Impact of Azaproline on Peptide Conformation

The amino acid analog azaproline (azPro) contains a nitrogen atom in place of the Cα of proline. Peptides containing azPro were shown to stabilize the cis-amide conformer for the acyl-azPro bond and prefer type VI β-turns both in crystals and in organic solvents by NMR. The increased stability for cis-amide conformers was relatively minor with respect to the trans-conformers. Further, their conformational preferences were depended on solvent. To elucidate the impact of azPro substitution on amide cis−trans isomerism and peptide conformation, this paper reports ab initio studies on azPro derivatives and a comparison with their cognate Pro derivatives:  1-acetyl-2methyl pyrrolidine (1), 1-acetyl-2-methyl pyrazolidine (2), Ac-Pro-NHMe (3), Ac-azPro-NHMe (4), Ac-azPro-NMe2 (5), Ac-azAzc-NHMe (6), and Ac-azPip-NHMe (7). Conformational preferences were explored at the MP2/6-31+G** level of theory in vacuo. Solvation effects for 1 and 2 were studied implicitly using the polarizable continuum model and explicitly represented by interactions with a single water molecule. An increase in the conformational preference for the cis-amide conformer of azPro was clearly seen. An intramolecular hydrogen bond occurred solely in the trans-amide conformer that reduced the preference for the cis-conformer by 2.2 kcal/mol. The larger ring homolog aza-pipecolic acid (azPip), in which this internal hydrogen bond was diminished, significantly augmented stabilization of the cis-amide conformer. In aqueous solution, the preference for the cis-amide conformers was greatly reduced, mainly as a result of interaction between water and the lone pair of the α-nitrogen in the trans-amide conformer that was 3.8 kcal/mol greater than that in the cis-conformer. In the azPro analog, the energy barrier for cis−trans amide isomerization was 6 kcal/mol less than that in the cognate Pro derivative. Because the azPro derivatives can stabilize the cis-amide bond and mimic a type VI β-turn without incorporation of additional steric bulk, such a simple chemical modification of the peptide backbone provides a useful conformational constraint when incorporated into the structure of selected bioactive peptides. Such modifications can scan receptors for biological recognition of reverse turns containing cis-amide bonds by the incorporation of type VI β-turn scaffolds with oriented appended side chains

Reactivities of the Front Pocket N‑Terminal Cap Cysteines in Human Kinases

The front pocket (FP) N-terminal cap (Ncap) cysteine is the most popular site of covalent modification in kinases. A long-standing hypothesis associates the Ncap position with cysteine hyper-reactivity; however, traditional computational predictions suggest that the FP Ncap cysteines are predominantly unreactive. Here we applied the state-of-the-art continuous constant pH molecular dynamics (CpHMD) to test the Ncap hypothesis. Simulations found that the Ncap cysteines of BTK/BMX/TEC/ITK/TXK, JAK3, and MKK7 are reactive to varying degrees; however, those of BLK and EGFR/ERBB2/ERBB4 possessing a Ncap+3 aspartate are unreactive. Analysis suggested that hydrogen bonding and electrostatic interactions drive the reactivity, and their absence renders the Ncap cysteine unreactive. To further test the Ncap hypothesis, we examined the FP Ncap+2 cysteines in JNK1/JNK2/JNK3 and CASK. Our work offers a systematic understanding of the cysteine structure–reactivity relationship and illustrates the use of CpHMD to differentiate cysteines toward the design of targeted covalent inhibitors with reduced chemical reactivities

Structural modeling of Compound 4 bound to the <i>P. aeruginosa</i> glyoxylate shunt enzymes supports the dual-targeting capability of lead compounds.

<p>Compound <b>4</b>, docked with ICL (A) or MS (B), is depicted in a cyan-carbon colored stick representation, with the active sites of ICL and MS shown as mesh surfaces, the protein backbones in a ribbon diagram, and magnesium as a green sphere.</p

Chemical structures of the 8 <i>P. aeruginosa</i> glyoxylate shunt-inhibiting compounds.

