Structure of HIV-1 protease with KNI-272, a tight-binding transition-state analog containing allophenylnorstatine.

BACKGROUND: HIV-1 protease (HIV PR), an aspartic protease, cleaves Phe-Pro bonds in the Gag and Gag-Pol viral polyproteins. Substrate-based peptide mimics constitute a major class of inhibitors of HIV PR presently being developed for AIDS treatment. One such compound, KNI-272, which incorporates allophenylnorstatine (Apns)-thioproline (Thp) in place of Phe-Pro, has potent antiviral activity and is undergoing clinical trials. The structure of the enzyme-inhibitor complex should lead to an understanding of the structural basis for its tight binding properties and provide a framework for interpreting the emerging resistance to this drug. RESULTS: The three-dimensional crystal structure of KNI-272 bound to HIV PR has been determined to 2.0 A resolution and used to analyze structure-activity data and drug resistance for the Arg8-->Gln and ILe84-->Val mutations in HIV PR. The conformationally constrained Apns-Thp linkage is favorably recognized in its low energy trans conformation, which results in a symmetric mode of binding to the active-site aspartic acids and also explains the unusual preference of HIV PR for the S, or syn, hydroxyl group of the Apns residue. The inhibitor recognizes the enzyme via hydrogen bonds to three bridging water molecules, including one that is coordinated directly to the catalytic Asp125 residue. CONCLUSIONS: The structure of the HIV PR/KNI-272 complex illustrates the importance of limiting the conformational degrees of freedom and of using protein-bound water molecules for building potent inhibitors. The binding mode of HIV PR inhibitors can be predicted from the stereochemical relationship between adjacent hydroxyl-bearing and side chain bearing carbon atoms of the P1 substituent. Our structure also provides a framework for designing analogs targeted to drug-resistant mutant enzymes.

Escape mutants of HIV-1 proteinase: enzymic efficiency and susceptibility to inhibition.

Genes encoding a number of mutants of HIV-1 proteinase were sub-cloned and expressed in E. coli. The proteinases containing mutations of single residues (e.g., G48V, V82F, I84V and L90M) were purified and their catalytic efficiencies relative to that of wild-type proteinase were examined using a polyprotein (recombinant HIV-1 gag) substrate and several series of synthetic peptides based on the -Hydrophobic * Hydrophobic-, -Aromatic * Pro- and pseudo-symmetrical types of cleavage junction. The L90M proteinase showed only small changes, whereas the activity of the other mutant enzymes was compromised more severely, particularly towards substrates of the -Aromatic * Pro- and pseudo-symmetrical types. The susceptibility of the mutants and the wild-type proteinase to inhibition by eleven different compounds was compared. The L90M proteinase again showed only marginal changes in its susceptibility to all except one of the inhibitors examined. The K(i) values determined for one inhibitor (Ro31-8959) showed that its potency towards the V82F, L90M, I84V and G48V mutant proteinases respectively was 2-, 3-, 17- and 27-fold less than against the wild-type proteinase. Several of the other inhibitors examined form a systematic series with Ro31-8959. The inhibition constants derived with these and a number of other inhibitors, including ABT-538 and L-735,524, are used in conjunction with the data on enzymic efficiency to assess whether each mutation in the proteinase confers an advantage for viral replication in the presence of any given inhibitor.

Human liver cathepsin D. Purification, crystallization and preliminary X-ray diffraction analysis of a lysosomal enzyme.

The two-chain form of active cathepsin D, a glycosylated, lysosomal aspartic proteinase, has been isolated from human liver. Isoelectric focusing revealed two major species of enzyme that differed by approximately 0.2 pI unit. Crystals suitable for X-ray diffraction analysis were prepared from acidic solutions using precipitation with ammonium sulfate. The hexagonal crystals diffracted X-rays to beyond 3.1 A resolution and belonged to space group P6(1) (or P6(5)) with cell constants a = b = 125.9 A, c = 104.1 A, gamma = 120.0 degrees. The crystals likely contain two molecules in the asymmetric unit, giving a solvent content of 56% (v/w). Biochemical analysis of crystals indicated that both isoforms were present in approximately equimolar proportions. Full structure determination of the enzyme is underway.

