Jerry Jeffrey

Mechanism of Action of 1-β-d-2,6-Diaminopurine Dioxolane, a Prodrug of the Human Immunodeficiency Virus Type 1 Inhibitor 1-β-d-Dioxolane Guanosine

ABSTRACT (−)-β-d-2,6-Diaminopurine dioxolane (DAPD), is a nucleoside reverse transcriptase (RT) inhibitor with activity against human immunodeficiency virus type 1 (HIV-1). DAPD, which was designed as a water-soluble prodrug, is deaminated by adenosine deaminase to give (−)-β-d-dioxolane guanine (DXG). By using calf adenosine deaminase a Km value of 15 ± 0.7 μM was determined for DAPD, which was similar to theKm value for adenosine. However, thekcat for DAPD was 540-fold slower than thekcat for adenosine. In CEM cells and peripheral blood mononuclear cells exposed to DAPD or DXG, only the 5′-triphosphate of DXG (DXG-TP) was detected. DXG-TP is a potent alternative substrate inhibitor of HIV-1 RT. Rapid transient kinetic studies show the efficiency of incorporation for DXG-TP to be lower than that measured for the natural substrate, 2′-deoxyguanosine 5′-triphosphate. DXG-TP is a weak inhibitor of human DNA polymerases α and β. Against the large subunit of human DNA polymerase γ aKi value of 4.3 ± 0.4 μM was determined for DXG-TP. DXG showed little or no cytotoxicity and no mitochondrial toxicity at the concentrations tested.

Carbamoyl pyridone HIV-1 integrase inhibitors 3. A diastereomeric approach to chiral nonracemic tricyclic ring systems and the discovery of dolutegravir (S/GSK1349572) and (S/GSK1265744).

We report herein the discovery of the human immunodeficiency virus type-1 (HIV-1) integrase inhibitors dolutegravir (S/GSK1349572) (3) and S/GSK1265744 (4). These drugs stem from a series of carbamoyl pyridone analogues designed using a two-metal chelation model of the integrase catalytic active site. Structure-activity studies evolved a tricyclic series of carbamoyl pyridines that demonstrated properties indicative of once-daily dosing and superior potency against resistant viral strains. An inherent hemiaminal ring fusion stereocenter within the tricyclic carbamoyl pyridone scaffold led to a critical substrate controlled diastereoselective synthetic strategy whereby chiral information from small readily available amino alcohols was employed to control relative and absolute stereochemistry of the final drug candidates. Modest to extremely high levels of stereochemical control were observed depending on ring size and position of the stereocenter. This approach resulted in the discovery of 3 and 4, which are currently in clinical development.

The use of oxadiazole and triazole substituted naphthyridines as HIV-1 integrase inhibitors. Part 1: Establishing the pharmacophore.

A series of HIV-1 integrase inhibitors containing a novel metal binding motif consisting of the 8-hydroxy-1,6-naphthyridine core and either an oxadiazole or triazole has been identified. The design of the key structural components was based on a two-metal coordination pharmacophore. This report presents initial structure-activity data that shows the new chelation architecture delivers potent inhibition in both enzymatic and antiviral assays.

1,3,4-Oxadiazole substituted naphthyridines as HIV-1 integrase inhibitors. Part 2: SAR of the C5 position

The use of a 1,3,4-oxadiazole in combination with an 8-hydroxy-1,6-naphthyridine ring system has been shown to deliver potent enzyme and antiviral activity through inhibition of viral DNA integration. This report presents a detailed structure-activity investigation of the C5 position resulting in low nM potency for several analogs with an excellent therapeutic index.

Allosteric Inhibition of Human Immunodeficiency Virus Integrase: LATE BLOCK DURING VIRAL REPLICATION AND ABNORMAL MULTIMERIZATION INVOLVING SPECIFIC PROTEIN DOMAINS*

Background: New antiviral agents bind to a site on HIV integrase protein also bound by the cellular protein LEDGF/p75. Results: Compound GSK1264 binds to this site, but it has surprising properties; it inhibits late during HIV replication, not early during integration, and it promotes abnormal multimerization. Conclusion: GSK1264 provides new insight into HIV replication. Significance: These observations inform the design of improved antiviral agents. HIV-1 replication in the presence of antiviral agents results in evolution of drug-resistant variants, motivating the search for additional drug classes. Here we report studies of GSK1264, which was identified as a compound that disrupts the interaction between HIV-1 integrase (IN) and the cellular factor lens epithelium-derived growth factor (LEDGF)/p75. GSK1264 displayed potent antiviral activity and was found to bind at the site occupied by LEDGF/p75 on IN by x-ray crystallography. Assays of HIV replication in the presence of GSK1264 showed only modest inhibition of the early infection steps and little effect on integration targeting, which is guided by the LEDGF/p75·IN interaction. In contrast, inhibition of late replication steps was more potent. Particle production was normal, but particles showed reduced infectivity. GSK1264 promoted aggregation of IN and preformed LEDGF/p75·IN complexes, suggesting a mechanism of inhibition. LEDGF/p75 was not displaced from IN during aggregation, indicating trapping of LEDGF/p75 in aggregates. Aggregation assays with truncated IN variants revealed that a construct with catalytic and C-terminal domains of IN only formed an open polymer associated with efficient drug-induced aggregation. These data suggest that the allosteric inhibitors of IN are promising antiviral agents and provide new information on their mechanism of action.

