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We have designed and synthesized a series of HIV protease inhibitors (PIs) with enamino-oxindole substituents optimized to interact with the S2' subsite of the HIV protease binding pocket. Several of these inhibitors have sub-nanomolar K(i) and antiviral IC(50) in the low nM range against WT HIV and against a panel of multi-drug resistant (MDR) strains.

We have developed a novel procedure to monitor the real-time cleavage of natural unmodified peptides (dark substrates). In the competition-based assay, the initial cleavage rate of a fluorogenic peptide substrate is measured in the presence of a second substrate that is not required to exhibit any optical property change upon cleavage. Using a unique experimental design and steady-state enzyme kinetics for a two-substrate system, we were able to determine both Km and k(cat) values for cleavage of the dark substrate. The method was applied to HIV-1 protease and to the V82F/I84V drug resistant mutant enzyme. Using two different substrates, we showed that the kinetic parameters derived from the competition assay are in good agreement with those determined independently using standard direct assay. This method can be applied to other enzyme systems as long as they have one substrate for which catalysis can be conveniently monitored in real time.

This review describes current approaches to HIV protease inhibitor design, with a focus on improving their profile against drug-resistant mutants. Potential explanations for the flat resistance profile of some potent protease inhibitors and discrepancies between the apparent fold change of potency at the enzyme level and in cell-based assays are discussed.Despite new ideas and a clear rationale for designing inhibitors that bind outside the enzyme active site, all current protease inhibitors with potent antiviral activity target this site. Several bis-tetrahydrofuran-containing compounds including darunavir, brecanavir, GS-8374, and Sequoia protease inhibitors exhibit excellent potency against mutant HIV strains that are resistant to clinically used protease inhibitors. The apparently flat resistance profiles of these and some other protease inhibitors may, at least in part, be explained by their high potency against wild-type enzyme. The substrate envelope and solvent-anchoring hypotheses have been used to design and/or rationalize improved resistance profiles. Traditional approaches yielded a lysine sulfonamide PL-100 with a unique resistance profile.Several theories on how to design HIV protease inhibitors with improved resistance profiles have been proposed during the review period. The general concepts that are incorporated into most design strategies include maximizing the interactions with the backbone and conserved side chains of the enzyme while minimizing inhibitor size and maintaining conformational flexibility to allow for modified binding modes.

Plasmepsin II is one of the four catalytically active plasmepsins found in the food vacuole of Plasmodium falciparum. These enzymes initiate hemoglobin degradation by cleavage at the alpha-chain between Phe33 and Leu34. The crystal structures of Ser205 mutant plasmepsin II from P. falciparum in complex with two inhibitors have been refined at a resolution of 1.8 A in the space group I222 and to R factors of 19.9 and 19.5%. Each crystal contains one monomer in the asymmetric unit. Both inhibitors have a Phe-Leu core and incorporate tetrahedral transition-state mimetic hydroxypropylamine. The inhibitor rs367 possesses a 2,6-dimethylphenyloxyacetyl group at the P2 position and 3-aminobenzamide at the P2' position, while rs370 has the same P2 group but 4-aminobenzamide in the P2' position. These complexes reveal key conserved hydrogen bonds between the inhibitor and the binding-cavity residues, notably with the flap residues Val78 and Ser79, the catalytic dyad Asp34 and Asp214 and the residues Ser218 and Gly36 that are in proximity to the catalytic dyad. The structures also show unexpected conformational variability of the binding cavity of plasmepsin II and may reflect the mode of binding of the hemoglobin alpha-chain for cleavage.

Plasmepsin-2 is a malarial aspartic proteinase that has been implicated in the initial steps of hemoglobin degradation in parasites and thus represents an attractive antimalarial target. Escherichia coli expressed proplasmepsin-2 is capable of activation at acidic pH by autocatalytic cleavage of the pro part region, which results in products of different length. We designed a 10-amino-acid deletion in the pro part region that allows faster generation of homogeneous enzyme upon activation. Incorporation of a (His)6 tag onto the N-terminus of the pro part enables on-column refolding of proplasmepsin-2 and simplifies proenzyme purification and pro part separation after activation. The proposed purification procedure results in highly pure and easily crystallizable enzyme.