Increased fitness of drug resistant HIV-1 protease as a result of acquisition of compensatory mutations during suboptimal therapy.

OBJECTIVE: It is thought as a consequence of continuous replication, HIV-1 has acquired an optimal fitness state and that suboptimal antiretroviral therapy selects for drug resistant variants which show impaired fitness in the absence of the drug. In this paper we studied the evolution and fitness of viral populations appearing in a patient who received protease monotherapy. METHODS: Two factors contributing to fitness, drug resistance and protease catalytic activity, were studied at the enzymatic and virological level. RESULTS: The first drug resistant viral variants that were selected in vivo harboured one to three protease substitutions. These mutants showed reduced protease activity and consequently a reduction in viral replication capacity. During continued in vivo replication of these viruses in the presence of the drug, novel variants harbouring additional substitutions in the viral protease appeared. These variants did not display any further increase in drug resistance but demonstrated clearly increased protease activity. Consequently the replication capacity of these viruses was raised to a level at which they replicated better than the original wild-type virus. CONCLUSION: This study indicates that the viral population in the patient does not have to represent the fittest possible variants, and thus antiretroviral therapy may drive the viral population first through a lower fitness level and then to a higher fitness level.

Structure-based subsite specificity mapping of human cathepsin D using statine-based inhibitors.

Human cathepsin D is a lysosomal aspartic protease that has been implicated in breast cancer metastasis and Alzheimer's disease. Based on a crystal structure of a human cathepsin D-pepstatin A complex, a series of statine-containing inhibitors was designed, synthesized, and tested for inhibitory activity toward the enzyme in vitro. The compounds were modified systematically at individual positions (P4, P3, P2, P1, and P2t) with the aim of mapping the cathepsin D subsite preferences. The experimentally obtained SAR data were correlated on the basis of molecular modeling. Side-chain preferences for the peptidomimetic inhibitors differed from those found previously using peptide substrates (Scarborough PE et al., 1993, Protein Sci 2:264-276). In addition, the effects of single side-chain modifications were often nonadditive. Structure-activity relationships, modeling, and thermodynamic analysis indicated that entropy plays a major stabilizing role in inhibitor binding to cathepsin D.

Drug resistance mutations can effect dimer stability of HIV-1 protease at neutral pH.

The monomer-dimer equilibrium for the human immunodeficiency virus type 1 (HIV-1) protease has been investigated under physiological conditions. Dimer dissociation at pH 7.0 was correlated with a loss in beta-sheet structure and a lower degree of ANS binding. An autolysis-resistant mutant, Q7K/L33I/L63I, was used to facilitate sedimentation equilibrium studies at neutral pH where the wild-type enzyme is typically unstable in the absence of bound inhibitor. The dimer dissociation constant (KD) of the triple mutant was 5.8 microM at pH 7.0 and was below the limit of measurement (approximately 100 nM) at pH 4.5. Similar studies using the catalytically inactive D25N mutant yielded a KD value of 1.0 microM at pH 7.0. These values differ significantly from a previously reported value of 23 nM obtained indirectly from inhibitor binding measurements (Darke et al., 1994). We show that the discrepancy may result from the thermodynamic linkage between the monomer-dimer and inhibitor binding equilibria. Under conditions where a significant degree of monomer is present, both substrates and competitive inhibitors will shift the equilibrium toward the dimer, resulting in apparent increases in dimer stability and decreases in ligand binding affinity. Sedimentation equilibrium studies were also carried out on several drug-resistant HIV-1 protease mutants: V82F, V82F/I84V, V82T/I84V, and L90M. All four mutants exhibited reduced dimer stability relative to the autolysis-resistant mutant at pH 7.0. Our results indicate that reductions in drug affinity may be due to the combined effects of mutations on both dimer stability and inhibitor binding.

A novel putative transcription factor protein MYT2 that preferentially binds supercoiled DNA and induces DNA synthesis in quiescent cells.

