Dolutegravir Interactions with HIV-1 Integrase-DNA: Structural Rationale for Drug Resistance and Dissociation Kinetics

<div><p>Signature HIV-1 integrase mutations associated with clinical raltegravir resistance involve 1 of 3 primary genetic pathways, Y143C/R, Q148H/K/R and N155H, the latter 2 of which confer cross-resistance to elvitegravir. In accord with clinical findings, in vitro drug resistance profiling studies with wild-type and site-directed integrase mutant viruses have shown significant fold increases in raltegravir and elvitegravir resistance for the specified viral mutants relative to wild-type HIV-1. Dolutegravir, in contrast, has demonstrated clinical efficacy in subjects failing raltegravir therapy due to integrase mutations at Y143, Q148 or N155, which is consistent with its distinct in vitro resistance profile as dolutegravir’s antiviral activity against these viral mutants is equivalent to its activity against wild-type HIV-1. Kinetic studies of inhibitor dissociation from wild-type and mutant integrase-viral DNA complexes have shown that dolutegravir also has a distinct off-rate profile with dissociative half-lives substantially longer than those of raltegravir and elvitegravir, suggesting that dolutegravir’s prolonged binding may be an important contributing factor to its distinct resistance profile. To provide a structural rationale for these observations, we constructed several molecular models of wild-type and clinically relevant mutant HIV-1 integrase enzymes in complex with viral DNA and dolutegravir, raltegravir or elvitegravir. Here, we discuss our structural models and the posited effects that the integrase mutations and the structural and electronic properties of the integrase inhibitors may have on the catalytic pocket and inhibitor binding and, consequently, on antiviral potency in vitro and in the clinic.</p> </div

2D structures of (A) dolutegravir, (B) raltegravir and (C) elvitegravir.

<p>Red ovals encircle the oxygen atoms that chelate the divalent metal cations in the active site; green ovals encircle the halobenzyl groups; and blue boxes encircle the approximate regions of the scaffolds that can accommodate positive charge after chelation of the metals. The purple circles at (B) encircle raltegravir’s gem-dimethyl (small circle) and oxadiazole groups, and the purple oval at (C) encircles elvitegravir’s 1-hydroxymethyl-2-methylpropyl group.</p

Structural model of Q148R HIV-1 integrase with U5 LTR DNA.

<p>The side chain of residue Q148R was modeled interacting with the side chain of E152 and in this conformation the residue may interfere with the binding of elvitegravir. Molecular representations and coloring schemes as described in Figure 2.</p

Structural models of HIV-1 integrase with U5 LTR DNA and (A, B) raltegravir, (C) elvitegravir or (D) dolutegravir.

<p>For raltegravir, the terminal 3′ adenylate is depicted in 2 distinct conformations: panel 2A shows the published conformer and panel 2B shows an alternative conformer that is also consistent with the observed electron density. Raltegravir, elvitegravir and dolutegravir are in stick representation with carbon, nitrogen, oxygen, fluorine and chlorine atoms colored gray, blue, red, cyan and green, respectively. A select subset of amino acids and nucleotides is depicted and labeled with residue type and number (numbering schemes as listed in Figure S1); all residues are in stick representation with carbon atoms colored by secondary structure/chain and nitrogen and oxygen atoms colored blue and red, respectively. The Mg²⁺ ions are represented as small yellow spheres with coordinate bonds to the inhibitors depicted as dashed yellow lines.</p

Structural models of (A) Q148H/G140S and (B) N155H HIV-1 integrase with U5 LTR DNA and dolutegravir.

<p>(A) The Q148H/G140S mutations are predicted to disrupt the structure of the flexible active-site loop, displacing the 3₁₀ helix away from the DDE motif and weakening the H-bond interaction between the backbone CO of Q148H and the backbone NH of E152. (B) The N155H mutation is predicted to disrupt the structure of the α4 helix, widen the base of the catalytic pocket, alter the placement of at least the Mg²⁺ ion coordinated to residues D64 and E152 and alter the conformation of the terminal 3′ adenosine forming part of the pocket. Molecular representations and coloring schemes are described in Figure 2.</p

Conformational Restriction Approach to β‑Secretase (BACE1) Inhibitors: Effect of a Cyclopropane Ring To Induce an Alternative Binding Mode

Improvement of a drug’s binding activity using the conformational restriction approach with sp³ hybridized carbon is becoming a key strategy in drug discovery. We applied this approach to BACE1 inhibitors and designed four stereoisomeric cyclopropane compounds in which the ethylene linker of a known amidine-type inhibitor <b>2</b> was replaced with chiral cyclopropane rings. The synthesis and biologic evaluation of these compounds revealed that the <i>cis</i>-(1<i>S</i>,2<i>R</i>) isomer <b>6</b> exhibited the most potent BACE1 inhibitory activity among them. X-ray structure analysis of the complex of <b>6</b> and BACE1 revealed that its unique binding mode is due to the apparent CH−π interaction between the rigid cyclopropane ring and the Tyr71 side chain. A derivatization study using <b>6</b> as a lead molecule led to the development of highly potent inhibitors in which the structure–activity relationship as well as the binding mode of the compounds clearly differ from those of known amidine-type inhibitors