Enzyme targets in rapidly replicating systems, such as retroviruses, commonly respond to drug-selective pressure with mutations arising in the active site pocket that limit inhibitor effectiveness by introducing steric hindrance or by eliminating essential molecular interactions. However, these primary mutations are disposed to compromising pathogenic fitness. Emerging secondary mutations, which are often found outside of the binding cavity, may or can restore fitness while maintaining drug resistance. The accumulated drug-pressure selected mutations could have an indirect effect in the development of resistance, such as altering protein flexibility or the dynamics of protein-ligand interactions. Here, we show that accumulation of mutations in a drug-resistant variant, D30N/M36I/A71V, HIV-1 protease (HIV-1 PR) changes the fractional occupancy of the equilibrium conformational sampling ensemble. Correlations are made among populations of the conformational states; namely, closed-like, semi-open, and open-like, with inhibition constants, as well as kinetic parameters. Mutations that stabilize a closed-like conformation correlate with enzymes of lowered activity and with higher affinity for inhibitors, which is corroborated by a further increase in the fractional occupancy of the closed state upon addition of inhibitor or substrate-mimic. Cross resistance is found to correlate with combinations of mutations that increase the population of the open-like conformations at the expense of the closed-like state while retaining native-like occupancy of the semi-open population. These correlations suggest that at least three states are required in the conformational sampling model to establish the emergence of drug-resistance in HIV-1 PR. More importantly, these results shed light on a possible mechanism whereby mutations combine to impart drug resistance while maintaining catalytic activity.

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Insects are the largest and most diverse group of organisms on earth, with over 1,000,000 species identified to date. Stick insects (“walkingsticks” or “phasmids”, Order Phasmatodea) are well known for and name derived from their uncanny stereotypical use of camouflage as a primary line of defense from predation. However, many species also possess a potent chemical defense spray. Recently we discovered that the defensive spray of Asceles glaber contains spiroketals (confirmed major component: (2S,6R)-(−)(E)-2-methyl-1,7-dioxaspiro[5.5]undecane and tentative minor component: 2-ethyl-1,6-dioxaspiro[4.5]decane) and glucose. In this paper we 1) illustrate the identification of spiroketals and glucose in the defense spray of A. glaber using Nuclear Magnetic Resonance (NMR), Gas Chromatography/Mass Spectrometry (GC/MS), and comparison with a synthetic reference sample, 2) provide the elucidation of the absolute configuration of the major spiroketal in that defense spray and 3) demonstrate the effect of this compound and its enantiomer on both fire ants (Solenopsis invicta) and mosquitoes (Aedes aegypti).