Myelin transcription factor 2 (MYT2), a putative transcription factor found in the human central nervous system, was cloned from an expression cDNA library from human T-cells. MYT2 shares weak similarity to bacterial type I topoisomerases and shares 63% sequence identity to a replicase from Leuconostoc mesenteroides. MYT2 preferentially binds supercoiled DNA (scDNA). Incubation of MYT2 and scDNA at or above equal molar ratios generated topoisomer-like patterns that were abolished by deproteination. Thus, MYT2 appears to relax scDNA via a non-enzymatic mechanism. The banding pattern of MYT2-scDNA complexes was shown to be quantisized, saturable and sequence-independent. Microinjection of MYT2 mRNA induced G(o) growth-arrested NIH 3T3 cells to enter the S phase of the cell cycle.

Design of sensitive fluorogenic substrates for human cathepsin D.

Cathepsin D is a lysosomal aspartic proteinase that has been implicated in several pathological processes such as breast cancer and Alzheimer's disease. We designed and synthesized a number of quenched fluorogenic substrates with P2 variations in the series AcEE(EDANS)KPIXFFRLGK(DABCYL)E-NH2, where X=cysteine, methylcysteine, ethylcysteine, tert-butylcysteine, carboxymethylcysteine, methionine, valine or isoleucine. Most of the fluorogenic substrates exhibited greater k(cat)/Km ratios than the best cathepsin D substrates described so far. Differences in kinetic constants, which were rationalized using structure-based modeling, might make certain substrates useful for particular applications, such as active site titrations or initial velocity determination using a fluorescent plate reader.

Cyclopropane-derived peptidomimetics. Design, synthesis, evaluation, and structure of novel HIV-1 protease inhibitors.

Toward establishing the general efficacy of using trisubstituted cyclopropanes as peptide mimics to stabilize extended peptide structures, the cyclopropanes 20a-d were incorporated as replacements into 9-13, which are analogues of the known HIV-1 protease inhibitors 14 and 15. The syntheses of 20a-d commenced with the Rh2[5(S)-MEPY]4-catalyzed cyclization of the allylic diazoesters 16a-d to give the cyclopropyl lactones 17a-d in high enantiomeric excess. Opening of the lactone moiety using the Weinreb protocol and straightforward refunctionalization of the intermediate amides 18a-d gave 20a-d. A similar sequence of reactions was used to prepare the N-methyl-2-pyridyl analogue 28. Coupling of 20a-d and 28 with the known diamino diol 22 delivered 9-13. Pseudopeptides 9-12 were found to be competitive inhibitors of wild-type HIV-1 protease in biological assays having Kis of 0.31-0.35 nM for 9, 0.16-0.21 nM for 10, 0.47 nM for 11, and 0.17 nM for 12; these inhibitors were thus approximately equipotent to the known inhibitor 14(IC50 = 0.22 nM) from which they were derived. On the other hand 13 (Ki = 80 nM) was a weaker inhibitor than its analogue 15 (Ki = 0.11 nM). The solution structures of 9 and 10 were analyzed by NMR spectroscopy and simulated annealing procedures that included restraints derived from homo- and heteronuclear coupling constants and NOEs; because of the molecular symmetry of9 and 10, a special protocol to treat the NOE data was used. The final structure was checked by restrained and free molecular dynamic calculations using an explicit DMSO solvent box. The preferred solution conformations of 9 and 10 are extended structures that closely resemble the three-dimensional structure of 10 bound to HIV-1 protease as determined by X-ray crystallographic analysis of the complex. This work convincingly demonstrates that extended structures of peptides may be stabilized by the presence of substituted cyclopropanes that serve as peptide replacements. Moreover, the linear structure enforced in solution by the two cyclopropane rings in the pseudopeptides 9-12 appears to correspond closely to the biologically active conformation of the more flexible inhibitors 14 and 15. The present work, which is a combination of medicinal, structural, and quantum chemistry, thus clearly establishes that cyclopropanes may be used as structural constraints to reduce the flexibility of linear pseudopeptides and to help enforce the biologically active conformation of such ligands in solution.

4-hydroxy-5,6-dihydropyrones. 2. Potent non-peptide inhibitors of HIV protease.

