Wild-type Protease Research Articles

Small-molecule dimerization inhibitors of wild-type and mutant HIV protease: a focused library approach.

We demonstrate that a focused library based on truncated, cross-linked interfacial peptides of HIV-1 protease produces effective dimerization inhibitors of the enzyme. By combining individual changes of the library into a single compound, we obtained a significantly more potent agent and found that an additive increase in inhibitor efficacy was obtained. The good activity of library members against an active-site drug-resistant protease mutant bodes well for dimerization inhibition as a complementary method to targeting the active site.

Efficiency of a second-generation HIV-1 protease inhibitor studied by molecular dynamics and absolute binding free energy calculations.

A subnanomolar inhibitor of human immunodeficiency virus type 1 (HIV-1) protease, designated QF34, potently inhibits the wild-type and drug-resistant enzyme. To explain its broad activity, the binding of QF34 to the wild-type HIV-1 protease is investigated by molecular dynamics simulations and compared to the binding of two inhibitors that are used clinically, saquinavir (SQV) and indinavir (IDV). Analysis of the flexibility of protease residues and inhibitor segments in the complex reveals that segments of QF34 were more mobile during the dynamics studies than the segments of SQV and IDV. The dynamics of hydrogen bonding show that QF34 forms a larger number of stable hydrogen bonds than the two inhibitors that are used clinically. Absolute binding free energies were calculated with molecular mechanics-generalized Born surface area (MM-GBSA) methodology using three protocols. The most consistent results were obtained using the single-trajectory approach, due to cancellation of errors and inadequate sampling in the separate-trajectory protocols. For all three inhibitors, energy components in favor of binding include van der Waals and electrostatic terms, whereas polar solvation and entropy terms oppose binding. Decomposition of binding energies reveals that more protease residues contribute significantly to the binding of QF34 than to the binding of SQV and IDV. Moreover, contributions from protease main chains and side chains are balanced in the case of QF34 (52:48 ratio, respectively), whereas side chain contributions prevail in both SQV and IDV (main-chain:side-chain ratios of 41:59 and 45:55, respectively). The presented results help explain the ability of QF34 to inhibit multiple resistant mutants and should be considered in the design of broad-specificity second-generation HIV-1 protease inhibitors.

Novel Arylsulfonamides Possessing Sub‐Picomolar HIV Protease Activities and Potent anti‐HIV Activity against Wild‐Type and Drug‐Resistant Viral Strains.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Communication between the active sites and dimer interface of a herpesvirus protease revealed by a transition-state inhibitor.

Structurally diverse organophosphonate inhibitors targeting the active site of the enzyme were used to investigate the relationship of the active site and the dimer interface of wild-type protease in solution. Positional scanning synthetic combinatorial libraries revealed Kaposi's sarcoma-associated herpesvirus protease to be highly specific, even at sites distal to the peptide bond undergoing hydrolysis. Specificity results were used to synthesize a hexapeptide diphenylphosphonate inhibitor of Kaposi's sarcoma-associated herpesvirus protease. The transition state analog inhibitors covalently phosphonylate the active site serine, freezing the enzyme structure during catalysis. An NMR-based assay was developed to monitor the native monomer-dimer equilibrium in solution and was used to demonstrate the effect of protease inhibition on the quaternary structure of the enzyme. NMR, circular dichroism, and size exclusion chromatography analysis showed that active site inhibition strongly regulates the binding affinity of the monomer-dimer equilibrium at the spatially separate dimer interface of the protease, shifting the equilibrium to the dimeric form of the enzyme. Furthermore, inhibitor studies revealed that the catalytic cycles of the spatially separate active sites are independent. These results (i) provide direct evidence that peptide bond hydrolysis is integrally linked to the quaternary structure of the enzyme, (ii) establish a molecular mechanism of protease activation and stabilization during catalysis, and (iii) highlight potential implications of substoichiometric inhibition of the viral protease in developing herpesviral therapeutics.

