Probing Resistance Mutations in Retroviral Integrases by Direct Measurement of Dolutegravir Fluorescence

Abstract

FDA-approved integrase strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir) efficiently inhibit HIV-1 replication. Here, we present fluorescence properties of these inhibitors. Dolutegravir displays an excitation mode particularly dependent on Mg2+ chelation, allowing to directly probe its Mg2+-dependent binding to the prototype foamy virus (PFV) integrase. Dolutegravir-binding studied by both its fluorescence anisotropy and subsequent emission enhancement, strictly requires a preformed integrase/DNA complex, the ten terminal base pairs from the 3′-end of the DNA reactive strand being crucial to optimize dolutegravir-binding in the context of the ternary complex. From the protein side, mutation of any catalytic residue fully abolishes dolutegravir-binding. We also compared dolutegravir-binding to PFV F190Y, G187R and S217K mutants, corresponding to HIV-1 F121Y, G118R and G140S/Q148K mutations that confer low-to-high resistance levels against raltegravir/dolutegravir. The dolutegravir-binding properties derived from fluorescence-based binding assays and drug susceptibilities in terms of catalytic activity, are well correlated. Indeed, dolutegravir-binding to wild-type and F190Y integrases are comparable while strongly compromised with G187R and S217K. Accordingly, the two latter mutants are highly resistant to dolutegravir while F190Y shows only moderate or no resistance. Intrinsic fluorescence properties of dolutegravir are thus particularly suitable for a thorough characterization of both DNA-binding properties of integrase and resistance mutations.

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

“…The G118R mutant was initially identified in selection studies using MK-2048 . This mutant has been selected frequently in PLWH during initial DTG treatments, and it is considered to be a major IN resistant mutant for DTG. − G118R has been shown to cause significant resistance (17×) against CAB in our single-round infection assays. Although CAB is generally more susceptible to losing inhibitory potency against resistant mutants than DTG or BIC, both DTG and BIC lose potency against the G118 R mutant (9× and 4×, respectively).…”

Pro-Inflammatory Interactions of Dolutegravir with Human Neutrophils in an In Vitro Study

“…Given the physicochemical properties of dolutegravir, specifically the combination of lipophilicity and the ability to chelate divalent/trivalent metal ions [21,23,24], this agent is likely to possess ionophore-like properties. These, in turn, may enable dolutegravir to chelate extracellular Ca 2+ cations and transport these across the plasma membrane to the cell cytosol where they activate/augment pro-inflammatory intracellular signaling mechanisms, such as PLC.…”

Design of Light-Sensitive Triggers for Endothelial NO-Synthase Activation

“…The NT solutions with or without the nNOS substrate L-arginine or N-hydroxyarginine (NOHA) and with or without competitor NADPH were titrated by increasing concentrations of nNOS. Additional measurements of anisotropy of free NT and nNOS-bound NT were performed on the same apparatus as detailed in [28,29]. The steady-state fluorescence anisotropy was calculated using: r = (I VV − G × I VH )/(I VV + 2G × I VH ) where I VV and I VH correspond to vertical and horizontal components, respectively, when the sample is excited with vertically polarized light, G is the G-factor accounting for the difference in the monochromator transmission between // and ⊥ polarized components (G = I HV /I HH ).…”