase I studies. Improvement ofphysical properties and pharmacokinetics without lossof potency led to the design and synthesis of weaklybasic cyclic urea analog DMP450 2 4 , currently inclinical trials ([114, 115] and references therein; recentpublications [116-122]).

Haloperidol is one of the first non-peptide inhibitorof HIV-1 PR, discovered through a computationalscreen of 10,000 molecules in the CambridgeStructural Database (CSD) based on stericcomplementarity with HIV-1 PR active site conformation[123]. Haloperidol is toxic at elevated concentrations

HO N NOH

O

HOPh

OH Ph

DMP450 Ki=0.34 nM

24

Cl

N

S S

OHF

UCSF-8 Ki=15µM

25


N Cu2+

N

N

O O

H2O

H2O

O

O

NHNO

O

Cu++

N NH

O

O

O

O

X

X

SETCEZ Ki=480µM

2627 X=MeOH

IC50=1.0µM

and therefore is not itself a viable drug for the treatmentof AIDS. Replacement of the ketone functional centerwith a thioketal ring resulted in ucsf8 2 5 [124]. Incontrast to peptidomimetic inhibitors, ucsf8 interactsmainly with the flaps of the enzyme in an openconformation. The interaction of ucsf8 with the catalyticsite of the protease is mediated by a water moleculelocated at equal distance (3Å) from the two catalyticaspartates Asp25/25'. This water molecule is probablyin a similar position to the catalytic water molecule thatinitiates the hydrolysis of the substrate during theproteolytic process. In the presence of peptidomimeticinhibitors, this water molecule is replaced by hydroxylgroups belonging to the inhibitors and is therefore notobserved in the crystallographic structures. Based onthis observation, a pharmacophore was designedwhich takes into account this crucial catalytic watermolecule (Fig. (4C )). The model, which adressesmetal-organic compounds particularly, was used forscreening new HIV- PR inhibitors through the CSD.Among the structures fitting into the active site of theenzyme, compound Diaqua [bis (2-pyridylcarbonyl)amido] copper (II) Nitrate 2 6 (SETCEZ) [125] was foundto behave as a competitive inhibitor of HIV-1 proteasewith a Ki value of 480 µM [126].

antifungal antibiotic cerulenin [130]. The carboxylicgroups Asp 25 and Asp 25’ in the active site are a goodtarget for irreversible inhibition of HIV-1 PR since theirinactivation leads to complete loss of the catalyticactivity. To date, a few epoxide-based inhibitors of HIV-1 PR have been reported that inactivate HIV-1 PR withhigh selectivity (UCSF 84, 2 9 ) [131]. Haloperidolderivatives with an ynone or an α,β-unsaturated ketoneas alkylating functionality were studied but it wasdiscovered that these compounds also led to enzymeinactivation via a sulfhydryl alkylation of the Cys95belonging to the dimer interface [131-134]. Thetripeptidomimetic epoxide 3 0 , spanning the S3-S1’substrate-binding pockets, was reported as time-dependent, irreversible inhibitor of HIV-1 protease(Kinact=20 µM) [135]. An increase in the potency ofthese inhibitors was obtained by extending the peptidesequence [136-138]. NMR and topochemical studiesof irreversible inhibitors containing a cis-epoxide asamide isostere revealed that these compounds preferto adopt extended conformations similar to the β-strandin solution [139]. Novel irreversible inhibitorscontaining sulfonamide and sulfone as amide bondisosteres were designed and displayed rapid, time-dependent inactivation of HIV-1 protease in thenanomolar range, with high potency in cell culture[140].New copper coordination compounds, having an

octahedral geometry favorable for the orientation oftheir interacting substituents within the proteasesubsites were designed and inhibited HIV-1 proteasein the micromolar range (as exemplified by complex 2 7 )[127].

The chemical reactivity of epoxides diminish thepotential in vivo efficacy of these compounds as HIV-1PR inhibitors. It came to conclusion that usefulinactivating agents for HIV-1 PR should incorporate afunctionality with the reactivity of a cyclic cis-1,2-disubstituted epoxide, preferably one which, like theepoxide, is activated towards nucleophilic addition byspecific hydrogen bonding with one of the catalyticaspartate groups. Using haloperidol as a lead structure,compound 3 1 was designed. The non-peptidicepoxide is an irreversible, active-site directed inhibitorof HIV-1 protease (Kinact=65 µM) [133].

Irreversible Inhibitors

A strategy to suppress the development of resistantstrains is to design irreversible inhibitors, as earlysuggested by the fact that HIV-1 PR is inactivated by1,2-epoxy-3-(p-nitrophenoxy)propane 2 8 (EPNP,Kinact=9.9 mM) [128, 129] and by the epoxide


EPNP K inact=9900 µM

28

NO2

OO

Cl

N

OH

O

UCSF 84 Kinact =521 µM

29

K inact = 20 µM K inact = 65 µM

Cbz-Phe-AlaNH

O

N

O

O

O

30 31

Dimerization Inhibitors developed which was > 10000-fold more potent thanthe starting peptide [144].

The homodimeric nature of the enzyme led to thesuggestion that a novel approach to inhibitor designcould be considered. This approach, based on thedissociation of enzyme subunits, would ultimatelydestroy the active site and result in loss of biologicalactivity. In fact, the strategy of using interface peptidesto inhibit enzyme activity by interfering with the subunitinteraction has been used successfully in the case ofribonucleotide reductase from herpes simplex virus(HSV) [141, 142] and of HIV-1 reverse transcriptase[143]. In the first case, a peptidomimetic drug was

HIV protease dimers have significant subunit-subunit interactions in four regions: the 'firemen's grip'at the active site; the flap region with interactionsbetween residues Gly49(49’), Ile50(50’), Gly51(51’);the salt bridges involving Arg8, Asp29 and Arg87, and,the termini of the two subunits, which form a four-stranded antiparallel β-sheet (Fig. (5 )). A total of 34hydrogen bonds and four ionic interactions occurbetween the two subunits: two hydrogen bonds occurbetween the flaps, five occur between active site

N' 1' 2' 3' 4'

99 98 97 9669

96' 97' 98' 99'

4 3 2 1

69'

C

C'

N

T l Q P

+

T L N F

TLNF

P Q l T

C'

H

H_

_

+

Fig. (5) . Ionic interactions and hydrogen bonds in the four-stranded β-sheet formed by amino and carboxyl termini. In additionto the hydrogen bonds between main chain C=O and NH groups, there is a salt bridge between the amino terminus of onesubunit and the carboxyl terminus of the other subunit. The carboxyl terminus can also form an intersubunit hydrogen bondinteraction with His69. There is a network of hydrogen bond linking the side chains of Asn98 and Gly2 from both subunits.


residues 25-27; eight occur between residues 6-8, 29,and 87; and 19 hydrogen bonds and two ion pairs areformed by terminal residues 1-4 and 96-99. In factresidues 96-99 contribute 50% of the ionic and 56% ofthe hydrogen bond intersubunit interactions (whichrepresents 75% of the total Gibbs energy [145]). Thissuggests that using peptides as competitive inhibitorsof β-sheet formation by the amino and carboxyl terminimay efficiently inhibit dimer formation and thus reducePR activity [146].

Ki,dim values. Although consistent with Kd estimates by

Kettner et al [149] this procedure is still a controversialissue [150].

Peptidic Inhibitors

To target the dimerization interface by peptidicinhibitors, several approaches can be used. On onehand, a single strand peptide based on the C-terminusor the N-terminus sequences could interfere with onemonomer and induce a competition with thehomodimer formation (Fig. (6A )). On the other hand, adouble strand peptide could be designed tointerdigitate with the HIV protease monomer andreproduce the typical ß-sheet structure (Fig. (6B )).

There are several reasons for focusing on the dimerinterface as a site for antiproteolytic intervention. First,dimerization of the protease polypeptides is essentialfor activity and represents one of the initialposttranslational steps. Dimerization inhibitors may beable to bind the PR domain in uncleaved gag/polpolyprotein prior to particle assembly. Second, as morethan 50% of the intersubunit interactions are providedby the extended interface created by the C- and N-termini, this interface may be less vulnerable to

C-Terminus and N-Terminus Peptides andPeptidomimetic Inhibitors

Two octapeptides (3 2 and 3 3 ) derived from thesequence of the N- and C-termini of HIV-1 proteasewere tested for their ability to inhibit HIV-1

X-X-X-X-X

X-X-X-X-X

X-X-X-X-XHlV-1PRmonomer

HlV-1PRmonomer

A B.

