Peter J. Felock

Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes

The process of integrating the reverse-transcribed HIV-1 DNA into the host chromosomal DNA is catalyzed by the virally encoded enzyme integrase (IN). Integration requires two metal-dependent reactions, 3′ end processing and strand transfer. Compounds that contain a diketo acid moiety have been shown to selectively inhibit the strand transfer reaction of IN in vitro and in infected cells and are effective as inhibitors of HIV-1 replication. To characterize the molecular basis of inhibition, we used functional assays and binding assays to evaluate a series of structurally related analogs. These studies focused on investigating the role of the conserved carboxylate and metal binding. We demonstrate that an acidic moiety such as a carboxylate or isosteric heterocycle is not required for binding to the enzyme complex but is essential for inhibition and confers distinct metal-dependent properties on the inhibitor. Binding requires divalent metal and resistance is metal dependent with active site mutants displaying resistance only when the enzymes are evaluated in the context of Mg2+. The mechanism of action of these inhibitors is therefore likely a consequence of the interaction between the acid moiety and metal ion(s) in the IN active site, resulting in a functional sequestration of the critical metal cofactor(s). These studies thus have implications for modeling active site inhibitors of IN, designing and evaluating analogs with improved efficacy, and identifying inhibitors of other metal-dependent phosphotransferases.

Discovery of 3-{5-[(6-amino-1H-pyrazolo[3,4-b]pyridine-3-yl)methoxy]-2-chlorophenoxy}-5-chlorobenzonitrile (MK-4965): a potent, orally bioavailable HIV-1 non-nucleoside reverse transcriptase inhibitor with improved potency against key mutant viruses.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have been shown to be a key component of highly active antiretroviral therapy (HAART). The use of NNRTIs has become part of standard combination antiviral therapies producing clinical outcomes with efficacy comparable to other antiviral regimens. There is, however, a critical issue with the emergence of clinical resistance, and a need has arisen for novel NNRTIs with a broad spectrum of activity against key HIV-1 RT mutations. Using a combination of traditional medicinal chemistry/SAR analyses, crystallography, and molecular modeling, we have designed and synthesized a series of novel, highly potent NNRTIs that possess broad spectrum antiviral activity and good pharmacokinetic profiles. Further refinement of key compounds in this series to optimize physical properties and pharmacokinetics has resulted in the identification of 8e (MK-4965), which has high levels of potency against wild-type and key mutant viruses, excellent oral bioavailability and overall pharmacokinetics, and a clean ancillary profile.

STRUCTURE AND ABSOLUTE STEREOCHEMISTRY OF HIV-1 INTEGRASE INHIBITOR INTEGRIC ACID. A NOVEL EREMOPHILANE SESQUITERPENOID PRODUCED BY A XYLARIA SP.

Abstract HIV-1 integrase is critical for viral replication and is absent in the host, and therefore is a potential target for the development of non-toxic antiviral therapy. From the screening of natural product libraries we have discovered integric acid, a novel eremophilane sesquiterpenoid, from a Xylaria sp. It inhibited 3′ -end processing, strand transfer and disintegration reactions catalyzed by HIV-1 integrase with IC50 values of 3–10 μM. The isolation, structure elucidation, relative, and absolute stereochemistry of integric acid are described.

Isolation and characterization of novel human immunodeficiency virus integrase inhibitors from fungal metabolites.

We have identified a series of novel inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase by randomly screening natural product extracts using an in vitro biochemical assay designed to identify inhibitors of integrase-catalysed strand transfer. Equisetin recovered from the fungus Fusarium heterosporum and a novel enantiomeric homologue of equisetin from Phoma sp. were isolated as inhibitors of HIV-1 integrase in vitro. Two additional analogues, a novel decalin derivative, integric acid, and oteromycin were also discovered to be inhibitors of integrase. Equisetin and related compounds inhibit 3” end-processing and strand transfer as well as disintegration catalysed by either the full-length enzyme or the truncated integrase core domain (amino acids 50–212). These compounds also inhibit strand transfer reactions catalysed by stable complexes assembled in vitro and integration reactions catalysed by pre-integration complexes isolated from HIV-1-infected cells. The compounds described in this report are structurally novel and mechanistically distinct from many previously described inhibitors of HIV-1 integrase. These results demonstrate the utility of using an appropriately configured assay to identify compounds that are effective post-assembly and the potential of isolating novel integrase inhibitors from complex natural product extracts.

Design and synthesis of bicyclic pyrimidinones as potent and orally bioavailable HIV-1 integrase inhibitors.

