Designing and synthesizing peptide-based quorum sensing modulators.

Quorum sensing (QS) is a density-dependent bacterial communication system that uses small molecules as regulatory modulators. Synthetic changes to these molecules can up-or-down-regulate this system, leading to control of phenotypes, like competence and virulence factor production, that have implications in human health. In this chapter, a methodology for library design and screening of synthetic autoinducing peptides (AIPs) to uncover QS SARs is delineated. Additionally, procedures for the synthesis, purification and analysis of linear and cyclic AIPs are detailed. This includes solutions for potential synthetic challenges including diketopiperazine formation when using N-methyl amino acids and cyclization of peptides containing N-terminal cysteine residues. These procedures have and are currently being applied to develop potent QS modulators in Streptococcus pneumoniae, Bacillus cereus, Streptococcus gordonii and Lactiplantibacillus plantarum.

The importance of the PapR7 C-terminus and amide protons in mediating quorum sensing in Bacilluscereus.

The opportunistic human pathogen Bacillus cereus controls the expression of key infection-promoting phenotypes using bacterial quorum sensing (QS). QS signal transduction within the species is controlled by an autoinducing peptide, PapR7, and its cognate receptor, PlcR, indicating that the PlcR:PapR interface is a prime target for QS inhibitor development. The C-terminal region of the peptide (PapR7; ADLPFEF) has been successfully employed as a scaffold to develop potent QS modulators. Despite the noted importance of the C-terminal carboxylate and amide protons in crystallographic data, their role in QS activity has yet to be explored. In this study, an N-methyl scan of PapR7 was conducted in conjunction with a C-terminal modification of previously identified B. cereus QS inhibitors. The results indicate that the amide proton at Glu6 and the C-terminal carboxylate are important for effective QS inhibition of the PlcR regulon. Through ß-galactosidase and hemolysis assays, a series of QS inhibitors were discovered, including several capable of inhibiting QS with nanomolar potency. These inhibitors, along with the structure-activity data reported, will serve as valuable tools for disrupting the B. cereus QS pathway towards developing novel anti-infective strategies.

Optimizing CSP1 analogs for modulating quorum sensing in Streptococcus pneumoniae with bulky, hydrophobic nonproteogenic amino acid substitutions.

The prompt appearance of multiantibiotic-resistant bacteria necessitates finding alternative treatments that can attenuate bacterial infections while minimizing the rate of antibiotic resistance development. Streptococcus pneumoniae, a notorious human pathogen, is responsible for severe antibiotic-resistant infections. Its pathogenicity is influenced by a cell-density communication system, termed quorum sensing (QS). As a result, controlling QS through the development of peptide-based QS modulators may serve to attenuate pneumococcal infections. Herein, we set out to evaluate the impact of the introduction of bulkier, nonproteogenic side-chain residues on the hydrophobic binding face of CSP1 to optimize receptor-binding interactions in both of the S. pneumoniae specificity groups. Our results indicate that these substitutions optimize the peptide-protein binding interactions, yielding several pneumococcal QS modulators with high potency. Moreover, pharmacological evaluation of lead analogs revealed that the incorporation of nonproteogenic amino acids increased the peptides' half-life towards enzymatic degradation while remaining nontoxic. Overall, our data convey key considerations for SAR using nonproteogenic amino acids, which provide analogs with better pharmacological properties.

Harnessing Multiple, Nonproteogenic Substitutions to Optimize CSP:ComD Hydrophobic Interactions in Group†1 Streptococcus pneumoniae.

