N-Substituted Bicyclic Carbamoyl Pyridones: Integrase Strand Transfer Inhibitors that Potently Inhibit Drug-Resistant HIV-1 Integrase Mutants

Abstract

HIV-1 integrase (IN) is an important molecular target for the development of anti-AIDS drugs. A recently FDA-approved second-generation integrase strand transfer inhibitor (INSTI) cabotegravir (CAB, 2021) is being marketed for use in long-duration antiviral formulations. However, missed doses during extended therapy can potentially result in persistent low levels of CAB that could select for resistant mutant forms of IN, leading to virological failure. We report a series of N-substituted bicyclic carbamoyl pyridones (BiCAPs) that are simplified analogs of CAB. Several of these potently inhibit wild-type HIV-1 in single-round infection assays in cultured cells and retain high inhibitory potencies against a panel of viral constructs carrying resistant mutant forms of IN. Our lead compound, 7c, proved to be more potent than CAB against the therapeutically important resistant double mutants E138K/Q148K (>12-fold relative to CAB) and G140S/Q148R (>36-fold relative to CAB). A significant number of the BiCAPs also potently inhibit the drug-resistant IN mutant R263K, which has proven to be problematic for the FDA-approved second-generation INSTIs.

Keywords: HIV-1; INSTI; INSTI-resistance; bicyclic carbamoyl pyridone (BiCAP); cabotegravir; one-pot synthesis; resistant mutants.

Figure 1

Chemical structures of FDA-approved INSTIs with company and year of FDA approval shown in parentheses. Metal-chelating heteroatom triads are highlighted in blue and magenta for the first- and second-generation INSTIs, respectively.

Figure 2

DTG, CAB, their simplified BiCAP congeners 6 and 7,^,, and the key synthetic precursor 8.^,

Figure 3

Antiviral potencies of INSTIs in single-round infection assays employing HIV vectors with WT HIV-1 and with IN mutants S230N, G118R, R263K, N155H, and G140S/Q148H. The potencies of DTG, BIC, and CAB have been previously reported. Previously reported data are shown to facilitate comparison with the data for the new compounds. Error bars represent the standard deviations of independent experiments, n = 4, performed in triplicate. EC₅₀ values shown in the graph have a maximum value of 300 nM.

Figure 4

Antiviral potencies of BiCAPs against a panel of selected IN double and triple mutants. EC₅₀ values were determined using a vector that carries the IN mutant in a single-round infection assay. Error bars represent the standard deviations of independent experiments, n = 4, the assays were performed in triplicate in each experiment. The EC₅₀ values shown in the graph have a maximum value of 400 nM.

Figure 5

In vitro enzymatic activities of CAB, 6c, and 7c. (A) Half site and concerted integrations were determined for CAB, 6c, and 7c using serial dilutions ranging from 0.00256 to 1000 μM as shown above the lanes. The IN-strand transfer reaction products were separated by agarose gel electrophoresis and visualized by both fluorescence scanning and ethidium bromide staining as indicated. The bands corresponding to the half site integration and concerted integration products are indicated on the fluorescence-scanned gels and the nicked plasmid DNA, linearized plasmid DNA, or supercoiled plasmid DNA are shown on the ethidium bromide-stained gels. (B) Using the data from the fluorescence-scanned gel, densitometry was used to generate the dose response curves to determine the potency of the inhibition (nM). The curves for CAB (blue marked with circles), 6c (magenta marked with squares), and 7c (orange marked with triangles) are indicated.