Interfacial peptide inhibitors of HIV-1 integrase activity and dimerization

Peptides derived from the interfacial region of dimeric HIV-1 integrase were evaluated as inhibitors of integrase's 3'-endonuclease activity. Three peptides were found to be moderately potent inhibitors with IC(50) values in the low micromolar range. The mode of inhibition was probed through protein crosslinking experiments. Active interfacial peptides were found to inhibit crosslinking of the dimeric form of integrase. Interfacial peptides that were poor inhibitors had no effect on integrase crosslinking.     

Discovery of novel non-cytotoxic salicylhydrazide containing HIV-1 integrase inhibitors

The previously discovered salicylhydrazide class of compounds displayed potent HIV-1 integrase (IN) inhibitory activity. The development of this class of compounds as antiretroviral agents was halted due to cytotoxicity in the nanomolar to sub-micromolar range. We identified a novel class of non-cytotoxic hydrazide IN inhibitors utilizing the minimally required salicylhydrazide substructure as a template in a small-molecule database search. The novel hydrazides displayed low micromolar IN inhibitory activity and are several hundred-fold less cytotoxic than previously disclosed salicylhydrazide IN inhibitors.     

Potent inhibition of HIV-1 replication by novel non-peptidyl small molecule inhibitors of protease dimerization

Dimerization of HIV-1 protease subunits is essential for its proteolytic activity, which plays a critical role in HIV-1 replication. Hence, the inhibition of protease dimerization represents a unique target for potential intervention of HIV-1. We developed an intermolecular fluorescence resonance energy transfer-based HIV-1-expression assay employing cyan and yellow fluorescent protein-tagged protease monomers. Using this assay, we identified non-peptidyl small molecule inhibitors of protease dimerization. These inhibitors, including darunavir and two experimental protease inhibitors, blocked protease dimerization at concentrations of as low as 0.01 microm and blocked HIV-1 replication with IC(50) values of 0.0002-0.48 microm. These agents also inhibited the proteolytic activity of mature protease. Other approved anti-HIV-1 agents examined except tipranavir, a CCR5 inhibitor, and soluble CD4 failed to block the dimerization event. Once protease monomers dimerize to become mature protease, mature protease is not dissociated by this dimerization inhibition mechanism, suggesting that these agents block dimerization at the nascent stage of protease maturation. The proteolytic activity of mature protease that managed to undergo dimerization despite the presence of these agents is likely to be inhibited by the same agents acting as conventional protease inhibitors. Such a dual inhibition mechanism should lead to highly potent inhibition of HIV-1.     

Development of an AlphaScreen-based HIV-1 integrase dimerization assay for discovery of novel allosteric inhibitors

In recent years, HIV-1 integrase (IN) has become an established target in the field of antiretroviral drug discovery. However, its sole clinically approved inhibitor, the integrase strand transfer inhibitor (INSTI) raltegravir, has a surprisingly low genetic barrier for resistance. Furthermore, the only two other integrase inhibitors currently in advanced clinical trials, elvitegravir and dolutegravir, share its mechanism of action and certain resistance pathways. To maintain a range of treatment options, drug discovery efforts are now turning toward allosteric IN inhibitors, which should be devoid of cross-resistance with INSTIs. As IN requires a precise and dynamic equilibrium between several oligomeric species for its activities, the modulation of this equilibrium presents an interesting allosteric target. We report on the development, characterization, and validation of an AlphaScreen-based assay for high-throughput screening for modulators of HIV-1 IN dimerization. Compounds identified as hits in this assay proved to act as allosteric IN inhibitors. Additionally, the assay offers a flexible platform to study IN dimerization.     

Discovery of structurally diverse HIV-1 integrase inhibitors based on a chalcone pharmacophore

Recently, we reported small-molecule chalcones as a novel class of HIV-1 integrase (IN) inhibitors. The most potent compound showed an IC50 value of 2 microM for both IN-mediated 3'-processing and strand transfer reactions. To further utilize the chalcones, we developed pharmacophore models to identify chemical signatures important for biological activity. The derived models were validated with a collection of published inhibitors, and then were applied to screen a subset of our small molecule database. We tested 71 compounds in an in vitro assay specific for IN enzymatic activity. Forty-four compounds showed inhibitory potency<100 microM, and four of them exhibited IC50 values<10 microM. One compound, 62, with an IC50 value of 0.6 microM, displayed better potency than the original chalcone 2 against the strand transfer process. This study demonstrates the systematic use of pharmacophore technologies to discover novel structurally diverse inhibitors based on lead molecules that would exhibit poor characteristics in vivo. The identified compounds have the potential to exhibit favorable pharmacokinetic and pharmacodynamic profiles.     

Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol: part II. integrase inhibition

We report molecular modeling and functional confirmation of Ole and HT binding to HIV-1 integrase. Docking simulations identified two binding regions for Ole within the integrase active site. Region I encompasses the conserved D64-D116-E152 motif, while region II involves the flexible loop region formed by amino acid residues 140-149. HT, on the other hand, binds to region II. Both Ole and HT exhibit favorable interactions with important amino acid residues through strong H-bonding and van der Waals contacts, predicting integrase inhibition. To test and confirm modeling predictions, we examined the effect of Ole and HT on HIV-1 integrase activities including 3'-processing, strand transfer, and disintegration. Ole and HT exhibit dose-dependent inhibition on all three activities, with EC(50)s in the nanomolar range. These studies demonstrate that molecular modeling of target-ligand interaction coupled with structural-activity analysis should facilitate the design and identification of innovative integrase inhibitors and other therapeutics.     

Dihydroxypyridopyrazine-1,6-dione HIV-1 integrase inhibitors

A series of potent novel dihydroxypyridopyrazine-1,6-dione HIV-1 integrase inhibitors was identified. These compounds inhibited the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 6 is active against replication of HIV with a CIC(95) of 0.31 microM and exhibits no shift in potency in the presence of 50% normal human serum. It displays a good pharmacokinetic profile when dosed in rats and no covalent binding with microsomal proteins in both in vitro and in vivo models.     

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.     

Substituted 2-pyrrolinone inhibitors of HIV-1 integrase

The beta-diketoacid class of HIV-1 integrase (IN) inhibitors represent the first potent class of compounds specific for the strand transfer catalytic activity of the viral enzyme. Previously, utilizing a beta-diketoacid pharmacophore as a search query, we identified a substituted 2-pyrrolinone with modest IN inhibitory activity from a database of small-molecules [Dayam, R.; Sanchez, T.; Neamati, N. J. Med. Chem.2005, 48, 8009]. In efforts to optimize this class of IN inhibitors, we carried out a structure-activity relationship analysis around the 2-pyrrolinone core. Here, we present a new class of 2-pyrrolinone IN inhibitors.     

Fragment based search for small molecule inhibitors of HIV-1 Tat-TAR

Basic molecular building blocks such as benzene rings, amidines, guanidines, and amino groups have been combined in a systematic way to generate ligand candidates for HIV-1 TAR RNA. Ranking of the resulting compounds was achieved in a fluorimetric Tat-TAR competition assay. Although simple molecules such as phenylguanidine are inactive, few iteration steps led to a set of ligands with IC₅₀ values ranging from 40 to 150 μM. 1,7-Diaminoisoquinoline 17 and 2,4,6-triaminoquinazoline 22 have been further characterized by NMR titrations with TAR RNA. Compound 22 is bound to TAR at two high affinity sites and shows slow exchange between the free ligand and the RNA complex. These results encourage investigations of dimeric ligands built from two copies of compound 22 or related heterocycles.     

Azido-containing aryl beta-diketo acid HIV-1 integrase inhibitors

Aryl beta-diketo acids (ADK) comprise a general class of potent HIV-1 integrase (IN) inhibitors, which can exhibit selective inhibition of strand transfer reactions in extracellular recombinant IN assays and provide potent antiviral effects in HIV-infected cells. Recent studies have shown that polycyclic aryl or aryl rings bearing aryl-containing substituents are components of potent members of this class. Reported herein is the first use of azido functionality as an aryl replacement in beta-diketo acid IN inhibitors. The ability of azido-containing inhibitors to exhibit potent inhibition of IN and antiviral protection in HIV-infected cells, renders the azide group of potential value in the further development of ADK-based IN inhibitors.     

Novel furocoumarins as potential HIV-1 integrase inhibitors

A series of seven novel, rationally designed N-substituted 3-{3,5-dimethylfuro[3,2-g]coumarin-6-yl}propanamides have been prepared as potential HIV-1 integrase (IN) inhibitors via a five-step pathway commencing with resorcinol and diethyl 2-acetylglutarate, and the HIV-1 IN inhibition potential of these compounds has been examined relative to raltegravir, a known HIV-1 IN inhibitor.     

