Structural basis for inhibition of the drug efflux pump NorA from Staphylococcus aureus.

Membrane protein efflux pumps confer antibiotic resistance by extruding structurally distinct compounds and lowering their intracellular concentration. Yet, there are no clinically approved drugs to inhibit efflux pumps, which would potentiate the efficacy of existing antibiotics rendered ineffective by drug efflux. Here we identified synthetic antigen-binding fragments (Fabs) that inhibit the quinolone transporter NorA from methicillin-resistant Staphylococcus aureus (MRSA). Structures of two NorA-Fab complexes determined using cryo-electron microscopy reveal a Fab loop deeply inserted in the substrate-binding pocket of NorA. An arginine residue on this loop interacts with two neighboring aspartate and glutamate residues essential for NorA-mediated antibiotic resistance in MRSA. Peptide mimics of the Fab loop inhibit NorA with submicromolar potency and ablate MRSA growth in combination with the antibiotic norfloxacin. These findings establish a class of peptide inhibitors that block antibiotic efflux in MRSA by targeting indispensable residues in NorA without the need for membrane permeability.

Novel hybrid nocodazole analogues as tubulin polymerization inhibitors and their antiproliferative activity.

We describe the design, synthesis and SAR profiling of a series of novel combretastatin-nocodazole conjugates as potential anticancer agents. The thiophene ring in the nocodazole moiety was replaced by a substituted phenyl ring from the combretastatin moiety to design novel hybrid analogues. The hydroxyl group at the ortho position in compounds 2, 3 and 4 was used as the conformationally locking tool by anticipated six-membered hydrogen bonding. The bioactivity profiles of all compounds as tubulin polymerization inhibitors and as antiproliferative agents against the A-549 human lung cancer cell line were investigated Compounds 1 and 4 showed µM IC50 values in both assays.

Correlation of hydrogen-bonding propensity and anticancer profile of tetrazole-tethered combretastatin analogues.

A series of 1,5-disubstituted tetrazole-tethered combretastatin analogues with extended hydrogen-bond donors at the ortho-positions of the aryl A and B rings were developed and evaluated for their antitubulin and antiproliferative activity. We wanted to test whether intramolecular hydrogen-bonding used as a conformational locking element in these analogues would improve their activity. The correlation of crystal structures with the antitubulin and antiproliferative profiles of the modified analogues suggested that hydrogen-bond-mediated conformational control of the A ring is deleterious to the bioactivity. In contrast, although there was no clear evidence that intramolecular hydrogen bonding to the B ring enhanced activity, we found that increased substitution on the B ring had a positive effect on antitubulin and antiproliferative activity. Among the various analogues synthesized, compounds 5d and 5e, having hydrogen-bonding donor groups at the ortho and meta-positions on the 4-methoxy phenyl B ring, are strong inhibitors of tubulin polymerization and antiproliferative agents having IC50 value in micromolar concentrations.

Hydrogen bond surrogate helices as minimal mimics of protein α-helices.

Examination of complexes of proteins with biomolecular ligands reveals that proteins tend to interact with partners via folded sub-domains, in which the backbone possesses secondary structure. α-Helices comprising the largest class of protein secondary structures, play fundamental roles in a multitude of highly specific protein-protein and protein-nucleic acid interactions. We have demonstrated a unique strategy for stabilization of the α-helical conformation that involves replacement of one of the main chain i and i+4 hydrogen bonds in the target α-helix with a covalent bond. We termed this synthetic strategy a hydrogen bond surrogate (HBS) approach. Two salient features of this approach are: (1) the internal placement of the crosslink allows development of helices such that none of the solvent-exposed surfaces are blocked by the constraining element, i.e., all side chains of the constrained helices remain available for molecular recognition. (2) This approach can be deployed to constrain very short peptides (<10 amino acid residues) into highly stable α-helices. This chapter presents the biophysical basis for the development of the hydrogen bond surrogate approach, as well as methods for the synthesis and conformational analysis of the artificial helices.

Angiotensin II analogs comprised of Pro-Amb (γ-turn scaffold) as angiotensin II type 2 (AT(2)) receptor agonists.

We describe herein the design, synthesis and conformational investigation of Pro-Amb (proline-3-amino-2-methoxybenzoic acid) incorporated Angiotensin II and its truncated analogues. Solution-state NMR and CD studies suggest γ-turn-like conformation in Pro-Amb analogs in aqueous solution. Furthermore, Pro-Amb analogs have been shown to act as AT2 receptor agonists.