Fig. (6). Approches to inhibit the protease dimerization using peptide inhibitors. Interaction of the PR monomers with A. singlesequence peptide B. double sequence peptide mimicking the ß-sheet structure.

mutational escape than the active site or the substratebinding regions of the protease and thereforemaintained in a greater number of evolving viral strains.In fact, comparison of the 3D structure and amino acidresidues involved in the intermolecular interface of HIV-1 PR in numerous resistant strains are conserved andthe fold is nearly superimposable. Therefore, targetingthe interface may result in the design of broader actinginhibitors capable of blocking dimerization of HIVprotease [147]. Zhang et al have analyzed the kineticsof dimerization inhibition for HIV-1 protease, based onthe assumption that the equilibrium betweenmonomers and dimers is rapid (Kd = 3.6 ± 1.9 nM atpH=5.0, 0.1 M sodium acetate, 1 M NaCl, 1 mM EDTAbuffer, 37 °C) [148]. Their method leads to a distinctionbetween competitive (active-site) inhibitors anddimerization inhibitors as well as the determination of

reproduction, but weak inhibitory activity was foundwith each of the two peptides [151]. Interestingly, thetoxicity of such peptides was very low (CC50 > 197 and181 µM).

Several other structures derived from the terminalsegments showed comparable inhibition [152, 153].Systematic study of the length requirements for the N-and C-terminal peptide needed for optimum HIV-1protease inhibition were conducted [154]. The bestinhibitory activity was obtained with sequencescorresponding exactly to the full length β-strands in thefour-stranded β-sheet region of the protease, lendingsupport to the hypothesis that these peptides mayinterfere with the dimerization interface. It was furtherobserved that inhibitors derived from the C-terminususually have better inhibitory properties than those

Peptide 32 (PR 92-99): Ac-Q-I-G-M-T-L-N-F-NH2 ED50 (HIV reproduction) = 27.1 µM

Peptide 33 (PR 1-8): Ac-P-Q-I-T-L-W-Q-R-NH2 ED50 (HIV reproduction) = 58.4 µΜ


Table 2. Inhibitory Activity of Some Peptides Derived from the Protease Terminal Segments [156]

C-terminal peptides Ki,dim(µM)

34 Y E L 2.35

35 S Y E W 0.32

36 S Y E L 1.27

37 Ac S Y E L 0.29

38 I S Y E L 0.39

39 Ac I S Y E L 0.27

40 Pam T V S Y E L 0.16

N-terminal peptide

41 L Q I T L W 0.59

from the N-terminus. This can be explained by thestronger binding of this part of the β-sheet to the rest ofthe molecule (in terms of hydrogen bonds) [152].

be viewed as a pharmacophore for ulteriorpeptidomimetic or non-peptidic inhibitor design [157](Table 3).

In order to improve their inhibitory potency, thosepeptides were systematically modified on the basis ofpublished PR structures, mutation studies andsequence analysis of retroviral proteases, and weremodeled into their binding sites. The best-fittingpeptides were synthesized and tested in the PRenzyme assay [155, 156]. Several new peptides withimproved inhibitory potency were found (see examplesin Table 2) Among them, the short peptide Ac-T-V-S-F-N-F (IC50 = 80 µM) mimics the C-terminal part of the gag-pol frame shift protein p6*. This suggests a regulatoryfunction of p6* as a dimerization inhibitor of HIVprotease in the virion [155].

Double Sequence Peptide

Since short peptides derived from both PR terminiwere found active and were designed to target distinctbinding sites, attempts were made to combine bothinhibitory motifs. Peptides were constructed byconnecting the N- and C- terminal sequences withlinkers consisting of natural and unnatural amino acids[153, 158]. Tested for inhibitory activity, some of thebifunctional peptides showed an IC50 lower than onemicromolar [158].

42 PQITL-(Gly)3-CTLNF IC50 = 40 µΜ

43 H-LEITLWER-XX-ISYEL-OH IC50 = 0.5 µΜThe fact that the inhibition constant could be

improved so much (about 1000-fold) on the basis ofsimple rational design supports the hypothesis thatthese peptides act as dimerization inhibitors. Based onthese results, a consensus sequence of potentpeptidic inhibitors was established for positions 94-99of the protease [156]. This consensus sequence can

44 H-LEITLEN-XXX-ISYEL-OH IC50 = 0.5 µM

X = 6-aminohexanoic acid

However, these peptides possibly might notinterdigitate a single monomer using both halves as inFig. (6B ), but would rather act as monofunctional

Table 3. Consensus Sequence for the Dimerization Inhibition by C-terminal Peptide Based Inhibitors[156]

94 95 96 97 98 99 OH

T V S Y D L

I T E Y

N W

medium/largehydrophobic

smallhydrophobic

OHH bonded with the active sitesegment : D25’-T26’-G27’

charged interactionwith H67’ and P1’

large hydrophobic interaction with H69’and P1’


N

( )n

O

P-Q-l-T-L-W-OH

HN

O

S-T-L-N-F-OH

F-N-L-T...

T-l-Q-P

HlV-1PRmonomer

n=13

n=14

IC50 = 2.5 µM

IC50 = 2.0 µM

45

46

Fig. (7). Interaction mode between cross-linked peptides and the monomer of HIV-1 protease [159].

peptides as no activity improvement was observedcompared to the single peptides. It was proposed thatthese compounds would dissociate a protease dimerby binding half of the agent into each proteasemonomer, inherent to the explicit linking of the N- andC- termini of the monomer terminal sequences (seeFig(5 )).

peptidic strands. Inhibitions (submicromolecular range)were obtained with molecular tongs containingtripeptidic or tetrapetidic arms linked to pyridinediol- ornaphthalenediol-based scaffold (Fig. (8 )).

Kinetic studies are in agreement with an interfaceinhibition mechanism. The more potent inhibitordescribed by Zutshi et al. (IC50 = 2 µM) was obtainedwith two different peptidic sequences reproducing theN- and C-termini of HIV-1 PR monomer (PQITLW-OHand STLNF-OH, respectively, cross-linked with a 14-methylene flexible spacer). With two identical andshorter peptidic strands (VLV-OMe and the rigidnaphtalene spacer, 4 9 ), a comparable inhibitoryefficiency was obtained (Kid = 0.56 µM).

The strategy used to reproduce the ß-sheetstructure capable of forming 1:1 complexes with theprotease monomer was to covalently crosslink the N-termini of the protease peptides instead of the N- andC- termini as described in the previous paragraph. Suchpeptides were shown to inhibit the protease activityand decrease the amount of protease dimer in solution[159]. In HIV-1 PR, the N-terminal ends of residuesPro1 and Cys95 are held at a distance of approximately10 Å. Different agents were designed to bridge these10 Å gap while allowing the N- and C- terminal peptidesto bind into the appropriate four-stranded β-sheetarrangement with a protease monomer. Agents withtether containing 13-14 methylene groups producedIC50 values in the low micromolar range (Fig. (7 )).

Non Peptidic Inhibitors

Triterpenes-Steroids

The structural consensus features of the dockedpeptides (Table 3.) were transformed intopharmacophoric elements and used for the design ofnon-peptide inhibitors [157, 161]. A virtual screen ofthe Cambridge Structural Database was conductedusing one of the pharmacophore distances (distancerange of 1.2 to1.4 nm for OH 97' to COOH 99'). Bycomputer docking, several triterpenes were identifiedthat could fit into the hydrophobic interface site of therelaxed monomer. The IC50 of these compounds wasdetermined to be in the low micromolar range. The use

The major drawback of the interfacial peptides cross-linked with polyalkyl chains is related to theirconformational freedom which induces an unfavorableentropy term in the interaction energy of the inhibitor-HIV-1 PR monomer complex. Bouras et al. [160]designed more rigid "molecular tongs" based on aconformationally constrained scaffold attached to two

OX1

O

OX2

O

47

48

49

50

Pyridine GLL-OMe 4.5

Pyridine TIV-OMe 1.4

Naphthalene VLV-OMe 0.56

Naphthalene TLNF-OMe 1

Arom. X1=X2 Ki,dim (µM)

Arom.

Fig. (8) . Some “molecular tongs” and their inhibitory activities [160].


