Nouri Neamati

Hydrazide-containing inhibitors of HIV-1 integrase

Inhibitors of HIV integrase are currently being sought as potential new therapeutics for the treatment of AIDS. A large number of inhibitors discovered to date contain the o-bis-hydroxy catechol structure. In an effort to discover structural leads for the development of new HIV integrase inhibitors which do not rely on this potentially cytotoxic catechol substructure, NSC 310217 was identified using a three-point pharmacophore search based on its assigned structure N-(2-hydroxybenzoyl)-N-(2-hydroxy-3-phenoxypropyl)hydrazine (1). When a sample of NSC 310217 was obtained from the NCI repository, it was shown to exhibit potent inhibition of HIV-1 integrase (3-processing IC50 = 0.6 microgram/mL). In work reported herein, we demonstrate that NSC 310217, rather than containing 1, which has no inhibitory potency against HIV-1 integrase, is comprised of roughly a 1:1 mixture of N-(2-hydroxybenzoyl)-N-(2-hydroxy-3-phenoxypropyl)hydrazine (6) and N,N-bis-salicylhydrazine 7, with all inhibitory potency residing with compound 7(IC50 = 0.7 microM for strand transfer). In subsequent structure-activity studies on 7, it is shown that removing a single amide carbonyl (compound 14, IC50 = 5.2 microM) or replacing one aromatic ring system with a naphthyl ring (compound 19, IC50 = 1.1 microM) can be accomplished with little loss of inhibitory potency. Additionally, replacing a single hydroxyl with a sulfhydryl (compound 23, IC50 = 5.8 microM) results in only moderate loss of potency. All other modifications examined, including the replacement of a single hydroxyl with an amino group (compound 22), resulted in complete loss of potency. Being potent, structurally simple, and non-catechol-containing, compounds such as 7 and 14 may provide useful leads for the development of a new class of HIV integrase inhibitor.

Inhibitors of human immunodeficiency virus integrase.

Integration of the viral DNA into a host cell chromosome is an essential step for HIV replication and maintenance of persistent infection. Two viral factors are essential for integration: the viral DNA termini (the att sites) and IN. Accruing knowledge of the IN structure, catalytic mechanisms, and interactions with other proteins can be used to design strategies to block integration. A large number of inhibitors have been identified that can be used as leads for the development of potent and selective anti-IN drugs with antiviral activity.

HIV-1 Integrase as a Target for Antiviral Drugs:

Retrovirus integration requires at least two viral components, one of the three retroviral enzymes, integrase, and cis-acting sequences at the ends of the retroviral DNA termini U3 and U5 ends of the long terminal repeats. Because the virus cannot replicate without integration into a host chromosome, integrase is a logical therapeutic target. Therapeutic inhibitors already exist for reverse transcriptase and protease, and attacking the virus on these sites together has already proven effective for combination therapy. Thus, the discovery of integrase inhibitors should provide an additional benefit. Screening and pharmacology of anti-integrase drugs is facilitated by the cloning and expression of recombinant retroviral integrases and their use in a series of in vitro assays that mimic integration in vivo. This review first describes the integration reactions in the retrovirus life cycle and the integrase protein. Then we provide a comprehensive review of the inhibitors identified to date.

Solution Structure of Anti-HIV-1 and Anti-Tumor Protein MAP30: Structural Insights into Its Multiple Functions

We present the solution structure of MAP30, a plant protein with anti-HIV and anti-tumor activities. Structural analysis and subsequent biochemical assays lead to several novel discoveries. First, MAP30 acts like a DNA glycosylase/apurinic (ap) lyase, an additional activity distinct from its known RNA N-glycosidase activity toward the 28S rRNA. Glycosylase/ap lyase activity explains MAP30s apparent inhibition of the HIV-1 integrase, MAP30s ability to irreversibly relax supercoiled DNA, and may be an alternative cytotoxic pathway that contributes to MAP30s anti-HIV/anti-tumor activities. Second, two distinct, but contiguous, subsites are responsible for MAP30s glycosylase/ap lyase activity. Third, Mn2+ and Zn2+ interact with negatively charged surfaces next to the catalytic sites, facilitating DNA substrate binding instead of directly participating in catalysis.