Abstract: Structural Biochemistry Program, SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, P.O Box B, Frederick, MD 21702-1201. Present Address: Tibotec Inc., 1330 Piccard Dr., Rockville, MD, 20850. 1Present Address: Gilford Pharmaceuticals Inc., 6611 Tributary Street, Baltimore, MD, 21224. Plasmepsin 2 is a malarial aspartic proteinase that has been implicated in initial steps of hemoglobin degradation in parasite and thus represents an attractive antimalarial target. We designed and synthesized a number of quenched fluorogenic substrates and measured the kinetic constants of their cleavage by plasmepsin-2. High sensitivity and superior kinetic properties make the substrates suitable for different application including fluorescence plate reader.

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.

A series of novel unsymmetrical anthranilamide-containing HIV protease inhibitors was designed. The structure-activity studies revealed a series of potent P2–P3′ inhibitors that incorporate an anthranilamide group at the P2′ position. A reduction in molecular weight and lipophilicity is achieved by a judicious choice of P2 ligands (i.e., aromatic, heteroaromatic, carbamate, and peptidic). A systematic investigation led to the 5-thiazolyl carbamate analog 8m, which exhibited a favorable C max EC 50 ratio (>30), plasma half-life (>8 h), and potent in vitro antiviral activity (EC50 = 0.2 uM).

The catalytic activity and inhibitor binding energetics of enzymes are often pH-dependent properties. Aspartic proteases comprise an important class of enzyme targets for structure-based drug design. We have performed a complete thermodynamic study of pepstatin binding to plasmepsin II, an aspartic proteinase found in Plasmodium falciparum, using isothermal titration calorimetry and circular dichroism. Thermodynamic parameters (DeltaG, DeltaH, DeltaCp, and DeltaS) were measured as functions of both pH and temperature. In the pH range from 4.5 to 7.0, pepstatin binding is accompanied by proton transfer between the solvent and the complex. We used thermodynamic proton linkage theory to derive both the pH-independent binding energetics for pepstatin and the number and pKa values of ionizable residues whose pKa values change during ligand binding. These residues were identified as the two catalytic aspartates, with pKas of 6.5 and 3.0, and His 164, with a pKa of 7.5, based on the three-dimensional structure of the pepstatin-plasmepsin II complex. At pH 5.0, where the protease has optimum activity, the proton transfer process contributes almost 40% of the total binding free energy change and the total charge of the active-site aspartic acid residues is -1. These experimental results provide direct measurement for the protonation states of the catalytic aspartates in the presence of bound ligands. Comparison of the thermodynamic and structural data for pepstatin binding with human cathepsin D, a lysosomal aspartic protease that shares 35% sequence identity with plasmepsin II, suggests that the energetic differences between these two proteins are due to a higher interdomain flexibility in plasmepsin II.

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.

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.

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 to 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.

Guided by the structure of HIV PR complexed with 2S,3R,4S,5S-2,5-bis[N,N'-((3-hydroxy-2-methylphenyl)carbonyl)amino]-3,4-dihydroxy-1,6-diphenyl hexane (1), a novel, achiral, non-peptidic anthranil (Ant) group was designed as a P2/P2′ ligand. Symmetry-based inhibitors containing N-(2-pyridinylmethoxy-carbonyl)anthranil group are potent anti viral agents. Compounds 12 and 14 exhibited protease inhibitory activity of 60 and 70 pM, anti viral activity of 13 and 56 nM and cellular toxicity of > 10 uM respectively. We describe a HIV PR/1 structure-guided design of the novel, nonpeptidic, achiral anthranilamide group as a P2/P2′ ligand for the symmetry-based HIV PR inhibitors. X-ray crystallographic analysis of HIV PR/12 complex reveal that the anthranilamide group participates in both the polar and hydrophobic interactions that are commonly observed between the enzyme and peptidic inhibitor in the vicinity of the S2/S2′ subsites. The antiviral activity of compound 12 compares favorably with other potent HIV PR inhibitors currently undergoing clinical trials.

Readily accessible, non-peptidic, achiral 2-methylbenzamides were designed to serve as P2/P2′ ligands for symmetry-based inhibitors of HIV-1 Protease. Introduction of 3-hydroxy substituent provided a potent inhibitor 7 ( K i = 0.8 nM).