R1R2

COOHCH3

HOH3C CH3

CH3

CH3

ursolic acid IC50 = 2µM

oleanilic acid IC50 = 1µM

R1=H, R2=CH3 ursolic acidR1=CH3, R2=H oleanilic acid

Cl

NHO

O

UCSF 142 Kinact =10.7 µM

51

52

of triterpene compounds as cheap building units in thedesign of non peptide inhibitors presents severaladvantages: 1) many steroids are used as drugs andhave good bioavailability 2) the rigid polycyclic nucleusshould reduce the tendency for hydrophobic collapseand loss of entropy generally associated with peptide-protein interactions 3) triterpenes offer multiplepossibilities for side chain modifications.

of HIV infection. In summary, the use of defective PRmonomers as trans-dominant PR inhibitors offers anelegant way for a single or multifaceted gene therapyapproach in the clinical treatment of HIV-1 infection[164].

Metal-based Inhibitors

Another approach to the inhibition of the proteasewas suggested from studies on the metal binding toproteins. In fact, such binding could lead to the enzymeinactivation via a metal catalyzed oxidation [165], or byinterfering with the correct binding of the substrate. Inorder to determine if the protease possesses asequence susceptible to bind a metal ion, theinfluence on the protease activity of several metalcations have been studied. Several cations with hardacidic nature such as UO2

2+, Ti4+, V5+, Au3+ and Pb2+

[166] or Zn2+ [167] were found to inhibit the proteaseactivity with IC50 in the micromolar range. The inhibitoryeffect was attributed to the binding at, or near thecatalytic aspartic residues at the active site level. Unlikethose metal species, cupric and mercuric ions tend tobind the sulfhydryl group of cysteine residues andwere found to inhibit the protease with activities in themicromolar range [168]. Beside Cys95 which belongsto the dimer interface, the HIV-1 protease possessesanother cysteine, Cys67, which lies on the surface ofthe enzyme and is structurally susceptible to bind ametal cation [16].

Haloperidol Based Irreversible Inhibitors

The search for active site irreversible inhibitors ofHIV-1 protease has led to the discovery that somehaloperidol derivatives were also able to inactivate theenzyme via a sulfhydryl alkylation of the Cys95belonging to the dimer interface [131-133]. Cys95derivatization was observed with other sulfhydrylreagents such as DTNB, glutathione [162] N-ethylmaleimide and iodoacetamine [128] and couldinduce an inhibition by interfering with the proteasedimerization.

Heterodimer Formation

Another inhibition approach exploiting the dimericnature of HIV protease is the use of defectivemonomers or non identical subunits to exchange withwild type homodimers and to produce catalyticallydefective heterodimers [147, 163]. A structure-basedapproach was used to identify amino acid substitutionsat the dimer interface of HIV-1 PR that would facilitatepreferential association of the defective monomers.Expression of those designed PR monomers inhibitedactivity of the wild-type HIV-1 PR and viral infectivitywhen assayed in a ex vivo model system. These resultsshow that it is possible to design PR monomers asmacromolecular inhibitors that may provide analternative to small molecule inhibitors for the treatment

The essential role of cysteines in the proteaseinactivation by cupric and mercuric ions has beendemonstrated by investigation of a variant proteaselacking cysteine residues which were replaced by α-aminobutyric acid [168]. This variant enzyme, whichhad virtually the same three-dimensional structure and


specific activity as the wild-type protein, was notinhibited by copper and mercury. Only in the presenceof exogenous chelating agents such as dithiothreitol[168], ascorbic acid [169], bathocuproine disulfonicacid [170] or carboxamide ligands [126, 127] wasinhibition recovered. In those particular cases, it wasagain postulated that the synthetic protease wasinactivated via a competitive inhibition pattern, by theso-formed metal-organic complexes. Further studieson cupric ion led to the hypothesis that binding toCys67 could induce allosteric deformation interferingwith the correct motion of the flaps. A structural study ofHIV-1 PR by NMR has demonstrated that flap motionoccurs with compensatory changes in residues 59-75in a manner of a cantilever [171]. As the flaps closedown, the cantilever moves up. The use of twoprotease mutants, C67S and H69A, supported theimplication of Cys67 as a coordinating atom andpointed out the importance of His69 residue as asecond ligand for copper [172]. Cys67 and His69 weresoon recognized as part of a metal binding sequenceCys67-Gly68-His69-Lys70. In fact, the sequence Gly-His-Lys is a well characterized copper-binding growthfactor isolated from human plasma. This tripeptide,which forms a high affinity complex with Cu2+, appearsto function in the transport of copper, and has diversebiological actions [169]. The reactivity of Cys67 residuewould further be enhanced by the presence of His69and Lys70, which create an electrostatic environmentthat induces ionization to the thiolate form at acid pH[173].

These studies cast new light on an interesting siteon the protease surface. This site, including residues59-75, was first described as a cantilever by Harte et al.Among these residues, a short sequence has beenpointed out: Cys67-Gly68-His69-Lys70 which can beconsidered as a metal binding site. Since the binding ofcopper or the derivatization with DTNB causesinactivation, it seems likely that any molecule whichbinds to this region has the potential to change theenzyme activity via an oxidation mechanism and/orallosteric deformations. In addition, Cys67glutathionation experiments have demonstrated onepossible mechanism for regulation of the HIV-1protease through cysteine modification. So, anadditional target has been identified that affectsprotease activity other than the active site or thedimerization site.

Conclusion

Due to its massive threat to health on a global scale,HIV-1 protease has been the subject of extensivestudies and to date, the protein is probably one of themost studied enzyme, with more than 80 X-ray andNMR structures available in the PDB. It is estimated thatmore than 200 structures have been determined sincethe first crystallographic structure was released in 1989.HIV-1 PR can therefore be considered as an idealtarget for drug design methodologies. Most studieshave been carried out to design inhibitors targeting theactive site of the enzyme. Robust substrate-basedmethodologies, inspired from renin inhibitor projectsand coupled with rational design techniques, have ledto several approved drugs: Invirase (4 ) (saquinavirmesylate, Hoffmann-LaRoche), Norvir (1 8 ) (ritonavir,Abott Laboratories), Crixivan (6 ) (indinavir sulfate,Merck) and Viracept (5 ) (nelfinavir mesylate, AgouronPharmaceuticals) as well as Amprenavir (VX-478, GlaxoWellcome, currently in clinical trials). The most strikingsuccess using random screening is probably thediscovery of phenprocoumon (1 9 ) as a lead non-peptide inhibitor of HIV-1 PR, which was identified viabroad screening of the Pharmacia & Upjohn compoundlibrary. Iterative cycles of structure-based design led toTipranavir (2 0 ) (PNU-140690, clinical trials), a potentand orally bioavailable nonpeptidic protease inhibitor.Finally, the favorable pharmacokinetics of cyclic ureasled to the identification by rational drug design of thefirst cyclic urea candidate shortly after its initial designand synthesis. Improvement of physical properties andpharmacokinetics without loss of potency led to thedesign and synthesis of weakly basic cyclic urea analogDMP450 (2 4 ) currently in clinical development. Apromising trend in HIV-1 protease inhibition is thedevelopment of compounds interfering with theenzyme dimerization process. Peptides and

On the other hand, the presence of cysteineresidues renders the protease especially susceptibleto oxidation and disulfide formation. In fact, the additionof copper to the protease at pH 5.5 induce aggregationof the protein, via oxidation of cysteine residues todisulfides [169]. The ready modification of cysteines bycopper has led to the investigation of their reactivitywith DTNB [5,5'-dithiobis-(2-nitrobenzoic acid) orEllman's reagent], a sulfhydryl compound which reactswith the ionized form of the cysteine residues [162,169, 173]. By using three recombinant proteasemutants (C67A; C95A; C67A-C95A) it has beendemonstrated that each cysteine can be derivatized byDTNB, althought Cys95 derivatization seems to have agreater effect on the loss of activity [162]. The effect ofa physiologically relevant sulfhydryl agent, glutathionehas also been investigated. It has been shown that ifglutathionation of cysteine 95 abolishes activity, theenzyme modified at cysteine 67 with glutathione hasincreased activity over the unmodified protein. Inaddition, glutathionation at cysteine 67 markedlystabilized the enzyme activity presumably by reducingautoproteolysis. The greater stability of theglutathiolated enzyme in conjunction with an increasein activity may function to regulate polyproteinprocessing in vivo [162, 174].


peptidomimetics based on the terminal sequence ofthe enzyme, a site which is proposed to be lesssusceptible to mutations, have been shown to lead toHIV-1 PR inactivation. As with therapeutic proceduresthat successfully combine reverse transcriptase (RT)active site inhibitors and compounds targeting thehighly specific RT allosteric TIBO site, it is thought thatPR active-site and dimerization inhibitors could provideanother option in anti-HIV drug combination strategies.Finally, it is worth noting that an additional target hasbeen identified that affects protease activity, as cupricion was described to act on a metal-binding sequenceon the protease surface, which includes cysteine andhistidine residues, leading to the inhibition of theenzyme.