Crystal structures of HIV protease V82A and L90M mutants reveal changes in the indinavir-binding site.

The crystal structures of the wild-type HIV-1 protease (PR) and the two resistant variants, PR(V82A) and PR(L90M), have been determined in complex with the antiviral drug, indinavir, to gain insight into the molecular basis of drug resistance. V82A and L90M correspond to an active site mutation and nonactive site mutation, respectively. The inhibition (K(i)) of PR(V82A) and PR(L90M) was 3.3- and 0.16-fold, respectively, relative to the value for PR. They showed only a modest decrease, of 10-15%, in their k(cat)/K(m) values relative to PR. The crystal structures were refined to resolutions of 1.25-1.4 A to reveal critical features associated with inhibitor resistance. PR(V82A) showed local changes in residues 81-82 at the site of the mutation, while PR(L90M) showed local changes near Met90 and an additional interaction with indinavir. These structural differences concur with the kinetic data.

High Resolution Crystal Structures of HIV-1 Protease with a Potent Non-peptide Inhibitor (UIC-94017) Active Against Multi-drug-resistant Clinical Strains

The compound UIC-94017 (TMC-114) is a second-generation HIV protease inhibitor with improved pharmacokinetics that is chemically related to the clinical inhibitor amprenavir. UIC-94017 is a broad-spectrum potent inhibitor active against HIV-1 clinical isolates with minimal cytotoxicity. We have determined the high-resolution crystal structures of UIC-94017 in complexes with wild-type HIV-1 protease (PR) and mutant proteases PR V82A and PR I84V that are common in drug-resistant HIV. The structures were refined at resolutions of 1.10–1.53 Å. The crystal structures of PR and PR I84V with UIC-94017 ternary complexes show that the inhibitor binds to the protease in two overlapping positions, while the PR V82A complex had one ordered inhibitor. In all three structures, UIC-94017 forms hydrogen bonds with the conserved main-chain atoms of Asp29 and Asp30 of the protease. These interactions are proposed to be critical for the potency of this compound against HIV isolates that are resistant to multiple protease inhibitors. Other small differences were observed in the interactions of the mutants with UIC-94017 as compared to PR. PR V82A showed differences in the position of the main-chain atoms of residue 82 compared to PR structure that better accommodated the inhibitor. Finally, the 1.10 Å resolution structure of PR V82A with UIC-94017 showed an unusual distribution of electron density for the catalytic aspartate residues, which is discussed in relation to the reaction mechanism.

A Phenylnorstatine Inhibitor Binding to HIV-1 Protease: Geometry, Protonation, and Subsite−Pocket Interactions Analyzed at Atomic Resolution

The X-ray structure of a complex of HIV-1 protease (PR) with a phenylnorstatine inhibitor Z-Pns-Phe-Glu-Glu-NH(2) has been determined at 1.03 A, the highest resolution so far reported for any HIV PR complex. The inhibitor shows subnanomolar K(i) values for both the wild-type PR and the variant representing one of the most common mutations linked to resistance development. The structure comprising the phenylnorstatine moiety of (2R,3S)-chirality displays a unique pattern of hydrogen bonding to the two catalytic aspartate residues. This high resolution makes it possible to assess the donor and acceptor relations of this hydrogen bonding and to indicate a proton shared by the two catalytic residues. A structural mechanism for the unimpaired inhibition of the protease Val82Ala mutant is also suggested, based on energy calculations and analyses.

Novel arylsulfonamides possessing sub-picomolar HIV protease activities and potent anti-HIV activity against wild-type and drug-resistant viral strains

A novel series of P1′ chain-extended arylsufonamides was synthesiszed and evaluated for wild-type HIV protease inhibitory activity and in vitro antiviral activity against wild type virus and two protease inhibitor-resistant mutant viruses. All of the compounds showed dramatic increases in enzyme activity as compared to the currently marketed HIV protease inhibitors amprenavir, indinavir, and nelfinavir. In addition, significant improvements in antiviral potencies against wild type and the two mutant viruses were also realized.

