Structure-activity relationships of novel HIV-1 protease inhibitors containing the 3-amino-2-chlorobenzoyl-allophenylnorstatine structure

A series of peptidomimetic human immunodeficiency virus (HIV) protease inhibitors containing substituted allophenylnorstatine (Apns: (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid) were designed and synthesized. From the structure-activity relationship of this series of compounds, SM-309515 was found to have potent antiviral activity against wild-type and resistant HIV-1s and to possess a desirable pharmacokinetic profile in dogs.     

HIV-1 protease inhibitors containing an N-hydroxyamino acid core structure

Two series of peptidomimetics containing an N-hydroxyamino acid core structure were prepared by mixed solution solid-phase synthesis and tested for inhibitory activity against the human immunodeficiency virus (HIV-1) protease (Pr) and the virus in cell culture. In general, N-hydroxy Gly containing pseudopeptides displayed modest HIV Pr inhibition (IC50 > or = 930 nM). In the N-hydroxy Phe derivatives, Fmoc-Phe-psi[CO-N(OH)]-Phe-Pro-NHtBu was the best inhibitor of the series (IC50 = 144nM) showing satisfactory inhibition of HIV replication in cell culture (ED50 = 98 nM) and remarkable stability against cell culture and plasma enzymes.     

Evaluation of triazolamers as active site inhibitors of HIV-1 protease

Proteases typically recognize their peptide substrates in extended conformations. General approaches for designing protease inhibitors often consist of peptidomimetics that feature this conformation. Herein we discuss a combination of computational and experimental studies to evaluate the potential of triazole-linked β-strand mimetics as inhibitors of HIV-1 protease activity.     

HIV-1 protease inhibitors containing statine: inhibitory potency and antiviral activity.

Several series of chemically different inhibitors of the HIV-1 aspartyl protease have been described. Nevertheless despite the high in vitro potency showed, in most cases these inhibitors are unable to inhibit viral replication in infected cells. Penetration of the inhibitors across the cell membrane might account for their low antiviral activity. The relationship between inhibitory potency, antiviral activity and chemical structures of a series of oligopeptides containing statine or statine derivatives are presented here.     

Non-active site changes elicit broad-based cross-resistance of the HIV-1 protease to inhibitors.

Three high level, cross-resistant variants of the HIV-1 protease have been analyzed for their ability to bind four protease inhibitors approved by the Food and Drug Administration (saquinavir, ritonavir, indinavir, and nelfinavir) as AIDS therapeutics. The loss in binding energy (DeltaDeltaG(b)) going from the wild-type enzyme to mutant enzymes ranges from 2.5 to 4.4 kcal/mol, 40-65% of which is attributed to amino acid substitutions away from the active site of the protease and not in direct contact with the inhibitor. The data suggest that non-active site changes are collectively a major contributor toward engendering resistance against the protease inhibitor and cannot be ignored when considering cross-resistance issues of drugs against the HIV-1 protease.     

Structure-activity studies of FIV and HIV protease inhibitors containing allophenylnorstatine

The interaction of P1 and P3 side chains with the combining S1 and S3 hydrophobic subsites of HIV and FIV proteases has been explored using asymmetric competitive inhibitors. The inhibitors evaluated contained (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid (allophenylnorstatine) as the hydroxymethylcarbonyl isostere, (R)-5,5-dimethyl-1, 3-thiazolidine-4-carbonyl as P1', Val as P2 and P2' residues, and a variety of amino acids at the P3 and P3' positions. All inhibitors showed competitive inhibition of both enzymes with higher potency against the HIV protease in vitro. Within this series, 31 (VLE776) is the most effective inhibitor against FIV protease, and it contains Phe at P3, but no P3' residue. VLE776 also exhibited potent antiviral activities against the drug-resistant HIV mutants (G48V and V82F) and the TL3-resistant HIV mutants. Explanation of the inhibition activities was described. In addition, a new strategy was described for development of bifunctional inhibitors, which combine the protease inhibitor and another enzyme inhibitor in one molecule.     