[11] Schäfer, W., Friebe, W.-G., Leinert, H., Mertens, A., Poll, T., vonder Saal, W., Zilch, H., Nuber, B., and Ziegler, M.L. J. Med.Chem. 1993, 36, 726.

[12] Smerdon, S.J., Jäger, J., Wang, J., Kohlstaedt, L.A., Chirino, A.J.,Friedman, J.M., Rice, P.A., and Steitz, T.A. Proc. Natl. Acad. Sci.USA 1994, 91, 3911.

[13] Seelmeier, S., Schmidt, H., Turk, V., and von der Helm, K. Proc.Natl. Acad. Sci. USA 1988, 85, 6612.

[14] Palella, F.J., Delaney, K.M., Moorman, A.C., Loveless, M.O.,Fuhrer, J., Satten, G.A., Aschman, D.J., Holmberg, S.D. NewEngl. J. Med. 1998, 338, 853.

[15] Navia, M.A., Fitzgerald, P.M.D., McKeever, B.M., Leu, C.T.,Heimbach, J.C., Herber, W.K., Sigal, I.S., Darke, P.L., andSpringer, J.P. Nature 1989, 337, 615.

[16] Wlodawer, A., Miller, M., Jaskolski, M., Sathyanarayana, B.K.,Baldwin, E., Weber, I.T., Selk, L.M., Clawson, L., Schneider, J.,and Kent, S.B.H. Science 1989, 245, 616.Acknowledgments

[17] Lapatto, R., Blundell, T., Hemmings, A., Overington, J.,Wilderspin, A., Wood, S., Merson, J.R., Whittle, P.J., Danley,D.E., Geoghegan, K.F., Hawrylik, S.J., Lee, S.E., Scheld, K.G.,and Hobart, P.M. Nature 1989, 342, 299.

Very special thanks to Michèle Reboud-Ravaux,François Durant, Hans-Joachim Schramm and SamesSicsic for proofreading of the manuscript. We wish tothank the European Communities EC for grant CT96-0823 and the National Belgian Foundation for ScientificResearch (FNRS), IBM-Belgium and the FacultésUniversitaires Notre-Dame de la Paix (FUNDP) for theuse of the Namur Scientific Computing Facility. F. L.and M. L. thank the National Belgian Foundation forScientific Research (FNRS) and the Industry andAgriculture Research Foundation (FRIA) for financialsupport.

[18] Toh, H., Ono, M., Saigo, K., and Miyata, T. Nature 1985, 315, 691.

[19] Layman, P. C&EN 1993, 3.

[20] Friedman, S.H., DeCamp, D.L., Sijbesman, R.P., Srdanov, G.,Wudl, F., and Kenyon, G.L. J. Am. Chem. Soc. 1993, 115, 6506.

[21] Sijbesma, R., Srdanov, G., Wudl, F., Castoro, J.A., Wilkins, C.,Friedman, S.H., DeCamp, D.L., and Kenyon, G.L. J. Am. Chem.Soc. 1993, 115, 6510.

[22] Friedman, S.H., Ganapathi, P.S., Rubin, Y., and Kenyon, G.L. J.Med. Chem. 1998, 41, 2424.

References [23] Marshall, G.R. Tetrahedron 1993, 49, 3547.

[1] Barré-Sinoussi, F., Chermann, J.C., Rey, F., Nugeyre, M.T.,Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vézinet-Brun, F., Rouzioux, C., Rozenbaum, W., and Montagnier, L.Science 1983, 220, 868.

[24] Gante, J. Angew Chem Int Ed 1994, 33, 1699.

[25] Mills, J.S. Antiviral Chemistry & Chemotherapy 1996, 7, 281.

[26] Oliyaï, R. advanced drug delivery reviews 1996, 19, 275.

[2] Gallo, R.C., Salahuddin, S.Z., Popovic, M., Shearer, G.M.,Kaplan, M., Haynes, B.F., Palker, T.J., Redfield, R., Oleske, J.,Safai, B., White, G., Foster, P., and Markham, P.D. Science1984, 224, 500.

[27] Roberts, N.A., Martin, J.A., Kinchington, D., Broadhurst, A.V.,Craig, J.C., Duncan, I.B., Galpin, S.A., Handa, B.K., Kay, J.,Kröhn, A., Lambert, R.W., Merret, J.H., Mills, J.S., Parkes,K.E.B., Redshaw, S., Ritchie, A.J., Taylor, D.L., Thomas, G.J.,and Machin, P.J. Science 1990, 248, 358.[3] Levine, A.J., In VIRUSES; Scientific American Library: New

York, pp. 131.[28] Fitzgerald, P.M.D., McKeever, B.M., VanMiddlesworth, J.F.,

Springer, J.P., Heimbach, J.C., Leu, C.T., Herber, W.K., Dixon,R.A.F., and Darke, P.L. J. Biol. Chem. 1990, 265, 14209.

[4] Ratner, R. Perspective in Drug Discovery and Design 1993, 1, 3.

[5] Katz, R.A. and Skalka, A.M. Annu. Rev. Biochem. 1994, 63, 133.[29] Sakurai, M., Sugano, M., Handa, H., Komai, T., Yagi, R.,

Nishigaki, T., and Yabe, Y. Chem. Pharm. Bull. 1993, 41, 1369.[6] Johnson, V.A. Antivir. Res. 1996, 29, 35.

[7] De Clercq, E. J. Med. Chem. 1995, 38, 2491. [30] Baldwin, E.T., Bhat, T.N., Gulnik, S., Liu, B.S., Topol, I.A., Kiso,Y., Mimoto, T., Mitsuya, H., and Erickson, J.W. Structure 1995,3, 581.[8] De Clercq, E. Collect. Czech. Chem. Commun. 1998, 63, 449.

[9] Kohlstaedt, L.A., Wang, J., Friedman, J.M., Rice, P.A., and Steitz,T.A. Science 1992, 256, 1783.

[31] Kiso, Y. Biopolymers 1996, 40, 235.

[32] Chokekijchai, S., Shirasaka, T., Weinstein, J.N., and Mitsuya, H.Antiviral Research 1995, 28, 25.[10] Mui, P.W., Jacober, S.P., Hargrave, K.D., and Adams, J. J. Med.

Chem. 1992, 35, 201.


[33] Kiriyama, A., Nishiura, T., Ishino, M., Yamamoto, Y., Ogita, I.,Kiso, Y., and Takada, K. Biopharmaceutics & Drug Disposition1996, 17, 739.

[48] Partaledis, J.A., Yamaguchi, K., Tisdale, M., Blair, E.E., Falcione,C., Maschera, B., Myers, R.E., Pazhanisamy, S., Futer, O.,Cullinan, A.B., Stuver, C.M., Byrn, R.A., and Livingston, D.J. JVirol 1995, 69, 5228.

[34] Mueller, B.U., Anderson, B.D., Farley, M.Q., Murphy, R.,Zuckerman, J., Jarosinski, P., Godwin, K., Mccully, C.L.,Mitsuya, H., Pizzo, P.A., and Balis, F.M. Antimicrob AgentsChemother 1998, 42, 1815.

[49] Decker, C.J., Laitinen, L.M., Bridson, G.W., Raybuck, S.A., Tung,R.D., and Chaturvedi, P.R. J Pharm Sci 1998, 87, 803.

[50] Miller, M., Schneider, J., Sathyanarayana, B.K., Toth, M.V.,Marshall, G.R., Clawson, L., Selk, L., Kent, S.B.H., andWlodawer, A. Science 1989, 246, 1149.

[35] Mimoto, K., Kato, R., Takaku, H., Nojima, S., Terashima, K.,Misawa, S., Fukazawa, T., Ueno, T., Sato, H., Hintani, M., Kiso,Y., and Hayashi, H. J. Med. Chem. 1999, 42, 1789.

[51] Reich, S.H., Melnick, M., Davies, J.F., Appelt, K., Lewis, K.K.,Fuhry, M.A., Pino, M., Trippe, A.J., Nguyen, D., Dawson, H., Wu,B.W., Musick, L., Kosa, M., Kahil, D., Webber, S., Gehlhaar,D.K., Andrada, D., and Shetty, B. Proc. Natl. Acad. Sci. USA1995, 92, 3298.

[36] Kiso, Y., Yamaguchi, S., Matsumoto, H., Mimoto, T., Kato, R.,Nojima, S., Takaku, H., Fukazawa, T., Kimura, T., and Akaji, K.Arch. Pharm. Pharm. Med. Chem. 1998, 331, 87.