Chemical Trapping of Ternary Complexes of Human Immunodeficiency Virus Type 1 Integrase, Divalent Metal, and DNA Substrates Containing an Abasic Site IMPLICATIONS FOR THE ROLE OF LYSINE 136 IN DNA BINDING

We report a novel assay for monitoring the DNA binding of human immunodeficiency virus type 1 (HIV-1) integrase and the effect of cofactors and inhibitors. The assay uses depurinated oligonucleotides that can form a Schiff base between the aldehydic abasic site and a nearby enzyme lysine ε-amino group which can subsequently be trapped by reduction with sodium borohydride. Chemically depurinated duplex substrates representing the U5 end of the HIV-1 DNA were initially used. We next substituted an enzymatically generated abasic site for each of 10 nucleotides normally present in a 21-mer duplex oligonucleotide representing the U5 end of the HIV-1 DNA. Using HIV-1, HIV-2, or simian immunodeficiency virus integrases, the amount of covalent enzyme-DNA complex trapped decreased as the abasic site was moved away from the conserved CA dinucleotide. The enzyme-DNA complexes formed in the presence of manganese were not reversed by subsequent addition of EDTA, indicating that the divalent metal required for integrase catalysis is tightly bound in a ternary enzyme-metal-DNA complex. Both the N- and C-terminal domains of integrase contributed to efficient DNA binding, and mutation of Lys-136 significantly reduced Schiff base formation, implicating this residue in viral DNA binding.

Effect of HIV integrase inhibitors on the RAG1/2 recombinase

Assembly of functional Ig and T cell receptor genes by V(D)J recombination depends on site-specific cleavage of chromosomal DNA by the RAG1/2 recombinase. As RAG1/2 action has mechanistic similarities to DNA transposases and integrases such as HIV-1 integrase, we sought to determine how integrase inhibitors of the diketo acid type would affect the various activities of RAG1/2. Both of the inhibitors we tested interfered with DNA cleavage and disintegration activities of RAG1/2, apparently by disrupting interaction with the DNA motifs bound specifically by the recombinase. The inhibitors did not ablate RAG1/2s transposition activity or capture of nonspecific transpositional target DNA, suggesting this DNA occupies a site on the recombinase different from that used for specific binding. These results further underscore the similarities between RAG1/2 and integrase and suggest that certain integrase inhibitors may have the potential to interfere with aspects of B and T cell development.

Arylisothiocyanate-containing esters of caffeic acid designed as affinity ligands for HIV-1 integrase.

Integrase is an enzyme found in human immunodeficiency virus, which is required for the viral life cycle, yet has no human cellular homologue. For this reason, HIV integrase (IN) has become an important target for the development of new AIDS therapeutics. Irreversible affinity ligands have proven to be valuable tools for studying a number of enzyme and protein systems, yet to date there have been no reports of such affinity ligands for the study of IN. As an initial approach toward irreversible ligand design directed against IN, we appended isothiocyanate functionality onto caffeic acid phenethyl ester (CAPE), a known HIV integrase inhibitor. The choice of isothiocyanate as the reactive functionality, was based on its demonstrated utility in the preparation of affinity ligands directed against a number of other protein targets. Several isomeric CAPE isothiocyanates were prepared to explore the enzyme topography for reactive nitrogen and sulfur nucleophiles vicinal to the enzyme-bound CAPE. The preparation of these CAPE isothiocyanates, required development of new synthetic methodology which employed phenyl thiocarbamates as latent isothiocyanates which could be unmasked near the end of the synthetic sequence. When it was observed that beta-mercaptoethanol (beta-ME), which is required to maintain the catalytic activity of soluble IN (a F185KC280S mutant), reacted with CAPE isothiocyanate functionality to form the corresponding hydroxyethylthiocarbamate, a variety of mutant IN were examined which did not require the presence of beta-ME for catalytic activity. Although in these latter enzymes, CAPE isothiocyanate functionality was presumed to be present and available for acylation by IN nucleophiles, they were equally effective against Cys to Ser mutants. One conclusion of these studies, is that upon binding of CAPE to the integrase, nitrogen or sulfur nucleophiles may not be properly situated in the vicinity of the phenethyl aryl ring to allow reaction with and covalent modification of reactive functionality, such as isothiocyanate groups. The fact that introduction of the isothiocyanate group onto various positions of the phenethyl ring or replacement of the phenyl ring with naphthyl rings, failed to significantly affect inhibitory potency, indicates a degree of insensitivity of this region of the molecule toward structural modification. These findings may be useful in future studies concerned with the development and use of HIV-1 integrase affinity ligands.