Assessment of FIV-C infection of cats as a function of treatment with the protease inhibitor, TL-3

BackgroundThe protease inhibitor, TL-3, demonstrated broad efficacy in vitro against FIV, HIV and SIV (simian immunodeficiency virus), and exhibited very strong protective effects on early neurologic alterations in the CNS of FIV-PPR infected cats. In this study, we analyzed TL-3 efficacy using a highly pathogenic FIV-C isolate, which causes a severe acute phase immunodeficiency syndrome, with high early mortality rates.ResultsTwenty cats were infected with uncloned FIV-C and half were treated with TL-3 while the other half were left untreated. Two uninfected cats were used as controls. The general health and the immunological and virological status of the animals was monitored for eight weeks following infection. All infected animals became viremic independent of TL-3 treatment and seven of 20 FIV-C infected animals developed severe immunodepletive disease in conjunction with significantly (p ≤ 0.05) higher viral RNA loads as compared to asymptomatic animals. A marked and progressive increase in CD8+ T lymphocytes in animals surviving acute phase infection was noted, which was not evident in symptomatic animals (p ≤ 0.05). Average viral loads were lower in TL-3 treated animals and of the 6 animals requiring euthanasia, four were from the untreated cohort. At eight weeks post infection, half of the TL-3 treated animals and only one of six untreated animals had viral loads below detection limits. Analysis of protease genes in TL-3 treated animals with higher than average viral loads revealed sequence variations relative to wild type protease. In particular, one mutant, D105G, imparted 5-fold resistance against TL-3 relative to wild type protease.ConclusionsThe findings indicate that the protease inhibitor, TL-3, when administered orally as a monotherapy, did not prevent viremia in cats infected with high dose FIV-C. However, the modest lowering of viral loads with TL-3 treatment, the greater survival rate in symptomatic animals of the treated cohort, and the lower average viral load in TL-3 treated animals at eight weeks post infection is indicative of a therapeutic effect of the compound on virus infection.

Structural and Thermodynamic Basis of Resistance to HIV-1 Protease Inhibition: Implications for Inhibitor Design

One of the most serious side effects associated with the therapy of HIV-1 infection is the appearance of viral strains that exhibit resistance to protease inhibitors. At the molecular level, resistance to protease inhibition predominantly takes the form of mutations within the protease molecule that preferentially lower the affinity of protease inhibitors with respect to protease substrates, while still maintaining a viable catalytic activity. Mutations associated with drug resistance occur within the active site cavity as well as distal sites. Active site mutations affect directly inhibitor/protease interactions while non-active site mutations affect inhibitor binding through long range cooperative perturbations. The effects of mutations associated with drug resistance are compounded by the presence of naturally occurring polymorphisms, especially those observed in non-B subtypes of HIV-1. The binding thermodynamics of all clinical inhibitors against the wild type protease, drug resistant mutations and non-B subtype HIV-1 proteases has been determined by high sensitivity isothermal titration calorimetry. In conjunction with structural information, these data have provided a precise characterization of the binding mechanism of different inhibitors and their response to mutations. Inhibitors that exhibit extremely high affinity and low susceptibility to the effects of mutations share common features and binding determinants even if they belong to different chemical scaffolds. These binding determinants define a set of rules and constraints for the design of better HIV-1 protease inhibitors.