[37] Kalish, V., Kaldor, S., Shetty, B., Tatlock, J., Davies, J.,Hammond, M., Dressman, B., Fritz, J., Appelt, K., Reich, S.,Musick, L., Wu, B.W., and Su, K. Eur. J. Med. Chem. 1995, 30,S201.

[52] Jungheim, L.N., Shepherd, T.A., Baxter, A.J., Burgess, J., Hatch,S.D., Lubbehusen, P., Wiskerchen, M., and Muesing, M.A. J.Med. Chem. 1996, 39, 96.

[38] Reich, S.H., Melnick, M., Pino, M.J., Fuhry, M.A.M., Trippe, A.J.,Appelt, K., Davies, J.F., Wu, B.W., and Musick, L. J. Med. Chem.1996, 39, 2781.

[53] Sham, H.L., Betebenner, D.A., Wideburg, N.E., Saldivar, A.C.,Kohlbrenner, W.E., Vasavanonda, S., Kempf, D.J., Norbeck,D.W., Zhao, C., Clement, J.J., Erickson, J.E., and Plattner, J.J.Biochem. and Biophys. Res. Comm. 1991, 175, 914.

[39] Melnick, M., Reich, S.H., Lewis, K.K., Mitchell, L.J., Nguyen, D.,Trippe, A.J., Dawson, H., Davies, J.F., Appelt, K., Wu, B.W.,Musick, L., Gehlhaar, D.K., Webber, S., Shetty, B., Kosa, M.,Kahil, D., and Andrada, D. J. Med. Chem. 1996, 39, 2795.

[54] Sham, H.L., Wideburg, N.E., Spanton, S.G., Kohlbrenner, W.E.,Betebenner, D.A., Kempf, D.J., Norbeck, D.W., Plattner, J.J., andErickson, J.W. J. Chem. Soc., Chem. Commun. 1991, 110.

[40] Kaldor, S.W., Kalish, V.J., Davies, J.F., Shetty, B.V., Fritz, J.E.,Appelt, K., Burgess, J.A., Campanale, K.M., Chirgadze, N.Y.,Clawson, D.K., Dressman, B.A., Hatch, S.D., Khalil, D.A., Kosa,M.B., Lubbehusen, P.P., Muesing, M.A., Patick, A.K., Reich, S.H.,Su, K.S., and Tatlock, J.H. J Med Chem 1997, 40, 3979.

[55] Schirlin, D., Baltzer, S., Altenburger, J.M., Tarnus, C., andRemy, J.M. Tetrahedron 1996, 52, 305.

[56] Schirlin, D., Baltzer, S., Van Dorsselaer, V., Weber, F., Weill, C.,Altenburger, J.M., Neises, B., Flynn, G., Rémy, J.M., andTarnus, C. Bioorg. Medicinal Chem. 1993, 3, 253.

[41] Thompson, S.K., Eppley, A.M., Frazee, J.S., Darcy, M.G., Lum,R.T., Tomaszek, T.A., Ivanoff, L.A., Morris, J.F., Sternberg, E.J.,Lambert, D.M., Fernandez, A.V., Petteway, S.R., Meek, T.D.,Metcalf, B.W., and Gleason, J.G. Bioorg. Medicinal Chem. Letter1994, 4, 2441.

[57] Schirlin, D., Vandorsselaer, V., Tarnus, C., Taylor, D.L., Tyms,A.S., Baltzer, S., Weber, F., Remy, J.M., Brennan, T., Farr, R.,and Janowick, D. Bioorg. Medicinal Chem. Letter 1994, 4, 241.

[58] Vandorsselaer, V., Schirlin, D., Tarnus, C., Taylor, D.L., Tyms,A.S., Weber, F., Baltzer, S., Remy, J.M., Brennan, T., andJanowick, D. Bioorg. Medicinal Chem. Letter 1994, 4, 1213.

[42] Thompson, S.K., Murthy, K.H.M., Zhao, B., Winborne, E., Green,D.W., Fisher, S.M., Desjarlais, R.L., Tomaszek, T.A., Meek,T.D., Gleason, J.G., and Abdelmeguid, S.S. J. Med. Chem. 1994,37, 3100. [59] Taylor, D.L., Ahmed, P.S., Brennan, T.M., Bridges, C.G., Tyms,

A.S., Van Dorsselaer, V., tarnus, C., Hornsperger, J.-M., andSchirlin, D. Antiviral Chemistry & Chemotherapy 1997, 8, 205.[43] Vacca, J.P., Dorsey, B.D., Schleif, W.A., Levin, R.B., Mcdaniel,

S.L., Darke, P.L., Zugay, J., Quintero, J.C., Blahy, O.M., Roth, E.,Sardana, V.V., Schlabach, A.J., Graham, P.I., Condra, J.H.,Gotlib, L., Holloway, M.K., Lin, J., Chen, I.W., Vastag, K.,Ostovic, D., Anderson, P.S., Emini, E.A., and Huff, J.R. Proc.Natl. Acad. Sci. USA 1994, 91, 4096.

[60] Frogier, P.R.T., Tran, T.T., Viani, S., Condom, R., and Guedj, R.Antivir Chem Chemother 1994, 5, 372.

[61] Marastoni, M., Fantin, G., Bortolotti, F., and Tomatis, R. DrugResearch 1996, 46(II), 1099.

[44] Chen, Z.G., Li, Y., Chen, E., Hall, D.L., Darke, P.L., Culberson,C., Shafer, J.A., and Kuo, L.C. J Biol Chem 1994, 269, 26344.

[62] Shepherd, T.A., Jungheim, L.N., and Baxter, A.J. Bioorg.Medicinal Chem. Letter 1994, 4, 1391.

[45] Dorsey, B.D., Levin, R.B., Mcdaniel, S.L., Vacca, J.P., Guare,J.P., Darke, P.L., Zugay, J.A., Emini, E.A., Schleif, W.A.,Quintero, J.C., Lin, J.H., Chen, I.W., Holloway, M.K., Fitzgerald,P.M.D., Axel, M.G., Ostovic, D., Anderson, P.S., and Huff, J.R. J.Med. Chem. 1994, 37, 3443.

[63] Benatalah, Z., Trigui, N., Sicsic, S., Tonnaire, T., Derosny, E.,Boggetto, N., and Reboud, M. Eur. J. Med. Chem. 1995, 30, 891.

[64] Kim, B.M., Guare, J.P., Hanifin, C.M., Arfordbickerstaff, D.J.,Vacca, J.P., and Ball, R.G. Tetrahedron Lett 1994, 35, 5153.

[46] Kim, E.E., Baker, C.T., Dwyer, M.D., Murcko, M.A., Rao, B.G.,Tung, R.D., and Navia, M.A. J. Am. Chem. Soc. 1995, 117, 1181.

[65] Kim, B.M., Vacca, J.P., Fitzgerald, P.M.D., Darke, P.L.,Holloway, M.K., Guare, J.P., Hanifin, C.M., Arfordbickerstaff,D.J., Zugay, J.A., Wai, J.M., Anderson, P.S., and Huff, J.R.Bioorg. Medicinal Chem. Letter 1994, 4, 2199.[47] Livingston, D.J., Pazhanisamy, S., Porter, D.J.T., Partaledis,

J.A., Tung, R.D., and Painter, G.R. The Journal of InfectiousDiseases 1995, 172, 1238.


[66] Vacca, J.P., Fitzgerald, P.M.D., Holloway, M.K., Hungate, R.W.,Starbuck, K.E., Chen, L.J., Darke, P.L., Anderson, P.S., and Huff,J.R. Bioorg. Medicinal Chem. Letter 1994, 4, 499.

[83] Romines, K.R., Watenpaugh, K.D., Howe, W.J., Tomich, P.K.,Lovasz, K.D., Morris, J.K., Janakiraman, M.N., Lynn, J.C.,Horng, M.M., Chong, K.T., Hinshaw, R.R., and Dolak, L.A. J.Med. Chem. 1995, 38, 4463.

[67] Abell, A.D. and Foulds, G.J. J. Chem. Soc., Perkin Trans. 1997,1, 2475. [84] Thaisrivongs, S., Watenpaugh, K.D., Howe, W.J., Tomich, P.K.,

Dolak, L.A., Chong, K.T., Tomich, C.S.C., Tomasselli, A.G.,Turner, S.R., Strohbach, J.W., Mulichak, A.M., Janakiraman,M.N., Moon, J.B., Lynn, J.C., Horng, M.M., Hinshaw, R.R.,Curry, K.A., and Rothrock, D.J. J. Med. Chem. 1995, 38, 3624.

