A Structural and Energetic Model for the Slow-Onset Inhibition of the <i>Mycobacterium tuberculosis</i> Enoyl-ACP Reductase InhA

Slow-onset enzyme inhibitors are of great interest for drug discovery programs since the slow dissociation of the inhibitor from the drug–target complex results in sustained target occupancy leading to improved pharmacodynamics. However, the structural basis for slow-onset inhibition is often not fully understood, hindering the development of structure-kinetic relationships and the rational optimization of drug-target residence time. Previously we demonstrated that slow-onset inhibition of the <i>Mycobacterium tuberculosis</i> enoyl-ACP reductase InhA correlated with motions of a substrate-binding loop (SBL) near the active site. In the present work, X-ray crystallography and molecular dynamics simulations have been used to map the structural and energetic changes of the SBL that occur upon enzyme inhibition. Helix-6 within the SBL adopts an open conformation when the inhibitor structure or binding kinetics is substrate-like. In contrast, slow-onset inhibition results in large-scale local refolding in which helix-6 adopts a closed conformation not normally populated during substrate turnover. The open and closed conformations of helix-6 are hypothesized to represent the EI and EI* states on the two-step induced-fit reaction coordinate for enzyme inhibition. These two states were used as the end points for nudged elastic band molecular dynamics simulations resulting in two-dimensional potential energy profiles that reveal the barrier between EI and EI*, thus rationalizing the binding kinetics observed with different inhibitors. Our findings indicate that the structural basis for slow-onset kinetics can be understood once the structures of both EI and EI* have been identified, thus providing a starting point for the rational control of enzyme–inhibitor binding kinetics

Thiolactomycin-Based Inhibitors of Bacterial β‑Ketoacyl-ACP Synthases with in Vivo Activity

β-Ketoacyl-ACP synthases (KAS) are key enzymes involved in the type II bacterial fatty acid biosynthesis (FASII) pathway and are putative targets for antibacterial discovery. Several natural product KAS inhibitors have previously been reported, including thiolactomycin (TLM), which is produced by Nocardia spp. Here we describe the synthesis and characterization of optically pure 5<i>R</i>-thiolactomycin (TLM) analogues that show improved whole cell activity against bacterial strains including methicillin-resistant Staphylococcus aureus (MRSA) and priority pathogens such as Francisella tularensis and Burkholderia pseudomallei. In addition, we identify TLM analogues with in vivo efficacy against MRSA and Klebsiella pneumoniae in animal models of infection

Rational Optimization of Drug-Target Residence Time: Insights from Inhibitor Binding to the <i>Staphylococcus aureus</i> FabI Enzyme–Product Complex

Drug-target kinetics has recently emerged as an especially important facet of the drug discovery process. In particular, prolonged drug-target residence times may confer enhanced efficacy and selectivity in the open <i>in vivo</i> system. However, the lack of accurate kinetic and structural data for a series of congeneric compounds hinders the rational design of inhibitors with decreased off-rates. Therefore, we chose the <i>Staphylococcus aureus</i> enoyl-ACP reductase (saFabI)  an important target for the development of new anti-staphylococcal drugs  as a model system to rationalize and optimize the drug-target residence time on a structural basis. Using our new, efficient, and widely applicable mechanistically informed kinetic approach, we obtained a full characterization of saFabI inhibition by a series of 20 diphenyl ethers complemented by a collection of 9 saFabI–inhibitor crystal structures. We identified a strong correlation between the affinities of the investigated saFabI diphenyl ether inhibitors and their corresponding residence times, which can be rationalized on a structural basis. Because of its favorable interactions with the enzyme, the residence time of our most potent compound exceeds 10 h. In addition, we found that affinity and residence time in this system can be significantly enhanced by modifications predictable by a careful consideration of catalysis. Our study provides a blueprint for investigating and prolonging drug-target kinetics and may aid in the rational design of long-residence-time inhibitors targeting the essential saFabI enzyme

Clonal variation in OCH-CD1d tetramer binding by human iNKT cells is not related to TCR expression levels.

<p>Flow cytometric analysis of one representative CD4+ human Vα24+/Vβ11+ iNKT line (A) and three representative CD4+ human Vα24+/Vβ11+ iNKT clones from different donors (B) demonstrates clonal variation in binding to OCH-CD1d (upper row), but not K7-CD1d (lower row) tetramers. (C) K7- and OCH-CD1d tetramer staining in pure human iNKT lines (<i>n</i> = 68) and clones (<i>n</i> = 256) was related to expression levels of iNKT TCR Vα24 and Vβ11. The intensity (MFI) of K7- but not OCH-CD1d tetramer staining was strongly associated with Vα24 and Vβ11 expression, as determined by Pearson correlation analysis, but not with CD4+ (blue markers) or CD4−CD8− double negative (red markers) phenotype.</p

Differential autoreactive functional responses by human OCH^HIGH and OCH^LOW iNKT clones.