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 Asp 125 residue. Conclusion 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.

A series of novel unsymmetrical anthranilamide-containing HIV protease inhibitors was designed. The structure-activity studies revealed a series of potent P2–P3′ inhibitors that incorporate an anthranilamide group at the P2′ position. A reduction in molecular weight and lipophilicity is achieved by a judicious choice of P2 ligands (i.e., aromatic, heteroaromatic, carbamate, and peptidic). A systematic investigation led to the 5-thiazolyl carbamate analog 8m, which exhibited a favorable C max EC 50 ratio (>30), plasma half-life (>8 h), and potent in vitro antiviral activity (EC50 = 0.2 uM).

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.

Malaria remains a human disease of global significance and a major cause of high infant mortality in endemic nations. Parasites of the genus Plasmodium cause the disease by degrading human hemoglobin as a source of amino acids for their growth and maturation. Hemoglobin degradation is initiated by aspartic proteases, termed plasmepsins, with a cleavage at the alpha-chain between residues Phe33 and Leu34. Plasmepsin II is one of the four catalytically active plasmepsins that has been identified in the food vacuole of Plasmodium falciparum. Novel crystal structures of uncomplexed plasmepsin II as well as the complex with a potent inhibitor have been refined with data extending to resolution limits of 1.9A and 2.7A, and to R factors of 17% and 18%, respectively. The inhibitor, N-(3-[(2-benzo[1,3]dioxol-5-yl-ethyl)[3-(1-methyl-3-oxo-1,3-dihydro-isoindol-2-yl)-propionyl]-amino]-1-benzyl-2-(hydroxypropyl)-4-benzyloxy-3,5-dimethoxy-benzamide, belongs to a family of potent non-peptidic inhibitors that have large P1' groups. Such inhibitors could not be modeled into the binding cavity of the structure of plasmepsin II in complex with pepstatin A. Our structures reveal that the binding cavities of the new complex and uncomplexed plasmepsin II are considerably more open than that of the pepstatin A complex, allowing for larger heterocyclic groups in the P1', P2' and P2 positions. Both complexed and uncomplexed plasmepsin II crystallized in space group P2, with one monomer in the asymmetric unit. The structures show extensive interlocking of monomers around the crystallographic axis of symmetry, with areas in excess of 2300A(2) buried at the interface, and a loop of one monomer interacting with the binding cavity of the 2-fold related monomer. Electron density for this loop is only fully ordered in the complexed structure.

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 Go growth-arrested NIH 3T3 cells to enter the S phase of the cell cycle.

HIV protease is responsible for processing of the gag and gag-pol polyproteins during virion maturation. The activity of this enzyme is essential for virus infectivity, rendering the protein a major therapeutic target for AIDS treatment. This articles reviews the biochemical and biophysical properties of the enzyme. The clinical and in vitro observations of resistance to protease inhibitors are discussed from the perspective of drug resistance mechanisms of HIV protease mutants.

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.

To investigate the biochemical properties of the protease encoded by the human endogenous retrovirus, K10 (HERV-K), 213 amino acids of the 3'-end of the HERV-K protease (PR) open reading frame were expressed in Escherichia coli. Autocatalytic cleavage of the expressed polypeptide resulted in an 18.2 kDa protein which was shown to be proteolytically active against a fluorogenic peptide used as a substrate for HIV-1 protease. On the basis of sequence homology and molecular modeling, the 106 N-terminal amino acids of HERV-K PR were predicted to comprise a retroviral protease core domain. An 11.6 kDa protein corresponding to this region was expressed and shown to be a fully functional enzyme. The 11.6 kDa domain of HERV-K PR is unusually stable over a wide pH range, exhibits optimal catalytic activity between pH 4.0 and 5.0, and exists as a dimer at pH 7.0 with a Kd of 50 microM. Like HIV-1 PR, the HERV-K PR core domain is activated by high salt concentrations and processes HIV-1 matrix-capsid polyprotein at the authentic HIV-1 PR recognition site. However, both the 18.2 and 11.6 kDa forms of HERV-K PR were highly resistant to a number of clinically useful HIV-1 PR inhibitors, including ritonavir, indinavir, and saquinavir. This raises the possibility that HERV-K PR may complement HIV-1 PR during infection, and could have implications for protease inhibitor therapy and drug resistance.