[68] Kroemer, R.T., Ettmayer, P., and Hecht, P. J. Med. Chem. 1995,38, 4917.

[69] Chen, J.J., Coles, P.J., Arnold, L.D., Smith, R.A., MacDonald,I.D., Carrière, J., and Krantz, A. Bioorg. & Medicinal Chem. 1996,6, 435.

[85] Thaisrivongs, S., Janakiraman, M.N., Chong, K.T., Tomich, P.K.,Dolak, L.A., Turner, S.R., Strohbach, J.W., Lynn, J.C., Horng,M.M., Hinshaw, R.R., and Watenpaugh, K.D. J. Med. Chem.1996, 39, 2400.[70] Ettmayer, P., Billich, A., Hecht, P., Rosenwirth, B., and Gstach,

H. J. Med. Chem. 1996, 39, 3291.

[86] Romines, K.R., Morris, J.K., Howe, W.J., Tomich, P.K., Horng,M.M., Chong, K.T., Hinshaw, R.R., Anderson, D.J., Strohbach,J.W., Turner, S.R., and Mizsak, S.A. J. Med. Chem. 1996, 39,4125.

[71] Podlogar, B.L., Farr, R.A., Friedrich, D., Tarnus, C., Huber,E.W., Cregge, R.J., and Schirlin, D. J. Med. Chem. 1994, 37,3684.

[72] Abbenante, G., March, D.R., Bergman, D.A., Hunt, P.A.,Garnham, B., Dancer, R.J., Martin, J.L., and Fairlie, D.P. J. Am.Chem. Soc. 1995, 117, 10220.

[87] Janakiraman, M.N., Watenpaugh, K.D., Tomich, P.K., Chong,K.T., Turner, S.R., Tommasi, R.A., Thaisrivongs, S., andStrohbach, J.W. Bioorg Medicinal Chem Letter 1998, 8, 1237.

[73] Ripka, A.S., Bohacek, R.S., and Rich, D.H. Bioorg MedicinalChem Letter 1998, 8, 357.

[88] Turner, S.R., Strohbach, J.W., Tommasi, R.A., Aristoff, P.A.,Johnson, P.D., Skulnick, H.I., Dolak, L.A., Seest, E.P., Tomich,P.K., Bohanon, M.J., Horng, M.M., Lynn, J.C., Chong, K.-T.,Hinshaw, R.R., Watenpaugh, K.D., Janakiraman, M.N., andThaisrivongs, S. J. Med. Chem. 1998, 1998, 3467.

[74] Borman, S. C&EN 1993, 8, 27.

[75] Smith III, A.B., Hirschmann, R., Pasternak, A., Akaishi, R.,Guzman, M.C., Jones, D.R., Keenan, T.P., Sprengeler, P.A.,Darke, P.L., Emini, E.A., Holloway, M.K., and Schleif, W.A. J.Med. Chem. 1994, 37, 215.

[89] Prasad, J.V.N.V., Para, K.S., Lunney, E.A., Ortwine, D.F.,Dunbar, J.B., Ferguson, D., Tummino, P.J., Hupe, D., Tait, B.D.,Domagala, J.M., Humblet, C., Bhat, T.N., Liu, B.S., Guerin,D.M.A., Baldwin, E.T., Erickson, J.W., and Sawyer, T.K. J. Am.Chem. Soc. 1994, 116, 6989.

[76] SmithIII, A.B., Guzman, M.C., Sprengeler, P.A., Keenan, T.P.,Holcomb, R.C., Wood, J.L., Carroll, P.J., and Hirschmann, R. J.Am. Chem. Soc. 1994, 116, 9947.

[90] Lunney, E.A., Hagen, S.E., Domagala, J.M., Humblet, C.,Kosinski, J., Tait, B.D., Warmus, J.S., Wilson, M., Ferguson, D.,Hupe, D., Tummino, P.J., Baldwin, E.T., Bhat, T.N., Liu, B.S., andErickson, J.W. J. Med. Chem. 1994, 37, 2664.

[77] Smith III, A.B., Hirschmann, R., Pasternak, A., Yao, W., andSprengeler, P.A. J. Med. Chem. 1997, 40, 2440.

[78] Kempf, D.J., Norbeck, D.W., Codacovi, L.M., Wang, X.C.,Kohlbrenner, W.E., Wideburg, N.E., Paul, D.A., Knigge, M.F.,Vasavanonda, S., Craig-Kennard, A., Saldivar, A., Rosenbrook,W., Clement, J.J.J., Plattner, J.J., and Erickson, J. J. Med.Chem. 1990, 33, 2687.

[91] Prasad, J.V.N.V., Lunney, E.A., Ferguson, D., Tummino, P.J.,Rubin, J.R., Reyner, E.L., Stewart, B.H., Guttendorf, R.J.,Domagala, J.M., Suvorov, L.I., Gulnik, S.V., Topol, I.A., Bhat,T.N., and Erickson, J.W. J. Am. Chem. Soc. 1995, 117, 11070.

[92] Prasad, J.V.N., Para, K.S., Tummino, P.J., Ferguson, D.,Mcquade, T.J., Lunney, E.A., Rapundalo, S.T., Batley, B.L.,Hingorani, G., Domagala, J.M., Gracheck, S.J., Bhat, T.N., Liu,B.S., Baldwin, E.T., Erickson, J.W., and Sawyer, T.K. J. Med.Chem. 1995, 38, 898.

[79] Erickson, J. and Kempf, D. Arch Virol 1994, 19.

[80] Kempf, D.J., Sham, H.L., Marsh, K.C., Flentge, C.A.,Betenbenner, D., Green, B.E., McDonald, E., Vasavanonda, S.,Saldivar, A., Wideburg, N.E., Kati, W.M., Ruiz, L., Zhao, C., Fino,L., Patterson, J., Molla, A., Plattner, J.J., and Norbeck, D.W. J.Med. Chem. 1998, 41, 602. [93] Prasad, J.V.N.V., Tummino, P.J., Ferguson, D., Saunders, J.,

Vanderroest, S., Mcquade, T.J., Heldsinger, A., Reyner, E.L.,Stewart, B.H., Guttendorf, R.J., Para, K.S., Lunney, E.A.,Gracheck, S.J., and Domagala, J.M. Biochem. Biophys. Res.Commun. 1996, 221, 815.

[81] Thaisrivongs, S., Tomich, P.K., Watenpaugh, K.D., Chong, K.T.,Howe, W.J., Yang, C.P., Strohbach, J.W., Turner, S.R., Mcgrath,J.P., Bohanon, M.J., Lynn, J.C., Mulichak, A.M., Spinelli, P.A.,Hinshaw, R.R., Pagano, P.J., Moon, J.B., Ruwart, M.J.,Wilkinson, K.F., Rush, B.D., Zipp, G.L., Dalga, R.J., Schwende,F.J., Howard, G.M., Padbury, G.E., Toth, L.N., Zhao, Z.Y.,Koeplinger, K.A., Kakuk, T.J., Cole, S.L., Zaya, R.M., Piper, R.C.,and Jeffrey, P. J. Med. Chem. 1994, 37, 3200.

[94] Hamilton, H.W., Tait, B.D., Gajda, C., Hagen, S.E., Ferguson, D.,Lunney, E.A., Pavlovsky, A., and Tummino, P.J. Bioorg.Medicinal Chem. Letter 1996, 6, 719.

[95] Steinbaugh, B.A., Hamilton, H.W., Prasad, J.V.N.V., Para, K.S.,Tummino, P.J., Ferguson, D., Lunney, E.A., and Blankley, C.J.Bioorg. Medicinal Chem. Letter 1996, 6, 1099.

[82] Romines, K.R., Watenpaugh, K.D., Tomich, P.K., Howe, W.J.,Morris, J.K., Lovasz, K.D., Mulichak, A.M., Finzel, B.C., Lynn,J.C., Horng, M.M., Schwende, F.J., Ruwart, M.J., Zipp, G.L.,Chong, K.T., Dolak, L.A., Toth, L.N., Howard, G.M., Rush, B.D.,Wilkinson, K.F., Possert, P.L., Dalga, R.J., and Hinshaw, R.R. J.Med. Chem. 1995, 38, 1884.

[96] Tait, B.D., Hagen, S., Domagala, J., Ellsworth, E.L., Gajda, C.,Hamilton, H.W., Prasad, J.V.N.V., Ferguson, D., Graham, N.,Hupe, D., Nouhan, C., Tummino, P.J., Humblet, C., Lunney, E.A.,Pavlovsky, A., Rubin, J., Gracheck, S.J., Baldwin, E.T., Bhat,


T.N., Erickson, J.W., Gulnik, S.V., and Liu, B.S. J Med Chem1997, 40, 3781.