Streptococcus pneumoniae (pneumococcus) is a human pathobiont that causes drastic antibiotic-resistant infections and is responsible for millions of deaths universally. Pneumococcus pathogenicity relies on the competence-stimulating peptide (CSP)-mediated quorum-sensing (QS) pathway that controls competence development for genetic transformation and, consequently, the spread of antibiotic resistance and virulence genes. Modulation of QS in S. pneumoniae can therefore be used to enervate pneumococcal infectivity as well as minimize the susceptibility to resistance development. In this work, we sought to optimize the interaction of CSP1 with its cognate transmembrane histidine kinase receptor (ComD1) through substitution of proteogenic and nonproteogenic amino acids on the hydrophobic binding face of CSP1. The findings from this study not only provided additional structure-activity data that are significant in optimizing CSP1 potency, but also led to the development of potent QS modulators. These CSP-based QS modulators could be used as privileged scaffolds for the development of antimicrobial agents against pneumococcal infections.

n → π* Interactions in N-Acyl Homoserine Lactone Derivatives and Their Effects on Hydrolysis Rates.

N-Acyl homoserine lactones (AHLs) mediate population-wide behavioral changes in Gram-negative bacteria through quorum sensing (QS), a process shown to regulate virulence, biofilm formation, and other phenotypes that impact human health. AHLs have been proposed to contain an n → π* interaction that reduces the molecules' susceptibility to signal inactivation via lactone hydrolysis. In this work, seven AHL derivatives were modeled via gas-phase DFT calculations, implicit solvent DFT calculations, and MD simulations. Each derivative was then synthesized and hydrolyzed to probe the relationship between the strength of the orbital interaction and hydrolysis rate. The data obtained support that an increase in n → π* energy (En→π*) correlates to a decrease in the hydrolysis rate constant ( kobs). Further, the observed variation in these rates demonstrates that AHL hydrolysis can be modified by manipulating steric and electronic effects that alter the electrophilicity of the lactone carbonyl. These results help to elucidate nature's choice of AHLs as agents of density-dependent bacterial communication and could inform the design of AHL-based quorum sensing modulators.

Defining the hydrophobic interactions that drive competence stimulating peptide (CSP)-ComD binding in Streptococcus pneumoniae.

Quorum sensing (QS) is a cell-cell communication mechanism that enables bacteria to assess their population density and alter their behavior upon reaching high cell number. Many bacterial pathogens utilize QS to initiate an attack on their host, thus QS has attracted significant attention as a potential antivirulence alternative to traditional antibiotics. Streptococcus pneumoniae, a notorious human pathogen responsible for a variety of acute and chronic infections, utilizes the competence regulon and its associated signaling peptide, the competence stimulating peptide (CSP), to acquire antibiotic resistance and establish an infection. In this work, we sought to define the binding pockets within the ComD1 receptor used for binding the hydrophobic side-chain residues in CSP1 through the introduction of highly-conservative point mutations within the peptide. Optimization of these binding interactions could lead to the development of highly potent CSP-based QS modulators while the inclusion of non-natural amino acids within the CSP sequence would confer resistance to protease degradation, a requirement for drug candidates.

The Hydrocarbon Staple & Beyond: Recent Advances Towards Stapled Peptide Therapeutics that Target Protein-Protein Interactions.

Anomalous protein-protein interactions (PPIs) have been correlated to a variety of disease states, such as cancer, infectious disease, neurological disorders, diabetes, endocrine disorders and cardiovascular disease. Stapled peptides are an emerging intervention for these PPIs due to their improved structural rigidity and pharmacokinetic properties relative to unstapled peptides. This review details the most recent advances in the field of stapled peptide therapeutics, including the increasing variety of PPIs being targeted and types of peptide staples being employed.

Selective transamidation of 3-oxo-N-acyl homoserine lactones by hydrazine derivatives.

A method for the selective transamidation of the 3-oxo sub-family of N-acyl homoserine lactones (3-oxo-AHLs) under physiologically relevant conditions has been developed. The reaction has the potential to serve as a strategy for selective knockdown of key autoinducers in a multicellular environment.

Thiourea-catalyzed aminolysis of N-acyl homoserine lactones.

Thiourea catalysts accelerate aminolysis of N-acyl homoserine lactones (AHLs), molecules integral to bacterial quorum sensing. The catalysts afford rate enhancement of up to 10 times the control in CD(3)CN. Mild catalysis in other polar aprotic solvents is still observed, while the activity is attenuated in polar protic solvents.