<p>Matched pairs of human OCH^HIGH (red columns and markers) and OCH^LOW (blue columns and markers) iNKT clones were compared for their ability to proliferate, secrete cytokines, and exhibit cytotoxicity in response to lipid-pulsed or endogenous lipid presenting CD1d-positive antigen presenting cells. (A) Proliferation of three representative pairs of OCH^HIGH and OCH^LOW iNKT clones from different healthy donors in response to K7-, OCH-, or vehicle-pulsed human CD1d-expressing T2 cells (T2-CD1d) or to K7-pulsed CD1d negative T2 cells (T2-) is shown. OCH^HIGH clones consistently displayed greater proliferation than OCH^LOW clones in response to OCH or vehicle pulsed T2-CD1d. cpm, counts per minute. Mean values ± s.e.m. are shown. (B) Cytokine secretion profiles of a representative pair of matched OCH^HIGH and OCH^LOW iNKT clones in response to the strong agonist ligand K7 and the partial agonist ligand OCH, presented by T2-CD1d, are shown. OCH^HIGH iNKT clones exhibited much stronger cytokine secretion than OCH^LOW iNKT cells in response to OCH-pulsed T2-CD1d, while cytokine secretion was similar for both in response to K7-pulsed T2-CD1d. (C) Autoreactive cytokine release in response to T2-CD1d in the absence of added exogenous ligands is shown for four matched pairs of OCH^HIGH and OCH^LOW iNKT clones. OCH^HIGH but not OCH^LOW iNKT clones consistently exhibited substantial autoreactive cytokine secretion. (D) Specific lysis of K7- (filled markers) and OCH- (unfilled markers) pulsed T2-CD1d targets is shown for three matched pairs of OCH^HIGH and OCH^LOW iNKT clones from different donors.</p

Differential binding of OCH^HIGH and OCH^LOW iNKT clone derived TCR tetramers to endogenous lipid presenting CD1d molecules.

<p>PE-conjugated recombinant iNKT TCR tetramers derived from OCH^HIGH (4C1369; red lines) and OCH^LOW (4C12; blue lines) iNKT clones, at increasing concentrations, were used to stain T2-CD1d lymphoblasts. Clear staining of vehicle-pulsed T2-CD1d (unfilled markers) was only seen with the OCH^HIGH TCR tetramer, whereas both iNKT TCR tetramers strongly bound to K7-pulsed T2-CD1d (filled markers). The black bar shows background staining of T2- cells with iNKT TCR tetramers.</p

Characteristics of 7 different human iNKT TCRs.

<p>DN, double negative (CD4-CD8αβ-); Vα, Vβ, Variable α and β family; Jα, Jβ, Junctional α and β regions; N, N-region; Dβ, diversity region.</p

Differential binding of OCH^HIGH and OCH^LOW iNKT cells to βGC-CD1d tetramers.

<p>Ex vivo sorted human Vα24+/Vβ11+ iNKT clones were stained with different, α- or β-glycosylceramide loaded CD1d-tetramers. (A) A representative pair of CD4+ OCH^HIGH and OCH^LOW iNKT clones from one donor is shown. βGC-CD1d tetramers only stained OCH^HIGH but not OCH^LOW iNKT clones above background (as determined by PE-streptavidin binding). TCR Vα24 and Vβ11 surface expression levels for the two clones shown were very similar (for PE-conjugated anti-Vα24, MFI 2673 (OCH^HIGH) and 2710 (OCH^LOW); for FITC-conjugated anti-Vβ11, MFI 106 (OCH^HIGH) and 97 (OCH^LOW)). (B) βGC-CD1d tetramer staining intensity (MFI) of a panel of OCH-LOW (red markers), OCH-INT (green markers), and OCH-HIGH (blue markers) iNKT clones showed almost linear correlation with OCH-CD1d tetramer staining, but no correlation with either Vα24 or Vβ11 surface expression.</p

Binding of 7 human iNKT TCRs to different CD1d/ligand complexes.

<p>K_D, dissociation constant; T<b>½</b>, dissociation half-time; ND, not determined. All values given ± standard deviation.</p