Synthesis and antiviral activity of 7-benzyl-4-hydroxy-1,5-naphthyridin-2(1H)-one HIV integrase inhibitors.

The medicinal chemistry and structure-activity relationships for a novel series of 7-benzyl-4-hydroxy-1,5-naphthyridin-2(1H)-one HIV-integrase inhibitors are disclosed. Substituent effects were evaluated at the N-1, C-3, and 7-benzyl positions of the naphthyridinone ring system. Low nanomolar IC(50) values were achieved in an HIV-integrase strand transfer assay with both carboxylic ester and carboxamide groups at C-3. More importantly, several carboxamide congeners showed potent antiviral activity in cellular assays. A 7-benzyl substituent was found to be critical for potent enzyme inhibition, and an N-(2-methoxyethyl)carboxamide moiety at C-3 significantly reduced plasma protein binding effects in vitro. Pharmacokinetic data in rats for one carboxamide analogue demonstrated oral bioavailability and reasonable in vivo clearance.

In Vitro Combination of Amdoxovir and the Inosine Monophosphate Dehydrogenase Inhibitors Mycophenolic Acid and Ribavirin Demonstrates Potent Activity against Wild-Type and Drug-Resistant Variants of Human Immunodeficiency Virus Type 1

ABSTRACT Amdoxovir [(−)-β-d-2,6-diaminopurine dioxolane (DAPD)] is a nucleoside analogue reverse transcriptase inhibitor of human immunodeficiency virus type 1 (HIV-1) replication. DAPD is deaminated by adenosine deaminase to the guanosine analogue dioxolane guanosine (DXG), which is subsequently phosphorylated to the corresponding 5′ triphosphate (DXG-TP). DXG-TP competes with the natural substrate dGTP for binding to the enzyme-nucleic acid complex. Mycophenolic acid (MPA) and ribavirin (RBV), inhibitors of inosine monophosphate dehydrogenase (IMPDH), inhibit the de novo synthesis of guanine nucleotides, including dGTP. Reducing the intracellular levels of dGTP would be expected to augment the antiviral activity of analogues of deoxyguanosine. In this study we examined the effect of MPA and RBV on the anti-HIV activity of DAPD and DXG. When tested against wild-type virus, both MPA and RBV decreased the 50% effective concentration (EC50) for DXG by at least 10-fold. In contrast, both MPA and RBV increase the EC50 value for zidovudine. MPA and RBV completely reversed the resistance to DXG observed with HIV isolates containing mutations which confer partial resistance to DAPD and DXG. Similarly, when tested against a mutant virus fully resistant to inhibition by DAPD (K65R/Q151M), MPA and RBV reduced the EC50 for DAPD to within twofold of that for the wild type. The combination of MPA or RBV with DAPD or DXG did not result in increased cytotoxicity or reduced levels of mitochondrial DNA when tested at physiologically relevant concentrations. These studies suggest a potential role for the use of IMPDH inhibitors in combination therapy with amdoxovir in the treatment of HIV.

Structural Basis for Inhibitor-Induced Aggregation of HIV Integrase.

The allosteric inhibitors of integrase (termed ALLINIs) interfere with HIV replication by binding to the viral-encoded integrase (IN) protein. Surprisingly, ALLINIs interfere not with DNA integration but with viral particle assembly late during HIV replication. To investigate the ALLINI inhibitory mechanism, we crystallized full-length HIV-1 IN bound to the ALLINI GSK1264 and determined the structure of the complex at 4.4 Å resolution. The structure shows GSK1264 buried between the IN C-terminal domain (CTD) and the catalytic core domain. In the crystal lattice, the interacting domains are contributed by two different dimers so that IN forms an open polymer mediated by inhibitor-bridged contacts; the N-terminal domains do not participate and are structurally disordered. Engineered amino acid substitutions at the inhibitor interface blocked ALLINI-induced multimerization. HIV escape mutants with reduced sensitivity to ALLINIs commonly altered amino acids at or near the inhibitor-bound interface, and these substitutions also diminished IN multimerization. We propose that ALLINIs inhibit particle assembly by stimulating inappropriate polymerization of IN via interactions between the catalytic core domain and the CTD and that understanding the interface involved offers new routes to inhibitor optimization.

Naphthyridinone (NTD) integrase inhibitors: N1 Protio and methyl combination substituent effects with C3 amide groups

Substituent effects of a series of N1 protio and methyl naphthyridinone HIV-1 integrase strand-transfer inhibitors has been explored. The effects of combinations of the N1 substituent and C3 amide groups was extensively studied to compare enzyme inhibition, antiviral activity and protein binding effects on potency. The impact of substitution on ligand efficiency was considered and several compounds were advanced into in vivo pharmacokinetic studies ultimately leading to the clinical candidate GSK364735.

Combining symmetry elements results in potent naphthyridinone (NTD) HIV-1 integrase inhibitors

A series of naphthyridinone HIV-1 integrase strand-transfer inhibitors have been designed based on a psdeudo-C2 symmetry element present in the two-metal chelation pharmacophore. A combination of two distinct inhibitor binding modes resulted in potent inhibition of the integrase strand-transfer reaction in the low nM range. Effects of aryl and N1 substitutions are disclosed including the impact on protein binding adjusted antiviral activity.