Aberrant, persistent inclusion into lipid rafts limits the tumorigenic function of membrane type-1 matrix metalloproteinase in malignant cells

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a key enzyme in cell locomotion and tissue remodeling. Trafficking to the plasma membrane and internalization into the transient storage compartment both regulate the cell surface presentation of MT1-MMP. Our data indicate that mutant MT1-MMP lacking the cytoplasmic tail is recruited to the caveolae-enriched lipid raft membrane microdomains in breast carcinoma MCF7 cells. In contrast, the wild-type protease is not permanently associated with lipid rafts. Trafficking to lipid rafts correlated with poor internalization and the persistent presentation of MT1-MMP at the cell surface. The tail mutant efficiently functioned in inducing the activation of the latent proMMP-2 zymogen, matrix remodeling, and contraction of three-dimensional collagen lattices. Recruitment of the tail mutant to lipid raft antagonized, however, the cleavage of the plasma membrane-associated E-cadherin. These events limited the contribution of the tail mutant to cell locomotion and malignant growth. It is conceivable that the tail peptide sequence plays a crucial role in the translocations of MT1-MMP across the cell and contributes to coordinated cellular functions. It is tempting to hypothesize that the mechanisms involved in trafficking of MT1-MMP to caveolin-enriched lipid rafts may be targeted in a clinically advantageous manner.

Kinetics of the dimerization of retroviral proteases: the "fireman's grip" and dimerization.

All retroviral proteases belong to the family of aspartic proteases. They are active as homodimers, each unit contributing one catalytic aspartate to the active site dyad. An important feature of all aspartic proteases is a conserved complex scaffold of hydrogen bonds supporting the active site, called the "fireman's grip," which involves the hydroxyl groups of two threonine (serine) residues in the active site Asp-Thr(Ser)-Gly triplets. It was shown previously that the fireman's grip is indispensable for the dimer stability of HIV protease. The retroviral proteases harboring Ser in their active site triplet are less active and, under natural conditions, are expressed in higher enzyme/substrate ratio than those having Asp-Thr-Gly triplet. To analyze whether this observation can be attributed to the different influence of Thr or Ser on dimerization, we prepared two pairs of the wild-type and mutant proteases from HIV and myeloblastosis-associated virus harboring either Ser or Thr in their Asp-Thr(Ser)-Gly triplet. The equilibrium dimerization constants differed by an order of magnitude within the relevant pairs. The proteases with Thr in their active site triplets were found to be approximately 10 times more thermodynamically stable. The dimer association contributes to this difference more than does the dissociation. We propose that the fireman's grip might be important in the initial phases of dimer formation to help properly orientate the two subunits of a retroviral protease. The methyl group of threonine might contribute significantly to fixing such an intermediate conformation.

Design, synthesis, and biological evaluation of monopyrrolinone-based HIV-1 protease inhibitors.

The design, synthesis, and biological evaluation of a series of HIV-1 protease inhibitors [(-)-6, (-)-7, (-)-23, (+)-24] based upon the 3,5,5-trisubstituted pyrrolin-4-one scaffold is described. Use of a monopyrrolinone scaffold leads to inhibitors with improved cellular transport properties relative to the earlier inhibitors based on bispyrrolinones and their peptide counterparts. The most potent inhibitor (-)-7 displayed 13% oral bioavailability in dogs. X-ray structure analysis of the monopyrrolinone compounds cocrystallized with the wild-type HIV-1 protease provided valuable information on the interactions between the inhibitors and the HIV-1 enzyme. In each case, the inhibitors assumed similar orientations for the P2'-P1 substituents, along with an unexpected hydrogen bond of the pyrrolinone NH with Asp225. Interactions with the S2 pocket, however, were not optimal, as illustrated by the inclusion of a water molecule in two of the three inhibitor-enzyme complexes. Efforts to increase affinity by displacing the water molecule with second and third generation inhibitors did not prove successful. Lack of success with this venture is a testament to the difficulty of accurately predicting the many variables that influence and build binding affinity. Comparison of the inhibitor positions in three complexes with that of Indinavir revealed displacements of the protease backbones in the enzyme flap region, accompanied by variations in hydrogen bonding to accommodate the monopyrrolinone ring. The binding orientation of the pyrrolinone-based inhibitors may explain their sustained efficacy against mutant strains of the HIV-1 protease enzyme as compared to Indinavir.

