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Unambiguous Stereochemical Assignment of Cyclo(Phe-Pro), Cyclo(Leu-Pro), and Cyclo(Val-Pro) by Electronic Circular Dichroic Spectroscopy.

ABSTRACT: 2,5-diketopiperazines (DKPs) are cyclic dipeptides ubiquitously found in nature. In particular, cyclo(Phe-Pro), cyclo(Leu-Pro), and cyclo(Val-Pro) are frequently detected in many microbial cultures. Each of these DKPs has four possible stereoisomers due to the presence of two chirality centers. However, absolute configurations of natural DKPs are often ambiguous due to the lack of a simple, sensitive, and reproducible method for stereochemical assignment. This is an important problem because stereochemistry is a key determinant of biological activity. Here, we report a synthetic DKP library containing all stereoisomers of cyclo(Phe-Pro), cyclo(Leu-Pro), and cyclo(Val-Pro). The library was subjected to spectroscopic characterization using mass spectrometry, NMR, and electronic circular dichroism (ECD). It turned out that ECD can clearly differentiate DKP stereoisomers. Thus, our ECD dataset can serve as a reference for unambiguous stereochemical assignment of cyclo(Phe-Pro), cyclo(Leu-Pro), and cyclo(Val-Pro) samples from natural sources. The DKP library was also subjected to a biological screening using assays for E. coli growth and biofilm formation, which revealed distinct biological effects of cyclo(D-Phe-L-Pro).

SUBMITTER: Domzalski A

PROVIDER: S-EPMC8512403 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The anti-inflammatory, antiviral, and anti-cancer properties, as well as the mechanism of action of cyclo-[Pro-Pro-β3-HoPhe-Phe-] tetrapeptide (denoted as 4B8M), were recently described. The aim of this work was to synthesize and evaluate the immunosuppressive actions of the stereochemical variants of 4B8M by sequential substitution of L-amino acids by D-amino acids (a series of peptides denoted as P01-P07) using parent 4B8M as a reference compound. In addition, diverse available bioinformatics tools using machine learning and artificial intelligence were tested to find the bio-pharmacokinetic and polypharmacological attributes of analyzed stereomers. All peptides were non-toxic to human peripheral blood mononuclear cells (PBMCs) and only cyclo-[D-Pro-Pro-β3-HoPhe-Phe-] peptide (P03) was capable of inhibiting mitogen-induced PBMC proliferation. The peptides inhibited the lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-α) to various degrees, with P04 (cyclo-[Pro-Pro-D-β3-HoPhe-Phe-]) and P03 being the most potent. For further in vivo studies, P03 was selected because it had the combined properties of inhibiting cell proliferation and TNF-α production. P03 demonstrated a comparable ability to 4B8M in the inhibition of auricle edema and lymph node cell number and in the normalization of a distorted blood cell composition in contact sensitivity to the oxazolone mouse model. In the mouse model of carrageenan-induced inflammation of the air pouch, P03 exhibited a similar inhibition of the cell number in the air pouches as 4B8M, but its inhibitory effects on the percentage of neutrophils and eosinophils in the air pouches and blood, as well as on mastocyte degranulation in the air pouches, were stronger in comparison to 4B8M. Lastly, in a mouse model of dextran sulfate-induced colitis, similar effects to 4B8M regarding thymocyte number restoration and normalization of the blood cell pictures by P03 were observed. In summary, depending on either experimental findings or in silico predictions, P03 demonstrated comparable, or even better, anti-inflammatory and bio-pharmacokinetic properties to 4B8M and may be considered as a potential therapeutic. The possibility of P00 and P03 identification by circular dichroism measurements was tested by quantum-chemical calculations.

Project description:UnlabelledVibrio cholerae is an aquatic organism that causes the severe acute diarrheal disease cholera. The ability of V. cholerae to cause disease is dependent upon the production of two critical virulence determinants, cholera toxin (CT) and the toxin-coregulated pilus (TCP). The expression of the genes that encode for CT and TCP production is under the control of a hierarchical regulatory system called the ToxR regulon, which functions to activate virulence gene expression in response to in vivo stimuli. Cyclic dipeptides have been found to be produced by numerous bacteria, yet their biological function remains unknown. V. cholerae has been shown to produce cyclo(Phe-Pro). Previous studies in our laboratory demonstrated that cyclo(Phe-Pro) inhibited V. cholerae virulence factor production. For this study, we report on the mechanism by which cyclo(Phe-Pro) inhibited virulence factor production. We have demonstrated that exogenous cyclo(Phe-Pro) activated the expression of leuO, a LysR-family regulator that had not been previously associated with V. cholerae virulence. Increased leuO expression repressed aphA transcription, which resulted in downregulation of the ToxR regulon and attenuated CT and TCP production. The cyclo(Phe-Pro)-dependent induction of leuO expression was found to be dependent upon the virulence regulator ToxR. Cyclo(Phe-Pro) did not affect toxR transcription or ToxR protein levels but appeared to enhance the ToxR-dependent transcription of leuO. These results have identified leuO as a new component of the ToxR regulon and demonstrate for the first time that ToxR is capable of downregulating virulence gene expression in response to an environmental cue.ImportanceThe ToxR regulon has been a focus of cholera research for more than three decades. During this time, a model has emerged wherein ToxR functions to activate the expression of Vibrio cholerae virulence factors upon host entry. V. cholerae and other enteric bacteria produce cyclo(Phe-Pro), a cyclic dipeptide that we identified as an inhibitor of V. cholerae virulence factor production. This finding suggested that cyclo(Phe-Pro) was a negative effector of virulence factor production and represented a molecule that could potentially be exploited for therapeutic development. In this work, we investigated the mechanism by which cyclo(Phe-Pro) inhibited virulence factor production. We found that cyclo(Phe-Pro) signaled through ToxR to activate the expression of leuO, a new virulence regulator that functioned to repress virulence factor production. Our results have identified a new arm of the ToxR regulon and suggest that ToxR may play a broader role in pathogenesis than previously known.

Dataset Information

Unambiguous Stereochemical Assignment of Cyclo(Phe-Pro), Cyclo(Leu-Pro), and Cyclo(Val-Pro) by Electronic Circular Dichroic Spectroscopy.

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