Unveiling the Importance of Amide Protons in CSP:ComD Interactions in Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae is an opportunistic pathogen that can cause diseases ranging from mild respiratory infections to life-threatening conditions such as pneumonia, meningitis, and bacteremia. S. pneumoniae pathogenicity is dependent on the action of a 17-amino acid peptide pheromone, termed competence stimulating peptide (CSP) that controls the competence regulon, a quorum sensing (QS) circuit. Therefore, intercepting QS could have therapeutic implications in treating pneumococcal infections while avoiding emerging antimicrobial resistance. In this study, we set out to evaluate the impact of amide protons on CSP activity and metabolic stability through systematic Nmethylation. Our results indicate that the majority of amide protons are critical for CSP activity, either through direct interactions with the cognate receptor or by stabilizing the bioactive conformation. Importantly, we identified several N-methyl CSP analogs, namely, CSP1(15)-N-Me-K6 and CSP1(15)-N-Me-F7, that retain their biological activity while exhibiting enhanced metabolic stability. These analogs are privileged scaffolds for the design of CSP-based QS modulators with drug-like properties.

Development of Streptococcus pneumoniae Pan‐Group Quorum‐Sensing Modulators

“…β‐Galactosidase assay : The β‐gal assay was performed as we previously described (for full details of the assay protocols see the Supporting Information) …”

Biological evaluation of native streptococcal competence stimulating peptides reveals potential crosstalk between Streptococcus mitis and Streptococcus pneumoniae and a new scaffold for the development of S. pneumoniae quorum sensing modulators

“…The ability of the synthetic CSP analogues to activate the QS circuit was evaluated as previously described (see the ESI † for full experimental details). 27,28 EC 50 experiments. Analogues that exhibited high activity in the initial screening were further evaluated using a dosedependent assay in which peptide stock solutions were diluted with DMSO in serial dilutions (either 1:2, 1:3 or 1:5) and assayed as described in the ESI.…”

“…Previously, the Tal-Gan Laboratory had conducted a detailed structure-activity relationship (SAR) analysis of the two S. pneumoniae signal variants, CSP1 and CSP2, against the two S. pneumoniae ComD receptors, ComD1 and ComD2, and identified the critical residues involved in ComD1 and ComD2 receptor binding and activation. [27][28][29] Based on the SAR results of both the CSP1 and CSP2 scaffolds in S. pneumoniae, we designed ten S. mitis-CSP-2 analogues bearing a point modification in key positions to match the corresponding residue in S. pneumoniae CSP1 or CSP2 pheromones and examined their ability to modulate the competence regulon of S. pneumoniae (Table 2). Since S. mitis-CSP-2 and the two CSP signals of S. pneumoniae differ in many positions (Fig.…”

“…26,27,[31][32][33] All the previously developed CSP-based inhibitors share the same key modification, the replacement of glutamic acid at position 1 with alanine. [26][27][28][29][32][33][34][35] We therefore reasoned that the S. mitis-CSP-2 template can also be converted into a pneumococcus ComD inhibitor by incorporating the same E1A modification. To this end, we first replaced Glu1 with alanine in the native S. mitis-CSP-2 to afford S. mitis-CSP-2-E1A and found that the resulting analogue can effectively inhibit the pneumococcus ComD1 receptor, albeit at a 6-fold higher concentration than S. pneumoniae CSP1-E1A (IC 50 value of 497 nM), while losing all activity against the S. pneumoniae ComD2 receptor (Table 4).…”

Strategies to attenuate the competence regulon in Streptococcus pneumoniae

“…With this knowledge in mind, the truncated CSP1 sequence, CSP1-desK16K17 (aka CSP1 ( 15)), was used as template in this study. [62] A systematic N-methyl scan library was constructed, ComD1 receptor, however, expectedly, no activity was observed against the ComD2 receptor. Surprisingly, these CSP1-E1A analogs were also found to be more susceptible to Trypsin/Chymotrypsin digestion as they degraded faster than the truncated CSP1 (15) analogs (Table 1).…”