An ethylenamine inhibitor binds tightly to both wild type and mutant HIV-1 proteases. Structure and energy study.

An X-ray structure (resolution 2.2 A) of mutant HIV-1 protease (A71V, V82T, I84V) complexed with a newly developed peptidomimetic inhibitor with an ethylenamine isostere Boc-Phe-Psi[CH(2)CH(2)NH]-Phe-Glu-Phe-NH(2), denoted as OE, is described and compared with the complex of wild-type HIV-1 protease with the same inhibitor (resolution 2.5 A). OE shows tight binding to the wild type (K(i) = 1.5 nM) as well as mutant (K(i) = 4.1 nM) protease. The hydrogen bonds formed, in the case of hydroxyethylamine inhibitors, by a hydroxyl group are, in the case of OE inhibitors, replaced by a bifurcated hydrogen bond from the isosteric NH group to both catalytic aspartates Asp 25 and Asp 125. The binding modes of OE inhibitor to the wild type and mutant protease are similar. However, in the mutant protease, weaker van der Waals interactions of the mutated residues Val 84 and Val 184 with OE were found. This lack of interaction energy is compensated by a new aromatic hydrogen bond between the phenyl ring of the inhibitor in position P1 and the mutated residue Thr 182. Energy analysis based on molecular mechanics has been performed to distinguish between the static and dynamic backgrounds of disorder observed at the mutation sites Thr 82, Val 84, Thr 182, and Val 184.

Development and evaluation of a phenotypic assay monitoring resistance formation to protease inhibitors in HIV-1-infected patients

A novel phenotypic assay, based on recombinant expression of the HIV-1-protease was developed and evaluated; it monitors the formation of resistance to protease inhibitors. The HIV-1 protease-encoding region from the blood sample of patients was amplified, ligated into the expression vector pBD2, and recombinantly expressed in Escherichia coli TG1 cells. The resulting recombinant enzyme was purified by a newly developed one-step acid extraction protocol. The protease activity was determined in presence of five selected HIV protease inhibitors and the 50% inhibitory concentration (IC 50) to the respective protease inhibitors determined. The degree of resistance was expressed in terms of x-fold increase in IC 50 compared to the IC 50 value of an HIV-1 wild type protease preparation. The established test system showed a reproducible recombinant expression of each individual patients' HIV-1 protease population. Samples of nine clinically well characterised HIV-1-infected patients with varying degrees of resistance were analysed. There was a good correlation between clinical parameters and the results obtained by this phenotypic assay. For the majority of patients a blind genotypic analysis of the patients' protease domain revealed a fair correlation to the results of the phenotypic assay. In a minority of patients our phenotypic results diverged from the genotypic ones. This novel phenotypic assay can be carried out within 8–10 days, and offers a significant advantage in time to the current employed phenotypic tests.

Elucidation of HIV-1 protease resistance by characterization of interaction kinetics between inhibitors and enzyme variants

The kinetics of the interaction between drug-resistant variants of HIV-1 protease (G48V, V82A, L90M, I84V/L90M, and G48V/V82A/I84V/L90M) and clinically used inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) were determined using biosensor technology. The enzyme variants were immobilized on a biosensor chip and the association and dissociation rate constants ( k on and k off) and affinities ( K D) for interactions with inhibitors were determined. A unique interaction kinetic profile was observed for each variant/inhibitor combination. Substitution of single amino acids in the protease primarily resulted in reduced affinity through increased k off for the inhibitors. For inhibitors characterized by fast association rates to wild-type protease (ritonavir, amprenavir, and indinavir), additional substitutions resulted in a further reduction of affinity by a combination of decreased k on and increased k off. For inhibitors characterized by slow dissociation rates to wild-type enzyme (saquinavir and nelfinavir), the decrease of affinity conferred by additional mutations was attributed to increased k off values. Development of resistance thus appears to be associated with a change of the distinctive kinetic parameter contributing to high affinity. Further inhibitor design should focus on improving the “weak point” of the lead compound, that being either k on or k off.

