Elucidating a Relationship between Conformational Sampling and Drug Resistance in HIV‑1 Protease

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 HIV-1 protease (HIV-1 PR) variant, D30N/M36I/A71V, changes the fractional occupancy of the equilibrium conformational sampling ensemble. Correlations are made among populations of the conformational states, namely, closed-like, semiopen, 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 semiopen 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

SDS-PAGE analysis of time-resolved auto-maturation of pro<i>Pb</i>PM4.

<p>All of the auto-maturation assays were performed at 37°C by incubating 2.4 μg of pro<i>Pb</i>PM4 in buffer of pH 4.5 (A), pH 5.0 (B), pH 5.5 (C), and pH 6.0 (D). The molecular conversion from zymogen (~43 kDa) to mature enzyme (~36 kDa) was monitored at the time indicated above the gel images. M: molecular weight marker, unit: min, O/N = overnight.</p

Yields of expression and purification of the recombinant semi-pro<i>Pb</i>PM4 from one liter culture.

<p>^aThe product yield of each step was the result from three individual expressions, presented as mean ± SEM.</p><p>^bWeights were directly measured using a balance after centrifugation.</p><p>^cWeights were directly measured using a balance after purification.</p><p>^dThe concentration of soluble protein was determined using OD₂₈₀ (ε₂₈₀ = 41,510 M^-1 cm^-1, a theoretical value calculated from the sequence of semi-pro<i>Pb</i>PM4 using ProtParam [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141758#pone.0141758.ref058" target="_blank">58</a>]).</p><p>Yields of expression and purification of the recombinant semi-pro<i>Pb</i>PM4 from one liter culture.</p

Primary subsite preferences of <i>Pb</i>PM4.

<p>The initial velocities for hydrolysis of the P1 (A) and P1’ (B) library pools were determined spectroscopically and normalized to the highest cleavage velocities, which were set to 100 percent. Phenylalanine, leucine, and norleucine were the three most favored amino acid substitutes at P1; whereas norleucine, tyrosine, and phenylalanine were the three most favored at P1’.</p

The inhibition of peptidomimetic compounds on FV plasmepsins and hcatD.

<p>^aΨ = -CH2-NH-, nL = norleucine.</p><p>^b<i>Pf</i>PM1, <i>Pf</i>PM2 and <i>Pf</i>PM4, plasmepsins 1, 2 and 4 from <i>Plasmodium falciparum</i>, respectively; <i>Pv</i>PM4, plasmepsin 4 from <i>P</i>. <i>vivax</i>; <i>Po</i>PM4, plasmepsin 4 from <i>P</i>. <i>ovalae</i>; and <i>Pm</i>PM4, plasmepsin 4 from <i>P</i>. <i>malariae</i>.</p><p>^cThese dissociation constants were reported in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141758#pone.0141758.ref041" target="_blank">41</a>].</p><p>^dThese dissociation constants were reported in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141758#pone.0141758.ref042" target="_blank">42</a>].</p><p>The inhibition of peptidomimetic compounds on FV plasmepsins and hcatD.</p

Determination of the optimal conditions for the catalysis of <i>Pb</i>PM4.

<p>Experiments were performed at 37°C, in buffers of pH 3.5–6.0. The pro<i>Pb</i>PM4 was pre-incubated in buffers for the time indicated. Subsequently, enzyme-catalyzed initial hydrolysis rates of a chromogenic substrate were measured and normalized to the maximal initial velocity, which was set to 100 percent.</p

Kinetic analyses of the inhibition of <i>Pb</i>PM4.

<p>^aThe structures of the tested inhibitors are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141758#pone.0141758.s002" target="_blank">S2 Fig</a>.</p><p>Kinetic analyses of the inhibition of <i>Pb</i>PM4.</p

Secondary subsite preferences of <i>Pb</i>PM4 at S2’.

<p>The three most favored peptide pools, P1-phenylalanine (A), P1-leucine (B) and P1-norleucine (C), were used for analyzing the S2’ subsite preferences of <i>Pb</i>PM4. The relative abundances of tri-peptides varying at P2’ were determined using in-line LC/MS, and normalized to the quantity of the most abundant cleavage product, which was set to 100 percent. The peptide abundances were plotted against the P2’ amino acid substitutes.</p

SDS-PAGE analysis of over-expression and purification of recombinant semi-pro<i>Pb</i>PM4.

<p>M: molecular weight markers; 1: lysates of pre-IPTG-induced <i>E</i>. <i>coli</i> in 20 μL of cell suspension (OD₆₀₀ = 0.61); 2: lysate of post-IPTG-induced <i>E</i>. <i>coli</i> in 8.2 μL of cell suspension (OD₆₀₀ = 1.48); 3: purified, pro<i>Pb</i>PM4-enriched inclusion body (protein loading in lane: ~30 μg); 4: soluble dialysate following filtration of the <i>in vitro</i> refolding products (protein loading in lane: 20 μg); 5: anion exchange chromatography-purified pro<i>Pb</i>PM4 (protein loading in lane: 5 μg); 6: size exclusion chromatography-purified pro<i>Pb</i>PM4 (protein loading in lane: 5 μg). kDa: kilo-Daltons.</p

Hydroxyethylamine Based Phthalimides as New Class of Plasmepsin Hits: Design, Synthesis and Antimalarial Evaluation

<div><p>A novel class of phthalimides functionalized with privileged scaffolds was designed, synthesized and evaluated as potential inhibitors of plasmepsin 2 (K_i: 0.99 ± 0.1 μM for <b>6u</b>) and plasmepsin 4 (K_i: 3.3 ± 0.3 μM for <b>6t</b>), enzymes found in the digestive vacuole of the plasmodium parasite and considered as crucial drug targets. Three compounds were identified as potential candidates for further development. The listed compounds were also assayed for their antimalarial efficacy against chloroquine (CQ) sensitive strain (3D7) of <i>Plasmodium falciparum</i>. Assay of twenty seven hydroxyethylamine derivatives revealed four (<b>5e</b>, <b>6j</b>, <b>6o</b> and <b>6s</b>) as strongly active, which were further evaluated against CQ resistant strain (7GB) of <i>P</i>. <i>falciparum</i>. Compound <b>5e</b> possessing the piperidinopiperidine moiety exhibited promising antimalarial activity with an IC₅₀ of 1.16 ± 0.04 μM. Further, compounds <b>5e</b>, <b>6j</b>, <b>6o</b> and <b>6s</b> exhibited low cytotoxic effect on MCF-7 cell line. Compound <b>6s</b> possessing <i>C</i>_<i>2</i> symmetry was identified as the least cytotoxic with significant antimalarial activity (IC₅₀: 1.30 ± 0.03 μM). The combined presence of hydroxyethylamine and cyclic amines (piperazines and piperidines) was observed as crucial for the activity. The current studies suggest that hydroxyethylamine based molecules act as potent antimalarial agent and may be helpful in drug development.</p></div