The crystal structure of a catalytically inactive form of cathepsin D (CatDhi) has been obtained at pH 7.5. The N-terminal strand relocates by 30 A from its position in the interdomain beta-sheet and inserts into the active site cleft, effectively blocking substrate access. CatDhi has a five-stranded interdomain beta-sheet and resembles Intermediate 3, a hypothetical structure proposed to be transiently formed during proteolytic activation of the proenzyme precursor. Interconversion between active and inactive forms of CatD is reversible and may be regulated by an ionizable switch involving the carboxylate side chains of Glu 5, Glu 180, and Asp 187. Our findings provide a structural basis for the pH-dependent regulation of aspartic proteinase activity and suggest a novel mechanism for pH-dependent modulation of substrate specificity.

Aspartic proteases have been the subject of intensive physicochemical research in the areas of catalytic mechanism, three-dimensional structure, zymogen activation and substrate specificity.1,2 The important roles of various members of this family in human diseases, including hypertension,3 cancer,4 malaria5 and AIDS6 have made these enzymes the target of rational drug design efforts. Thermodynamic characterization of inhibitor binding is important for our understanding of the mechanism and energetics of enzyme-drug interactions. Recent developments in high sensitivity isothermal titration calorimetry (ITC) have made it possible to obtain extensive thermodynamic information for molecular interactions.9,10 The binding of pepstatin to three aspartic proteases, endothiapepsin from Endothia parasitica, plasmepsin II from Plasmodium falciparum and cathepsin D from human liver have been characterized using this technique.7,8

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.

A combination of structure-activity studies, kinetic analysis, X-ray crystallographic analysis, and modeling were employed in the design of a novel series of HIV-1 protease (HIV PR) inhibitors. The crystal structure of a complex of HIV PR with SRSS-2,5-bis[N-(tert-butyloxycarbonyl)amino]-3,4-dihydroxy-1, 6-diphenylhexane (1) delineated a crucial water-mediated hydrogen bond between the tert-butyloxy group of the inhibitor and the amide hydrogen of Asp29 of the enzyme. Achiral, nonpeptidic 2-hydroxyphenylacetamide and 3-hydroxybenzamide groups were modeled as novel P2/P2' ligands to replace the crystallographic water molecules and to provide direct interactions with the NH groups of the Asp29/129 residues. Indeed, the symmetry-based inhibitors 7 and 19, possessing 3-hydroxy and 3-aminobenzamide, respectively, as a P2/P2' ligand, were potent inhibitors of HIV PR. The benzamides were superior in potency to the phenylacetamides and have four fewer rotatable bonds. An X-ray crystal structure of the HIV PR/7 complex at 2.1 A resolution revealed an asymmetric mode of binding, in which the 3-hydroxy group of the benzamide ring makes the predicted interaction with the backbone NH of Asp29 on one side of the active site only. An unexpected hydrogen bond with the Gly148 carbonyl group, resulting from rotation of the aromatic ring out of the amide plane, was observed on the other side. The inhibitory potencies of the benzamide compounds were found to be sensitive to the nature and position of substituents on the benzamide ring, and can be rationalized on the basis of the structure of the HIV PR/7 complex. These results partly confirm our initial hypothesis and suggest that optimal inhibitor designs should satisfy a requirement for providing polar interactions with Asp29 NH, and should minimize the conformational entropy loss on binding by reducing the number of freely rotatable bonds in inhibitors.