[115] DeLucca, G.V., Erickson-Viitanen, S., and Lam, P.Y.S. DDT1997, 2, 6.

[97] Hagen, S.E., Prasad, J.V.N.V., Boyer, F.E., Domagala, J.M.,Ellsworth, E.L., Gajda, C., Hamilton, H.W., Markoski, L.J.,Steinbaugh, B.A., Tait, B.D., Lunney, E.A., Tummino, P.J.,Ferguson, D., Hupe, D., Nouhan, C., Gracheck, S.J., Saunders,J.M., and Vanderroest, S. J Med Chem 1997, 40, 3707.

[116] KaltenbachIII, R.F., Nugiel, D.A., Lam, P.Y.S., Klabe, R.M., andSeitz, S.P. J. Med. Chem. 1998, 41, 5113.

[117] Delucca, G.V., Kim, U.T., Liang, J., Cordova, B., Klabe, R.M.,Garber, S., Bacheler, L.T., Lam, G.N., Wright, M.R., Logue, K.A.,Ericksonviitanen, S., Ko, S.S., and Trainor, G.L. J Med Chem1998, 41, 2411.[98] Humber, D.C., Cammack, N., Coates, J.A.V., Cobley, K.N., Orr,

D.C., Storer, R., Weingarten, G.G., and Weir, M.P. J. Med.Chem. 1992, 35, 3080. [118] Patel, M., Kaltenbach, R.F., Nugiel, D.A., Mchugh, R.J., Jadhav,

P.K., Bacheler, L.T., Cordova, B.C., Klabe, R.M.,Ericksonviitanen, S., Garber, S., Reid, C., and Seitz, S.P. BioorgMedicinal Chem Letter 1998, 8, 1077.

[99] Holmes, D.S., Clemens, I.R., Cobley, K.N., Humber, D.C.,Kitchin, J., Orr, D.C., Patel, B., Paternoster, I.L., and Storer, R.Bioorg. Medicinal Chem. 1993, 3, 503.

[119] Patel, M., Bacheler, L.T., Rayner, M.M., Cordova, B.C., Klabe,R.M., Erickson-Viitanen, S., and Seitz, S.P. Bioorg. Med. Chem.Lett. 1998, 8, 823.

[100] Jhoti, H., Singh, O.M.P., Weir, M.P., Cooke, R., Murrayrust, P.,and Wonacott, A. Biochemistry 1994, 33, 8417.

[101] Humber, D.C., Bamford, M.J., Bethell, R.C., Cammack, N., Orr,D.C., Storer, R., Weingarten, G.G., and Wyatt, P.G. AntiviralChemistry & Chemotherapy 1994, 5, 197.

[120] Ala, P.J., DeLoskey, R.J., Huston, E.E., Jadhav, P.K., Lam,P.Y.S., Eyermann, C.J., Hodge, C.N., Schadt, M.C.,Lewandowski, F.A., Weber, P.C., McCabe, D.D., Duke, J.L., andChang, C.-H. J. Biol. Chem. 1998, 273, 12325.

[102] Kitchin, J., Bethell, R.C., Cammack, N., Dolan, S., Evans, D.N.,Holman, S., Holmes, D.S., Mcmeekin, P., Mo, C.L., Nieland, N.,Orr, D.C., Saunders, J., Shenoy, B.E.V., Starkey, I.D., and Storer,R. J. Med. Chem. 1994, 37, 3707.

[121] Rodgers, J.D., Johnson, B.L., Wang, H., Erickson-Viitanen, S.,Klabe, R.M., Bacheler, L., Cordova, B.C., and Chang, C.-H.Bioorg. Med. Chem. Lett. 1998, 8, 715.

[103] DeCamp, D.L., Babe, L.M., Salto, R., Lucich, J.L., Koo, M.-S.,Kahl, S.B., and Craik, C.S. J. Med. Chem. 1992, 35, 3426.

[122] DeLucca, G.V., Liang, J., and DeLucca, I. J. Med. Chem. 1999,42, 135.

[104] Otake, T., Mori, H., Morimoto, M., Ueba, N., Kusumoto, I.T., Lim,Y.A., Miyashiro, H., Hattori, M., Namba, T., Gupta, M.P., andCorrea, M. Journal of Traditional Medicinen 1994, 11, 188.

[123] Desjarlais, R.L., Seibel, G.L., Kuntz, I.D., Furth, P.S., Alvarez,J.C., Ortiz de Montellano, P.R., DeCamp, D.L., Babé, L.M., andCraik, C.S. Proc. Natl. Acad. Sci. USA 1990, 87, 6644.

[105] Otake, T., Mori, H., Morimoto, M., Ueba, N., Sutardjo, S.,Kusumoto, I.T., Hattori, M., and Namba, T. PhytotherapyResearch 1995, 9, 6.

[124] Rutenberg, E., Fauman, E.B., Keenan, R.J., Fong, S., Furth, P.S.,Ortiz de montellano, P.R., Meng, E., Kuntz, I.D., DeCamp, D.L.,Salto, A., Rosé, J.R., Craik, C.S., and Stroud, R.M. J. Biol.Chem. 1993, 268, 15343.

[106] Kusumoto, I.T., Nakabayashi, T., Kida, H., Miyashiro, H., Hattori,M., Namba, T., and Shimotohno, K. Phytother Res 1995, 9, 180. [125] Castro, I., Faus, J., Julve, M., Amigo, J.M., Sletten, J., and

Debaerdemaeker, T. J. Chem. Soc., Dalton Trans. 1990, 891.[107] Wan, M., Bloor, S., Foo, L.-Y., and Loh, B.-N. Phytotherapy

Research 1996, 10, 589. [126] Lebon, F., De Rosny, E., Reboud-Ravaux, M., and Durant, F.Eur. J. Med. Chem. 1998, 33, 733.

[108] Xu, H.-X., Wan, M., Loh, B.-N., Kon, O.-L., Chow, P.-W., andSim, K.-Y. Phytotherapy Research 1996, 10, 207. [127] Lebon, F., Ledecq, M., Benatallah, Z., Sicsic, S., Lapouyade, R.,

Kahn, O., Garçon, A., Reboud-Ravaux, M., and Durant, F. PerkinTrans. 2 1999, 4, 795.[109] Roggo, B.E., Petersen, F., Sills, M., Roesel, J.L., Moerker, T.,

and Peter, H.H. J. Antibiot. 1996, 49, 13.[128] Meek, T.D., Dayton, B.D., Metcalf, B.W., Dreyer, G.B., Strickler,

J.E., Gorniak, J.G., Rosenberg, M., Moore, M.L., Magaard, V.W.,and Debouck, C. Biochemistry 1989, 86, 1841.

[110] Chen, S.-X., Wan, M., and Loh, B.-N. Planta Medica 1996, 4, 381.

[111] Matsuse, I.T., Nakabayashi, T., Lim, Y.A., Hussein, G.M.E.,Miyashiro, H., Kakiuchi, N., Hattori, M., Stardjo, S., andShimotohno, K. Phytotherapy Research 1997, 11, 433.

[129] Rose, R.B., Rose, J.R., Salto, R., Craik, C.S., and Stroud, R.M.Biochemistry 1993, 32, 12498.

[112] Ichimura, T., Watanbe, O., and Maruyama, S. Biosci. Biotechnol.Biochem. 1998, 62, 575.

[130] Blumenstein, J.J., Copeland, T.D., Oroszlan, S., and Michejda,C.J. Biochemical and Biophysical Research Communications1989, 163, 980.

[113] Lam, P.Y.S., Jadhav, P.K., Eyermann, C.J., Hodge, C.N., Ru, Y.,Bacheler, L.T., Meek, J.L., Otto, M.J., Rayner, M.M., Wong, Y.N.,Chang, C.H., Weber, P.C., Jackson, D.A., Sharpe, T.R., andErickson-Viitanen, S. Science 1994, 263, 380.

[131] Salto, R., Babé, L.M., Li, J., Rosé, J.R., Yu, Z., Burlingame, A.,DeVoss, J.J., Sui, Z., Ortiz de Montellano, P., and Craik, C.S. J.Biol. Chem. 1994, 269, 10691.