A major role for a set of non-active site mutations in the development of HIV-1 protease drug resistance.

A major problem in the chemotherapy of HIV-1 infection is the appearance of drug resistance. In the case of HIV-1 protease inhibitors, resistance originates from mutations in the protease molecule that lower the affinity of inhibitors while still maintaining a viable enzymatic profile. Drug resistance mutations can be classified as active site or non-active site mutations depending on their location within the protease molecule. Active site mutations directly affect drug/target interactions, and their action can be readily understood in structural terms. Non-active site mutations influence binding from distal locations, and their mechanism of action is not immediately apparent. In this paper, we have characterized a mutant form of the HIV-1 protease, ANAM-11, identified in clinical isolates from HIV-1 infected patients treated with protease inhibitors. This mutant protease contains 11 mutations, 10 of which are located outside the active site (L10I/M36I/S37D/M46I/R57K/L63P/A71V/G73S/L90M/I93L) and 1 within the active site (I84V). ANAM-11 lowers the binding affinity of indinavir, nelfinavir, saquinavir, and ritonavir by factors of 4000, 3300, 5800, and 80000, respectively. Surprisingly, most of the loss in inhibitor affinity is due to the non-active site mutations as demonstrated by additional experiments performed with a protease containing only the 10 non-active site mutations (NAM-10) and another containing only the active site mutation (A-1). Kinetic analysis with two different substrates yielded comparable catalytic efficiencies for A-1, ANAM-11, NAM-10, and the wild-type protease. These studies demonstrate that non-active site mutations can be the primary source of resistance and that their role is not necessarily limited to compensate deleterious effects of active site mutations. Analysis of the structural stability of the proteases by differential scanning calorimetry reveals that ANAM-11 and NAM-10 are structurally more stable than the wild-type protease while A-1 is less stable. Together, the binding and structural thermodynamic results suggest that the non-active site mutants affect inhibitor binding by altering the geometry of the binding site cavity through the accumulation of mutations within the core of the protease molecule.

Lopinavir/ritonavir: a review of its use in the management of HIV infection.

Lopinavir/ritonavir: a review of its use in the management of HIV infection.

Rapid phenotypic assay for human immunodeficiency virus type 1 protease using in vitro translation

A rapid in vitro phenotyping method for human immunodeficiency virus type 1 (HIV-1) protease was developed. In this system, both HIV-1 protease and substrates are prepared using a rabbit reticulocyte based coupled in vitro transcription/translation system. The activity of protease is evaluated by the amount of cleaved substrate measured by ELISA. In this system, wild-type protease derived from strain HXB2 was specifically inhibited in a dose-dependent manner by the protease inhibitors, indinavir and nelfinavir. Three drug-resistant proteases carrying a single mutation, D30N, L90M, and V82F, were analyzed in the absence of the inhibitors. Reflecting their impaired fitness, they exhibited decreased protease activity compared with the wild type. The apparent protease activity was greater for a Gag-Pol substrate encompassing the Gag-protease-reverse transcriptase junctions than for a substrate only covering the Gag region. Using the Gag-Pol substrate as the target, the indinavir-resistant mutant V82F was evaluated further. V82F showed 9-fold resistance to its cognitive protease inhibitor, indinavir; however, it manifested only moderate (2-fold) resistance to a non-cognitive inhibitor, nelfinavir. This simple and rapid method may be useful for phenotyping of drug-resistant HIV-1 protease as well as for screening new inhibitors of HIV-1 protease.

Indinavir analogues with blocked metabolism sites as HIV protease inhibitors with improved pharmacological profiles and high potency against PI-Resistant viral strains

Indinavir analogues with blocked metabolism sites show highly improved pharmacokinetic profiles in animals. The cis-aminochromanol substituted analogues exhibited excellent potency against both the wild-type (NL4-3) virus and protease inhibitor-resistant HIV strains.