Eleven different recombinant, drug-resistant HIV-1 protease (HIV PR) mutants--R8Q, V32I, M46I, V82A, V82F, V82I, I84V, V32I/I84V, M46I/V82F, M46I/I84V, and V32I/K45I/F53L/A71V/I84V/L89M--were generated on the basis of results of in vitro selection experiments using the inhibitors A-77003, A-84538, and KNI-272. Kinetic parameters of mutant and wild-type (WT) enzymes were measured along with inhibition constants (Ki) toward the inhibitors A-77003, A-84538, KNI-272, L-735,524, and Ro31-8959. The catalytic efficiency, kcat/Km, for the mutants decreased relative to WT by a factor of 1.2-14.8 and was mainly due to the elevation of Km. The effects of specific mutations on Ki values were unique with respect to both inhibitor and mutant enzyme. A new property, termed vitality, defined as the ratio (Kikcat/Km)mutant/(Kikcat/Km)WT was introduced to compare the selective advantage of different mutants in the presence of a given inhibitor. High vitality values were generally observed with mutations that emerged during in vitro selection studies. The kinetic model along with the panel of mutants described here should be useful for evaluating and predicting patterns of resistance for HIV PR inhibitors and may aid in the selection of inhibitor combinations to combat drug resistance.

Cathepsin D (EC 3.4.23.5) (CatD) is an intracellular aspartic protease (AP) that is normally found in the lysosomes of higher eukaryotes. CatD shares two structural features with lysosomal enzymes that distinguish it from most secreted, extracellular APs. First, the mature enzyme from humans is found as a two-chain enzyme as a result of post-translational cleavage and removal of an insertion loop in the N-domain. Second, CatD contains phosphorylated, N-linked oligosaccharides that target the enzyme to lysosomes via man-nose-6-phosphate receptors (MPR). Interest in CatD as a target for drug design results from its association with several biological processes of therapeutic significance, particularly breast cancer and Alzheimer’s disease (Table 1). Recent studies have implicated CatD in the processing of (β-amyloid precursor protein to promote amyloid plaque formation in Alzheimer’s brain. Numerous studies of primary breast cancers have demonstrated that elevated levels of CatD were correlated with an increased risk of metastasis and shorter relapse-free survival. High levels of CatD produced in the vicinity of the growing tumor may degrade the extracellular matrix, either directly or indirectly by potentiating the activity of other tissue proteases, such as collagenase, and thereby promote tumor growth and metastasis. While a role for CatD or other APs in metastasis has been questioned by recent results that show a negative effect of pepstatin A in an in vitro invasion assay using breast cancer-derived MCF7 cells, new studies have revealed that elevated levels of CatD secretion in breast cancers correlate with tumor aggressiveness.

The structures of three aspartic proteases, penicillopepsin, endothiapepsin and rhizopuspepsin, have been determined in complexes with difluorostatone (DFS)-containing peptide-based inhibitors [1–3]. In all three structures, the hydrated core of the inhibitors display similar conformations and interactions with the active site aspartate groups. One of the gem-hydroxyls is positioned between both aspartates, the other one interacts with a single aspartate, and the fluorines do not interact with either of them. Since one of the hydroxyl groups is located near the position of the active site water observed in the native structures, these inhibitors have been proposed as analogs of transition state intermediates of peptide bond hydrolysis.

The HIV-1 Rev protein is an essential nuclear regulatory viral protein. Rev mutants that are able to block wild-type (WT) Rev activityin transhave been reported and used in antiviral approaches. Not only nuclear but also cytoplasmic Rev mutants were described and suspected to be transdominant by retaining WT Rev in the cytoplasm. To investigate their potential for cytoplasmic retention, we studied the localization, trafficking, and interactions of cytoplasmic Rev mutants containing mutations in the N-terminal multifunctional domain. Using a novel dual-color autofluorescent protein-tagging system, we found that coexpression of the nucleolar blue-tagged WT Rev protein together with green-labeled cytoplasmic Rev mutants did not result in the retention of WT Rev in the cytoplasm but, on the contrary, in colocalization of the mutants to the nucleolus. A combination of mutations abolished the interaction with WT Rev, defining two domains important for Rev protein interaction. The identified domains were also essential for specific Rev responsive element (RRE) RNA binding and nuclear retention. Inactivation of the nuclear export signal shifted the steady-state distribution of the mutants from the cytoplasm to the nucleus, indicating their capability for nucleo-cytoplasmic shuttling. The cytoplasmic mutants were not transdominant compared to the nuclear mutant RevM10BL. These results emphasize that efficient oligomerization with WT Rev combined with RRE-specific RNA binding are prerequisites for effective transdominance.

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 kcat/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.