Novel 3-amino-2-hydroxy acids containing protease inhibitors. Part 1: Synthesis and kinetic characterization as aminopeptidase P inhibitors

Novel, potent inhibitors of aminopeptidase P, containing a 3-amino-2-hydroxy acid and a proline or a proline analogues, have been prepared. One part of the bestatin-derived inhibitors was found to inhibit APP from Escherichia coli and from rat intestine according to a mixed-type mechanism, with Ki values up to 1.26 microM. The other compounds, 3-amino-2-hydroxy acyl prolines of a different configuration, inhibit APP competitively, according to a slow-binding mechanism, with Ki values in the nanomolar up to the micromolar range.     

Dimerization inhibitors of HIV-1 protease

By targeting the highly conserved antiparallel beta-sheet formed by the interdigitation of the N- and C-terminal strands of each monomer, dimerization inhibitors of HIV-1 protease may be useful to overcome the drug resistance observed with current active-site directed antiproteases. Sequestration of the monomer by the inhibitor (or disruption of the dimer interface) prevents the correct assembly of the inactive monomers to active enzyme. Strategies for the design of drugs targeting the dimer interface are described. Various dimerization inhibitors are reported including N- and C-terminal mimetics, lipopeptides and cross-linked interface peptides.     

P1' oxadiazole protease inhibitors with excellent activity against native and protease inhibitor-resistant HIV-1

HIV-1 protease inhibitors (PI's) bearing 1,3,4-oxadiazoles at the P1' position were prepared by a novel method involving the diastereoselective installation of a carboxylic acid and conversion to the P1' heterocycle. The compounds are picomolar inhibitors of native HIV-1 protease, with most of the compounds maintaining excellent antiviral activity against a panel of PI-resistant strains.     

A quantitative structure-activity relationship study on some HIV-1 protease inhibitors using molecular connectivity index

A quantitative structure-activity relationship (QSAR) study has been made on two different series of tetrahydropyrimidinones acting as HIV-1 protease inhibitors. A structural parameter, the first order valence molecular connectivity index ((1)chi(v)), has been used to account for the variation in the activity. The protease inhibition activity as well as the antiviral potency of the compounds are found to be significantly correlated with (1)chi(v) of P(2)/P(2') substituents attached to the two nitrogens N1 and N3, suggesting that substituents containing less electronegative and more saturated atoms, meaning thereby the less polar or more hydrophobic substituents, will be more advantageous. Further, if P(2) and P(2') are dissimilar, the former is found to be more effective than the latter. This difference is attributed to a conformational change in the enzyme that may be more favorable to P(2) binding than to P(2') binding.     

Lipopeptides as dimerization inhibitors of HIV-1 protease.

In AIDS therapy, attempts have been made to inhibit the virus-encoded enzymes, e.g. HIV-1 protease, using active site-directed inhibitors. This approach is questionable, however, due to virus mutations and the high toxicity of the drugs. An alternative method to inhibit the dimeric HIV protease is the targeting of the interface region of the protease subunits in order to prevent subunit dimerization and enzyme activity. This approach should be less prone to inactivation by mutation. A list of improved 'dimerization inhibitors' of HIV-1 protease is presented. The main structural features are a short 'interface' peptide segment, including non-natural amino acids, and an aliphatic N-terminal blocking group. The high inhibitory power of some of the lipopeptides [e.g. palmitoyl-Tyr-Glu-Leu-OH, palmitoyl-Tyr-Glu-(L-thyronine)-OH, palmitoyl-Tyr-Glu-(L-biphenyl-alanine)-OH] with low nanomolar Ki values in the enzyme test suggests that mimetics with good bio-availability can be derived for AIDS therapy.     

Conformational studies of irreversible HIV-1 protease inhibitors containing cis-epoxide as an amide isostere.