HIV integrase is one of the three enzymes encoded by HIV genome and is essential for viral replication, but integrase inhibitors as marketed drugs have just very recently started to emerge. In this study, we show the evolution from the N-methylpyrimidinone structure to bicyclic pyrimidinones. Introduction of a suitably substituted amino moiety modulated the physical-chemical properties of the molecules and conferred nanomolar activity in the inhibition of spread of HIV-1 infection in cell culture. An extensive SAR study led to sulfamide (R)- 22b, which inhibited the strand transfer with an IC50 of 7 nM and HIV infection in MT4 cells with a CIC95 of 44 nM, and ketoamide (S)- 28c that inhibited strand transfer with an IC50 of 12 nM and the HIV infection in MT4 cells with a CIC95 of 13 nM and exhibited a good pharmacokinetic profile when dosed orally to preclinical species.

The design and synthesis of diaryl ether second generation HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) with enhanced potency versus key clinical mutations.

Using a combination of traditional Medicinal Chemistry/SAR analysis, crystallography, and molecular modeling, we have designed and synthesized a series of novel, highly potent NNRTIs that possess broad antiviral activity against a number of key clinical mutations.

Distinct Mutation Pathways of Non-Subtype B HIV-1 during In Vitro Resistance Selection with Nonnucleoside Reverse Transcriptase Inhibitors

ABSTRACT Studies were conducted to investigate mutation pathways among subtypes A, B, and C of human immunodeficiency virus type 1 (HIV-1) during resistance selection with nonnucleoside reverse transcriptase inhibitors (NNRTIs) in cell culture under low-multiplicity of infection (MOI) conditions. The results showed that distinct pathways were selected by different virus subtypes under increasing selective pressure of NNRTIs. F227C and Y181C were the major mutations selected by MK-4965 in subtype A and C viruses during resistance selection. With efavirenz (EFV), F227C and V106M were the major mutations responsible for viral breakthrough in subtype A viruses, whereas a single pathway (G190A/V106M) accounted for mutation development in subtype C viruses. Y181C was the dominant mutation in the resistance selection with etravirine (ETV) in subtype A, and E138K/H221Y were the mutations detected in the breakthrough viruses from subtype C viruses with ETV. In subtype B viruses, on the other hand, known NNRTI-associated mutations (e.g., Y181C, P236L, L100I, V179D, and K103N) were selected by the NNRTIs. The susceptibility of the subtype A and B mutant viruses to NNRTIs was determined in order to gain insight into the potential mechanisms of mutation development. Collectively, these results suggest that minor differences may exist in conformation of the residues within the NNRTI binding pocket (NNRTIBP) of reverse transcriptase (RT) among the three subtypes of viruses. Thus, the interactions between NNRTIs and the residues in the NNRTIBPs of different subtypes may not be identical, leading to distinct mutation pathways during resistance selection in cell culture.

Four novel bis-(naphtho-γ-pyrones) isolated from Fusarium species as inhibitors of HIV-1 integrase

Abstract Integration of viral DNA into host cell DNA is an essential step in retroviral (HIV-1) replication and is catalyzed by HIV-1 integrase. HIV-1 integrase is a novel therapeutic target and is the focus of efforts to identify effective inhibitors that will prevent/or cure HIV infections. Four novel naphtho-γ-pyrones, belonging to the chaetochromin and ustilaginoidin family, were discovered as inhibitors of HIV-1 integrase from the screening of fungal extracts using a recombinant in vitro assay. These compounds inhibit both the coupled and strand transfer activity of HIV-1 integrase with IC 50 values of 1–3 and 4–12 μM, respectively. The discovery, structure elucidation, chemical modification and the structure–activity relationship of these compounds are described.

Biaryl Ethers as Novel Non-nucleoside Reverse Transcriptase Inhibitors with Improved Potency against Key Mutant Viruses

Biaryl ethers were recently reported as potent NNRTIs. Herein we disclose a detailed SAR study that led to the biaryl ether 6. This compound possessed excellent potency against WT RT and key clinically observed RT mutants and had an excellent pharmacokinetic profile in rats, dogs, and rhesus macaques. The compound also exhibited a clean safety profile in preclinical safety studies.

Integrastatins: structure and HIV-1 integrase inhibitory activities of two novel racemic tetracyclic aromatic heterocycles produced by two fungal species

Abstract Integrastatins are two novel aromatic natural products derived from fungal fermentations who possess a novel [6/6/6/6]-ring system and are racemic despite having two asymmetric centers. These compounds inhibited the strand transfer reaction of HIV-1 integrase with IC 50 values of 1.1–2.5 μM.