The 4-hydroxy-5,6-dihydropyrone template was utilized as a flexible scaffolding from which to build potent active site inhibitors of HIV protease. Dihydropyrone 1c (5,6-dihydro-4-hydroxy-6-phenyl-3-[(2-phenylethyl)thio]-2H-pyran-2-one) was modeled in the active site of HIV protease utilizing a similar binding mode found for the previously reported 4-hydroxybenzopyran-2-ones. Our model led us to pursue the synthesis of 6,6-disubstituted dihydropyrones with the aim of filling S1 and S2 and thereby increasing the potency of the parent dihydropyrone 1c which did not fill S2. Toward this end we attached various hydrophobic and hydrophilic side chains at the 6-position of the dihydropyrone to mimic the natural and unnatural amino acids known to be effective substrates at P2 and P2'. Parent dihydropyrone 1c (IC50 = 2100 nM) was elaborated into compounds with greater than a 100-fold increase in potency [18c, IC50 = 5 nM, 5-(3,6-dihydro-4-hydroxy-6-oxo-2-phenyl-5-[2-phenylethyl)thio] -2H-pyran-2-yl)pentanoic acid and 12c, IC50 = 51 nM, 5,6-dihydro-4-hydroxy-6-phenyl-6-(2-phenylethyl)-3- [(2-phenyl-ethyl)thio]-2H-pyran-2-one]. Optimization of the 3-position fragment to fill S1' and S2' afforded potent HIV protease inhibitor 49 [IC50 = 10 nM, 3-[(2-tert-butyl-5-methylphenyl)sulfanyl]-5,6-dihydro-4 -hydroxy-6-phenyl-6-(2-phenylethyl)-2H-pyran-2-one]. The resulting low molecular weight compounds (< 475) have one or no chiral centers and are readily synthesized.

5,6-Dihydropyran-2-ones possessing various sulfonyl functionalities: potent nonpeptidic inhibitors of HIV protease.

On the basis of previous SAR findings and molecular modeling studies, a series of compounds were synthesized which possessed various sulfonyl moieties substituted at the 4-position of the C-3 phenyl ring substituent of the dihydropyran-2-one ring system. The sulfonyl substituents were added in an attempt to fill the additional S(3)' pocket and thereby produce increasingly potent inhibitors of the target enzyme. Racemic and enantiomerically resolved varieties of selected compounds were synthesized. All analogues in the study displayed decent binding affinity to HIV protease, and several compounds were shown to possess very good antiviral efficacy and safety margins. X-ray crystallographic structures confirmed that the sulfonamide and sulfonate moieties were filling the S(3)' pocket of the enzyme. However, the additional substituent did not provide improved enzymatic inhibitory or antiviral activity as compared to the resolved unsubstituted aniline. The addition of the sulfonyl moiety substitution does not appear to provide favorable pharamacokinectic parameters. Selected inhibitors were tested for antiviral activity in clinical isolates and exhibited similar antiviral activity against all of the HIV-1 strains tested as they did against the wild-type HIV-1. In addition, the inhibitors exhibited good antiviral efficacies against HIV-1 strains that displayed resistance to the currently marketed protease inhibitors.

Design, synthesis, and resistance patterns of MP-134 and MP-167, two novel inhibitors of HIV type 1 protease.

Inhibitors of HIV-1 protease represent a new class of antiretroviral compounds. Here, we report the design and synthesis of two novel C2 symmetry-based inhibitors, MP-134 and MP-167, specifically targeted against HIV-1 variants with reduced sensitivity to another related protease inhibitor, A-77003. In addition, we describe the in vitro selection of viral variants with reduced sensitivity of these two protease inhibitors. An isoleucine-to-valine substitution at residue 84 (I84V) of the HIV-1 protease confers resistance to MP-134, whereas a glycine-to-valine substitution at residue 48 (G48V) confers resistance to MP-167. Testing other protease inhibitors against these variants has revealed specific overlapping patterns of resistance among these agents. These findings have important implications in the design of combination regimens using multiple protease inhibitors and underscore the need to develop non-cross-resistant compounds to be used toward this goal.