We have carried out NMR and molecular modeling studies of peptidomimetic HIV-1 protease inhibitors, LB71116: Qc-Asn-Phepsi[(1R,2S)-cis-epoxide]Gly-NH-CH(isopropyl)2 where Qc stands for quinaldic acid and LB71148: Qc-(SMe)Pen(O)2-Phepsi[(1R,2S)-cis-epoxide]Gly-NH-CH(isoprop yl)2 where (SMe)Pen(O)2 stands for S-methyl-S-dioxo-penicillamine. Through conformational calculations and NMR data analysis, we have obtained preferred conformations of the two inhibitors in solution. To our knowledge, this work is one of the first extensive conformational studies of peptidomimetics containing cis-epoxide amide isostere. The resulting preferred conformations contain extended structures. In these conformations, the psi of Phe(cep) is maintained about 130 degrees and the phi angle of (cep)Gly prefers +/- 150 degrees [where Phe(cep) and (cep)Gly are the residues generated by the replacement of the Phe-Gly peptide bond with cis-epoxide]. Two conformations were commonly observed in the preferred conformations of each inhibitor. Through restrained molecular dynamics simulating the hydrogen bond formation between our inhibitor and a water molecule ('flap water'), one of the conformations is assumed as the conformation which can bind to the enzyme without large conformational changes. Recently, we had the opportunity to compare the selected preferred conformation with the binding conformation of LB71116 observed from the X-ray studies of the complex between LB71116 and HIV-1 protease. These two conformations are surprisingly similar to each other. Thus, we can explain high activity and selectivity of our inhibitors to the HIV-1 protease by the similarity between the preferred conformations in solution and the binding conformation.     

Structure-activity relationship study of novel necroptosis inhibitors

Necroptosis is a regulated caspase-independent cell death mechanism that results in morphological features resembling necrosis. It can be induced in a FADD-deficient variant of human Jurkat T cells treated with TNF-alpha. 5-(1H-Indol-3-ylmethyl)-2-thiohydantoins and 5-(1H-indol-3-ylmethyl)hydantoins were found to be potent necroptosis inhibitors (called necrostatins). A SAR study revealed that several positions of the indole were intolerant of substitution, while small substituents at the 7-position resulted in increased inhibitory activity. The hydantoin ring was also quite sensitive to structural modifications. A representative member of this compound class demonstrated moderate pharmacokinetic characteristics and readily entered the central nervous system upon intravenous administration.     

Optimization of P1-P3 groups in symmetric and asymmetric HIV-1 protease inhibitors.

HIV-1 protease is an important target for treatment of AIDS, and efficient drugs have been developed. However, the resistance and negative side effects of the current drugs has necessitated the development of new compounds with different binding patterns. In this study, nine C-terminally duplicated HIV-1 protease inhibitors were cocrystallised with the enzyme, the crystal structures analysed at 1.8-2.3 A resolution, and the inhibitory activity of the compounds characterized in order to evaluate the effects of the individual modifications. These compounds comprise two central hydroxy groups that mimic the geminal hydroxy groups of a cleavage-reaction intermediate. One of the hydroxy groups is located between the delta-oxygen atoms of the two catalytic aspartic acid residues, and the other in the gauche position relative to the first. The asymmetric binding of the two central inhibitory hydroxyls induced a small deviation from exact C2 symmetry in the whole enzyme-inhibitor complex. The study shows that the protease molecule could accommodate its structure to different sizes of the P2/P2' groups. The structural alterations were, however, relatively conservative and limited. The binding capacity of the S3/S3' sites was exploited by elongation of the compounds with groups in the P3/P3' positions or by extension of the P1/P1' groups. Furthermore, water molecules were shown to be important binding links between the protease and the inhibitors. This study produced a number of inhibitors with Ki values in the 100 picomolar range.     

Structure-activity relationship within the serine protease inhibitors of the pacifastin family

The members of the Pacifastin family are serine protease inhibitors found in insects and crustacean. They are either small inhibitors (made of one consensus cysteine-rich motif) or proteins (4-9 motifs). Some of these inhibitors are characterized by a species selectivity for the trypsin inhibition. Structural data discriminate the small inhibitors that apparently look very similar into two groups. Interestingly, the inhibitors that display species selectivity fall in the same structural group.     