Identification of HIV-1 integrase inhibitors based on a four-point pharmacophore.

The rapid emergence of human immunodeficiency virus (HIV) strains resistant to available drugs implies that effective treatment modalities will require the use of a combination of drugs targeting different sites of the HIV life cycle. Because the virus cannot replicate without integration into a host chromosome, HIV-1 integrase (IN) is an attractive therapeutic target. Thus, an effective IN inhibitor should provide additional benefit in combination chemotherapy. A four-point pharmacophore has been identified based on the structures of quinalizarin and purpurin, which were found to be potent IN inhibitors using both a preintegration complex assay and a purified enzyme assay in vitro. Searching with this four-point pharmacophore in the ‘open” part of the National Cancer Institute three-dimensional structure database produced 234 compounds containing the pharmacophore. Sixty of these compounds were tested for their inhibitory activity against IN using the purified enzyme; 19 were found to be active against IN with IC50 values of less than 100 µM, among which 10 had IC50 values of less than 10 µM. These inhibitors can further serve as leads, and studies are in progress to design novel inhibitors based on the results presented in this study.

2-Mercaptobenzenesulphonamides as novel inhibitors of human immunodeficiency virus type 1 integrase and replication

An obligatory requirement in the retroviral life cycle is the integration of the viral dsDNA into the host chromosome, a process performed by viral integrase. The retroviral integrase is able to catalyse at least three discrete enzymatic steps. Two of these steps, 3′ processing and DNA strand transfer, can be measured in an in vitro assay in the presence of a duplex oligonucleotide corresponding to the viral long terminal repeat, recombinant integrase and the divalent cations, Mg2+ or Mn2+. This assay provides an efficient means of testing integrase inhibitors. As part of our continuous effort in developing novel inhibitors we examined a series of 2-mercaptobenzenesulphonamides (MBSAs) for their inhibitory activity against human immunodeficiency virus type 1 (HIV-1) integrase. From the list of compounds tested in an assay specific for HIV-1 integrase, 26 compounds inhibited the 3′ processing and strand transfer step with 50% inhibitory concentration (IC50) values below 25 μM. All the thioether derivatives were inactive. These results were further compared with the ability of MBSAs to protect HIV-1-infected T4 lymphocyte CEM cells. Among 68 compounds tested, 27 exhibited antiviral activity in cell-based assays with therapeutic indices of 1-16. All the MBSAs with antiviral activity were also effective inhibitors of recombinant HIV-1 integrase. Several aromatic disulphides were also tested and found to exhibit moderate antiviral and anti-integrase activities. These data demonstrate that MBSAs can be developed as inhibitors of HIV-1 integrase with the potential for antiviral activity.

Structure/activity studies of peptide library-based integrase inhibitors

The HIV virus encoded integrase (IN) enzyme is required for the integration of the viral genome into infected mammalian cells, and thus for the replication of the virus. Because the virus can not replicate without it, IN is a logical therapeutic target [1,2]. IN does not have an obvious cellular counterpart, therefore drugs that specifically inhibit integration may not be toxic for the cell. The mechanism of integration is well documented. After reverse transcription the IN enzyme removes two nucleotides from the 3 terminal end of the viral DNA, and the enzyme also functions in inserting the viral DNA through its 3-end into the genomic DNA of the cell. Several years ago Plasterk and coworkers identified a hexapeptide, 1, with sequence, H-C-K-F-W-W-amide, using synthetic peptide combinatorial library methods [3], that inhibited IN function at low micromolar levels. To capitalize on this finding we have carried out further structure activity studies with the aim of developing physiologically more stable analogs, to evaluate their specificities, and to develop a peptide based pharmacophore model for enzyme inhibition.