[114] Hodge, C.N., Aldrich, P.E., Bacheler, L.T., Chang, C.-H.,Eyermann, C.J., Garber, S., Grubb, M., Jackson, D.A., Jadhav,P.K., Korant, B., Lam, P.Y.S., Maurin, M.B., Meek, J.L., Otto,M.J., Rayner, M.M., Reid, C., Sharpe, T.R., Shum, L., Winslow,D.L., and Erickson-Vititanen, S. Current Biology 1996, 3, 301.

[132] De Voss, J.J., Sui, Z., DeCamp, D.L., Salto, R., Babe, L.M.,Craig, C.S., and Ortiz de Montellano, P.R. J. Med. Chem. 1994,37, 665.


[133] Yu, Z., Caldera, P., McPhee, F., DeVoss, J.J., Jones, P.R.,Burlingame, A.L., Kuntz, I.D., Craik, C.S., and Ortiz deMontellano, P.R. JACS 1996, 118, 5846.

[154] Franciskovich, J., Houseman, K., Mueller, R., and Chmielewski,J. Bioorg. Med. Chem. Lett. 1993, 3, 765.

[155] Schramm, H.J., Billig, A., Jaeger, E., Rücknagel, K.-P., Arnold,G., and Schramm, W. Biochem. Biophys. Res. Commun. 1993,194, 595.

[134] Mavri, J. Int. J. Quant. Chem. 1998, 69, 753.

[135] Grant, S.K., Moore, M.L., Fakhoury, S.A., Tomaszek Jr., T.A.,and Meek, T.D. Bioorg. Med. Chem. Lett. 1992, 2, 1441. [156] Schramm, H.J., Boetzel, J., Buttner, J., Fritsche, E., Gohring, W.,

Jaeger, E., Konig, S., Thumfart, O., Wenger, T., Nagel, N.E., andSchramm, W. Antivir. Res. 1996, 30, 155.[136] Lee, C.S., Choy, N., Park, C., Choi, H., Son, Y.C., Kim, S., Ok,

J.H., Yoon, H., and Kim, S.C. Bioorg. Medicinal Chem. Letter1996, 6, 589. [157] Quéré, L., Wenger, T., and Schramm, H.J. Biochem. Biophys.

Res. Commun. 1996, 227, 484.[137] Park, C., Koh, J.S., Son, Y.C., Choi, H.-i., Lee, C.S., Choy, N.,

Moon, K.Y., Jung, W.H., Kim, S.C., and Yoon, H. Bioorg. Med.Chem. Lett. 1995, 5, 1843.

[158] Arnold, G., Popinga, K., Schramm, H.J., Bell, M., Thumfart, O.,Wenger, T., and Schramm, W., In Peptide 1994 (H. L. S. Maia,ed.); ESCOM: Leiden; 1994, pp. 345.

[138] Abell, A.D., Hoult, D.A., Bergman, D.A., and Fairlie, D.P. BioorgMedicinal Chem Letter 1997, 7, 2853. [159] Zutshi, R., Franciskovich, J., Shultz, M., Scweitzer, B., Bishop,

P., Wilson, M., and Chmielewski, J. J. Am. Chem. Soc. 1997,119, 4841.[139] Ro, S., Baek, S.-G., Lee, B., Park, C., Choy, N., Lee, C.S., Son,

Y.C., Choi, H., Koh, J.S., Yoon, H., Kim, S.C., and Ok, J.H.Bioorg. Med. Chem. Lett. 1998, 8, 2423. [160] Bouras, A., Boggetto, N., Benatalah, Z., deRosny, E., Sicsic, S.,

and Reboud-Ravaux, M. J. Med. Chem. 1999.[140] Choy, N., Choi, H.-i., Jung, W.H., Kim, C.R., Yoon, H., Kim,

S.C., Lee, T.G., and Koh, J.S. Bioorg. Med. Chem. Lett. 1997, 7,2635.

[161] Schramm, H.J., Quéré, L., Büttner, J., Wenger, T., Dick, A., andSchramm, W. AIDS 1998, 12, 682.

[141] Cohen, E.A., Gaudreau, P., Brazeau, P., and Langelier, Y. Nature1986, 321, 441.

[162] Davis, D.A., Dorsey, K., Wingfield, P.T., Stahl, S.J., Kaufman, J.,Fales, H.M., and Levine, R.L. Biochemistry 1996, 35, 2482.

[142] Dutia, B.M., Frame, M.C., Subak-Sharpe, J.H., Clark, W.N., andMarsden, H.S. Nature 1986, 321, 439.

[163] Babé, L.M., Rosé, J., and Craik, C.S. Proc. Natl. Acad. Sci. USA1995, 92, 10069.

[143] Divita, G., Restle, T., Goody, R.S., Chermann, J.-C., and Baillon,J.G. J. Biol. Chem. 1994, 269, 13080.

[164] McPhee, F., Good, A., Kuntz, I.D., and Craik, C.S. Proc. Natl.Acad. Sci. USA 1996, 93, 11477.

[144] Liuzzi, M., Déziel, R., Moss, N., Bealieu, P., Bonneau, A.-M.,Bousquet, C., Chafouleas, J.G., Garneau, M., Jaramillo, J.,Krogsrud, R.L., Lagacé, L., McCollum, R.S., Namoot, S., andGuindon, Y. Nature 1994, 372, 695.

[165] Stadtman, E.R. and Oliver, C.N. J. Biol. Chem. 1991, 266, 2005.

[166] Woon, T.C., Brinkworth, R.I., and Fairlie, D.P. Int. J. Biochem.1992, 24, 911.

[167] Zhang, Z.-Y., Reardon, I.M., Hui, J.O., O'Connell, K.L., Poorman,R.A., Tomasselli, A.G., and Heinrikson, R.L. Biochemistry 1991,30, 8717.

[145] Todd, M.J., Semo, N., and Freire, E. J. Mol. Biol. 1998, 283, 475.

[146] Weber, I.T. J. Biol. Chem. 1990, 265, 10492.

[147] Babé, L.M., Pichuantes, S., and Craik, C.S. Biochemistry 1991,30, 106.

[168] Karlström, A.R.K. and Levine, R.L. Proc. Natl. Acad. Sci. USA1991, 88, 5552.

[148] Zhang, Z.-Y., Poorman, R.A., Maggiora, L.L., Heinrickson, R.L.,and Kezdy, F.J. J. Biol. Chem. 1991, 266, 15591.

[169] Karlström, A.R., Shames, B.D., and Levine, R.L. Arch. Biochem.Biophys. 1993, 304, 163.

[149] Cheng, Y.-S.E., Yin, F.H., Foundling, S., Blomstrom, D., andKettner, C.A. Proc. Natl. Acad. Sci. USA 1990, 87, 9660.

[170] Davis, D.A., Branca, A.A., Pallenberg, A.J., Marschner, T.M.,Patt, L.M., Chatlynne, L.G., Humphrey, R.W., Yarchoan, R., andLevine, R.L. Arch. Biochem. Biophys. 1995, 322, 127.

[150] Darke, P.L. and Huff, J.R., In Advances in Pharmacology, Vol 25(J. T. August, M. W. Anders, and F. Murad, eds.); AcademicPress Inc: 525 B Street, Suite 1900, San Diego, CA 92101-4495;1994, Vol. 25, pp. 399.

[171] Harte, W.E., Swaminathan, S., Mansuri, M.M., Martin, J.C.,Rosenberg, I.E., and Beveridge, D.L. Proc. Natl. Acad. Sci. USA1990, 87, 8864.

[151] Schramm, H.J., Nakashima, H., Schramm, W., Wakayama, H.,and Yamamoto, N. Bioch. Biophys. Res. Comm. 1991, 179, 847.

[172] Danielson, H., Lindgren, M.T., Markgren, P.-O., and Nillroth, U.Adv. Exp. Med. Biol. 1998, 436, 99.

[152] Schramm, H.J., Breipohl, G., Hansen, J., Henke, S., Jaeger, E.,Meichsner, C., Riess, G., Ruppert, D., Rücknagel, K.-P.,Schäfer, W., and Schramm, W. Biochem. Biophys. Res.Commun. 1992, 184, 980.

[173] D'Ettorre, C. and Levine, R.L. Arch Biochem Biophys 1994, 313,71.

[174] Davis, D.A., Yusa, K., Gillim, L.A., Newcombe, F.M., Mitsuya,H., and Yarchoan, R. Journal of Virology 1999, 73, 1156.

[153] Babé, L.M., Rosé, J., and Craik, C.S. Protein Science 1992, 1,1244.

Current Medicinal Chemistry Approaches to the Design of … · 2015. 2. 27. · Current Medicinal...

Documents

Transcript of Current Medicinal Chemistry Approaches to the Design of … · 2015. 2. 27. · Current Medicinal...