Novel pseudosymmetric inhibitors of HIV-1 protease

Compounds containing the easily accessible Phe[CH(OH)CH₂N(NH)Phe dipeptide isostere as a non-hydrolyzable replacement of the scissile amide bond in the natural substrate are potent inhibitors of HIV-1 protease. The expected symmetric binding pattern of the most potent inhibitor in this series (CGP 53280, IC₅₀ = 9 nM) is illustrated by the X-ray analysis performed with the corresponding enzyme-inhibitor complex.     

Sidechain-linked inhibitors of HIV-1 protease dimerization

There is a great need for alternative modes of inhibition for the design of anti-HIV therapies, due to the increased resistance of HIV to currently approved drugs. A novel strategy for generating potent dimerization inhibitors of HIV-1 protease is described based on sidechain-linked interfacial peptides. In a number of cases the activity of these agents against HIV-1 protease was found to be among the most potent reported, with inhibitory constants in the low nM range.     

Adaptive inhibitors of the HIV-1 protease

A significant obstacle to the efficacy of drugs directed against viral targets is the presence of amino acid polymorphisms in the targeted molecules. Amino acid polymorphisms may occur naturally due to the existence of variations within and between viral strains or as the result of mutations associated with drug resistance. An ideal drug will be one that is extremely effective against a primary target and maintains its effectiveness against the most important variations of the target molecule. A drug that simultaneously inhibits different variants of the target will lead to a faster suppression of the virus, retard the appearance of drug-resistant mutants and provide more efficacious and, in the long range, more affordable therapies. Drug molecules with the ability to inhibit several variants of a target with high affinity have been termed adaptive drugs (Nat. Biotechnol. 20 (2002) 15; Biochemistry 42 (2003) 8459; J. Cell. Biochem. S37 (2001) 82). Current drug design paradigms are predicated upon the lock-and-key hypothesis, which emphasizes shape complementarity as a way to attain specificity and improved binding affinity. Shape complementarity is accomplished by the introduction of conformational constraints in the drug molecule. While highly constrained molecules do well against a unique target, they lack the ability to adapt to target variations like those originating from naturally occurring polymorphisms or drug-resistant mutations. Targeting an array of closely related targets rather than a single one while still maintaining selectivity, requires a different approach. A plausible strategy for designing high affinity adaptive inhibitors is to engineer their most critical interactions (for affinity and specificity) with conserved regions of the target while allowing for adaptability through the introduction of flexible asymmetric functionalities in places facing variable regions of the target. The fundamental thermodynamics and structural principles associated with this approach are discussed in this chapter.     

Novel spirocyclic pyrrolidones as P2/P1 mimetics in potent inhibitors of HIV-1 protease

We have developed concise and efficient syntheses of novel spirocyclic pyrrolidones 1-3, which involve the alkylation of pyrrolidone precursor 13 with 1,5-dibromopentane, 16 and 15, followed by an in situ lactamization. Conjugates of 1 and 2 with P1'/P2' hydroxy-indanolamine moiety resulted in novel and potent inhibitors of HIV-1 protease 25 and 26, suggesting that 1 and 2 are novel P2/P1 HIV-PI mimetics.     

HIV-1 protease inhibitors with picomolar potency against PI-resistant HIV-1 by modification of the P1' substituent

Transposition of the pyridyl nitrogen from the P(3) substituent to the P(1)' substituent in HIV-1 protease inhibitors (PI) affords compounds such as 3 with an improved inhibitory profile against multiple P450 isoforms. These compounds also displayed increased potency, with 3 inhibiting viral spread (CIC(95)) at <8 nM for every strain of PI-resistant HIV-1 tested. The poor to modest bioavailability of these compounds may correlate in part to their aqueous solubility.