Inhibition of HIV-1 integrase dimerization and activity with crosslinked interfacial peptides

Alternative modes of inhibition for the design of anti-HIV therapies are sought due to the resistance of HIV to a number of the currently approved drugs. A non-active site strategy for generating potent inhibitors of HIV-1 integrase is described based on blocking protein association. Peptides α5 and α6 derived from the HIV-1 integrase dimeric interface have previously demonstrated efficacious dimerization inhibition of HIV-1 integrase. Due to the proximity of the termini of these peptides within the integrase structure, a focused library of tethered agents was designed based on crosslinking the peptides α5 and α6 to mimic a larger interfacial region. The best crosslinked inhibitors are approximately five-fold more potent against HIV-1 integrase than the individual peptides alone or in combination. The most active agents have an inhibitory constant in the mid-nM range and function via a dissociative mechanism of inhibition.     

Synthesis of novel thiazolothiazepine based HIV-1 integrase inhibitors

Thiazolothiazepines are among the smallest and most constrained inhibitors of human immunodeficiency virus type-1 integrase (HIV-1 IN) inhibitors (J. Med. Chem. 1999, 42, 3334). Previously, we identified two thiazolothiazepines lead IN inhibitors with antiviral activity in cell-based assays. Structural optimization of these molecules necessitated the design of easily synthesizable analogs. In order to design similar molecules with least number of substituent, herein we report the synthesis of 10 novel analogs. One of the new compounds (1) exhibited similar potency as the reference compounds, confirming that a thiazepinedione fused to a naphthalene ring system is the best combination for the molecule to accommodate into the IN active site. Thus, the replacement of sulfur in the thiazole ring with an oxygen does not seem considerably affect potency. On the other hand, the introduction of an extra methyl group at position 1 of the polycyclic system or the shift from a thiazepine to an oxazepine skeleton decreased potency. In order to understand their mode of interactions with IN active site, we docked all the compounds onto the previously reported X-ray crystal structure of IN. We observed that compounds 7-9 occupied an area close to D64 and Mg(2+) and surrounded by amino acid residues K159, K156, N155, E152, D116, H67, and T66. The oxygen atom of the oxazolo ring of 7 and 8 could chelate Mg(2+). These results indicate that the new analogs potentially interact with the highly conserved residues important for IN catalytic activities.     

Crucial amides for dimerization inhibitors of HIV-1 protease

An inhibitor based on crosslinked peptides from the interfacial region of HIV-1 protease, previously shown to act by dimerization inhibition, was modified by N-methylation to ascertain the importance of the amide hydrogens on inhibition. The effects of N-methylation on HIV-1 protease inhibition, as well as the effects on degradation by proteases are described.     

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.     

Development of tricyclic hydroxy-1H-pyrrolopyridine-trione containing HIV-1 integrase inhibitors

New tricyclic HIV-1 integrase (IN) inhibitors were prepared that combined structural features of bicyclic pyrimidinones with recently disclosed 4,5-dihydroxy-1H-isoindole-1,3(2H)-diones. This combination resulted in the introduction of a nitrogen into the aryl ring and the addition of a fused third ring to our previously described inhibitors. The resulting analogues showed low micromolar inhibitory potency in in vitro HIV-1 integrase assays, with good selectivity for strand transfer relative to 3′-processing.     

Discovery of the first antibacterial small molecule inhibitors of MurB

A series of imidazolinone analogues was synthesized and shown to possess potent MurB inhibitory as well as good antibacterial activity.     

Effect of substitution on novel tricyclic HIV-1 integrase inhibitors

A series of novel tricyclic inhibitors of HIV-1 integrase enzyme was prepared. The effect of substitution at C-6 of the 9-hydroxy-6,7-dihydropyrrolo[3,4-g]quinolin-8-one compounds was studied in vitro. Inhibitors with small side chains at C-6 were generally well tolerated by the enzyme, and the physicochemical properties of the inhibitors were improved by substitution of a small alkyl group at this position. A second series of analogs bearing a sulfamate at the C-5 position with various C-6 substituents were prepared to explore the interplay between the two groups. The SAR of the two classes are not parallel; modification at C-5 impacts the effect of substitutions at C-6.