<p>Chemical structures of the 8 <i>P. aeruginosa</i> glyoxylate shunt-inhibiting compounds.</p

MIC and IC₅₀ values (in μg ml⁻¹ and μM, respectively) for the 8 glyoxylate shunt-inhibiting compounds.

<p>MIC and IC₅₀ values (in μg ml⁻¹ and μM, respectively) for the 8 glyoxylate shunt-inhibiting compounds.</p

<i>P. aeruginosa</i> glyoxylate shunt mutants are deficient for growth both <i>in vitro</i> and <i>in vivo.</i>

<p>(A) The ability of wild-type <i>P. aeruginosa</i> PAO1 and its isogenic glyoxylate shunt mutants to utilize various sole carbon sources was assessed spectrophotometrically after overnight growth at 37°C. (B) The ability of these strains to colonize and persist in a murine lung model of infection was measured at 2- and 48-hours post-infection by lung homogenization and subsequent CFU ml⁻¹ determination. NR – no recoverable colonies.</p

Cyclic Peptide Design Guided by Residual Dipolar Couplings, <i>J</i>‑Couplings, and Intramolecular Hydrogen Bond Analysis

Cyclic peptides have long tantalized drug designers with their potential ability to combine the best attributes of antibodies and small molecules. An ideal cyclic peptide drug candidate would be able to recognize a protein surface like an antibody while achieving the oral bioavailability of a small molecule. It has been hypothesized that such cyclic peptides balance permeability and solubility using their solvent-dependent conformational flexibility. Herein we report a conformational deconvolution NMR methodology that combines residual dipolar couplings, J-couplings, and intramolecular hydrogen bond analysis along with conformational analysis using molecular dynamics simulations and density functional theory calculations for studying cyclic peptide conformations in both low-dielectric solvent (chloroform) and high-dielectric solvent (DMSO) to experimentally study the solvent-dependent conformational change hypothesis. Taken together, the combined experimental and computational approaches can illuminate conformational ensembles of cyclic peptides in solution and help identify design opportunities for better permeability

Siderophore Receptor-Mediated Uptake of Lactivicin Analogues in Gram-Negative Bacteria

Multidrug-resistant Gram-negative pathogens are an emerging threat to human health, and addressing this challenge will require development of new antibacterial agents. This can be achieved through an improved molecular understanding of drug–target interactions combined with enhanced delivery of these agents to the site of action. Herein we describe the first application of siderophore receptor-mediated drug uptake of lactivicin analogues as a strategy that enables the development of novel antibacterial agents against clinically relevant Gram-negative bacteria. We report the first crystal structures of several sideromimic conjugated compounds bound to penicillin binding proteins PBP3 and PBP1a from <i>Pseudomonas aeruginosa</i> and characterize the reactivity of lactivicin and β-lactam core structures. Results from drug sensitivity studies with β-lactamase enzymes are presented, as well as a structure-based hypothesis to reduce susceptibility to this enzyme class. Finally, mechanistic studies demonstrating that sideromimic modification alters the drug uptake process are discussed

Chemical and Computational Methods for the Characterization of Covalent Reactive Groups for the Prospective Design of Irreversible Inhibitors

Interest in drugs that covalently modify their target is driven by the desire for enhanced efficacy that can result from the silencing of enzymatic activity until protein resynthesis can occur, along with the potential for increased selectivity by targeting uniquely positioned nucleophilic residues in the protein. However, covalent approaches carry additional risk for toxicities or hypersensitivity reactions that can result from covalent modification of unintended targets. Here we describe methods for measuring the reactivity of covalent reactive groups (CRGs) with a biologically relevant nucleophile, glutathione (GSH), along with kinetic data for a broad array of electrophiles. We also describe a computational method for predicting electrophilic reactivity, which taken together can be applied to the prospective design of thiol-reactive covalent inhibitors