RESUMEN
Quorum sensing is a bacterial signaling system that involves the synthesis, secretion and detection of signal molecules called autoinducers. The main autoinducer in Gram-negative bacteria are acylated homoserine lactones, produced by the LuxI family of autoinducer synthases and detected by the LuxR family of autoinducer receptors. Quorum sensing allows for changes in gene expression and bacterial behaviors in a coordinated, cell density dependent manner. Quorum sensing controls the expression of virulence factors in some human pathogens, making quorum sensing an antibacterial drug target. Here we describe the design and synthesis of transition-state analogs of the autoinducer synthase enzymatic reaction and the evaluation of these compounds as inhibitors of the synthase CepI. One such compound potently inhibits CepI and constitutes a new type of inhibitor against this underdeveloped antibacterial target.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Lactonas/farmacología , Ligasas/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Lactonas/síntesis química , Lactonas/química , Ligasas/metabolismo , Estructura Molecular , Percepción de Quorum/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Bispecific antibodies represent an increasingly large fraction of biologics in therapeutic development due to their expanded scope in functional capabilities. Asymmetric monovalent bispecific IgGs (bsIgGs) have the additional advantage of maintaining a native antibody-like structure, which can provide favorable pharmacology and pharmacokinetic profiles. The production of correctly assembled asymmetric monovalent bsIgGs, however, is a complex engineering endeavor due to the propensity for non-cognate heavy and light chains to mis-pair. Previously, we introduced the DuetMab platform as a general solution for the production of bsIgGs, which utilizes an engineered interchain disulfide bond in one of the CH1-CL domains to promote orthogonal chain pairing between heavy and light chains. While highly effective in promoting cognate heavy and light chain pairing, residual chain mispairing could be detected for specific combinations of Fv pairs. Here, we present enhancements to the DuetMab design that improve chain pairing and production through the introduction of novel electrostatic steering mutations at the CH1-CL interface with lambda light chains (CH1-Cλ). These mutations work together with previously established charge-pair mutations at the CH1-CL interface with kappa light chains (CH1-Cκ) and Fab disulfide engineering to promote cognate heavy and light chain pairing and enable the reliable production of bsIgGs. Importantly, these enhanced DuetMabs do not require engineering of the variable domains and are robust when applied to a panel of bsIgGs with diverse Fv sequences. We present a comprehensive biochemical, biophysical, and functional characterization of the resulting DuetMabs to demonstrate compatibility with industrial production benchmarks. Overall, this enhanced DuetMab platform substantially streamlines process development of these disruptive biotherapeutics.
Asunto(s)
Anticuerpos Biespecíficos , Anticuerpos Biespecíficos/genética , Electricidad Estática , Disulfuros , Mutación , Inmunoglobulina G/genéticaRESUMEN
Stapled peptides have the ability to mimic α-helices involved in protein binding and have proved to be effective pharmacological agents for disrupting protein-protein interactions. DNA-binding proteins such as transcription factors bind their cognate DNA sequences via an α-helix interacting with the major groove of DNA. We previously developed a stapled peptide based on the bacterial alternative sigma factor RpoN capable of binding the RpoN DNA promoter sequence and inhibiting RpoN-mediated expression in Escherichia coli. We have elucidated a structure-activity relationship for DNA binding by this stapled peptide, improving DNA binding affinity constants in the high nM range. Lead peptides were shown to have low toxicity as determined by their low hemolytic activity at 100 µM and were shown to have anti-virulence activity in a Galleria mellonella model of Pseudomonas aeruginosa infection. These findings support further preclinical development of stapled peptides as antivirulence agents targeting P. aeruginosa.
RESUMEN
In response to environmental and other stresses, the σ54 subunit of bacterial RNA polymerase (RNAP) controls expression of several genes that play a significant role in the virulence of both plant and animal pathogens. Recruitment of σ54 to RNAP initiates promoter-specific transcription via the double-stranded DNA denaturation mechanism of the cofactor. The RpoN box, a recognition helix found in the C-terminal region of σ54, has been identified as the component necessary for major groove insertion at the -24 position of the promoter. We employed the hydrocarbon stapled peptide methodology to design and synthesize stapled σ54 peptides capable of penetrating Gram-negative bacteria, binding the σ54 promoter, and blocking the interaction between endogenous σ54 and its target DNA sequence, thereby reducing transcription and activation of σ54 response genes.