Versatile triblock peptides mimicking ABC-type heterotrimeric collagen with stabilizing salt bridges.

Type IV collagen, a principal constituent of basement membranes, consists of six distinct α chains that assemble into both ABC and AAB-type heterotrimers. While collagen-like peptides have been investigated for heterotrimer formation, the construction of ABC-type heterotrimeric collagen mimetic peptides remains a formidable challenge, primarily due to the intricate composition and arrangement of the chains. We have herein for the first time reported the development of a versatile triblock peptide system to mimic ABC-type heterotrimeric collagen stabilized by salt bridges. The triblock peptides A, B, and C incorporate functional natural type IV collagen sequences in the center, along with charged amino acids at their N and C-terminals. By leveraging electrostatic repulsion at these charged termini, the formation of homotrimers is effectively inhibited, while stable ABC-type heterotrimers are generated through the establishment of salt bridges between oppositely charged terminals. Circular dichroism (CD) spectroscopy demonstrated that peptides A, B, and C existed as individual monomers, while they effectively formed stable ABC-type heterotrimers upon being mixed at a molar ratio of 1:1:1. Additionally, fluorescence quenching results indicated that fluorescence-labeled peptides A', B', and C' formed ABC-type heterotrimer, exhibiting comparable thermal stability as determined by CD spectroscopy. Molecular dynamics simulations elucidated the role of salt bridges between arginine and aspartic acid residues at N- and C-terminals in maintaining a unique chain register in the ABC-type heterotrimers. These triblock peptides offer a robust approach for replicating the structural and functional characteristics of type IV collagen, with promising applications in elucidating the biological roles and pathologies associated with heterotrimeric collagen.

Druggable site near the upper vestibule determines the high affinity and P2X3 homotrimer selectivity of sivopixant/S-600918 and its analogue DDTPA.

BACKGROUND AND PURPOSE: The P2X3 receptor, a trimeric ionotropic purinergic receptor, has emerged as a potential therapeutic target for refractory chronic cough (RCC). Nevertheless, gefapixant/AF-219, the only marketed P2X3 receptor antagonist, might lead taste disorders by modulating the human P2X2/3 (hP2X2/3) heterotrimer. Hence, in RCC drug development, compounds exhibiting strong affinity for the hP2X3 homotrimer and a weak affinity for the hP2X2/3 heterotrimer hold promise. An example of such a molecule is sivopixant/S-600918, a clinical Phase II RCC candidate with a reduced incidence of taste disturbance compared to gefapixant. Sivopixant and its analogue, (3-(4-([3-chloro-4-isopropoxyphenyl]amino)-3-(4-methylbenzyl)-2,6-dioxo-3,6-dihydro-1,3,5-triazin-1(2H)-yl)propanoic acid (DDTPA), exhibit both high affinity and high selectivity for hP2X3 homotrimers, compared with hP2X2/3 heterotrimers. The mechanism underlying the druggable site and its high selectivity remains unclear. EXPERIMENTAL APPROACH: To analyse mechanisms that distinguish this drug candidate from other inhibitors of the P2X3 receptors we used a combination of chimera construction, site covalent occupation, metadynamics, mutagenesis and whole-cell recording. KEY RESULTS: The high affinity and selectivity of sivopixant/DDTPA for hP2X3 receptors was determined by the tri-symmetric site located close to the upper vestibule. Substitution of only four amino acids inside the upper body domain of hP2X2 with those of hP2X3, enabled the hP2X2/3 heterotrimer to exhibit a similar level of apparent affinity for sivopixant/DDTPA as the hP2X3 homotrimer. CONCLUSION AND IMPLICATIONS: From the receptor-ligand recognition perspective, we have elucidated the molecular basis of novel RCC clinical candidates' cough-suppressing properties and reduced side effects, offering a promising approach to the discovery of novel drugs that specifically target P2X3 receptors.

Toll/IL-1 receptor-containing proteins STIR-1, STIR-2 and STIR-3 synergistically assist Yersinia ruckeri SC09 immune escape.

Immune escape is a common feature of bacteria, viruses, parasites and even cancer cells. Our earlier work on an integrative and conjugative element (ICEr2) of Yersinia ruckeri SC09 demonstrated contributory roles of stir-1, stir-2 and stir-3 in bacterial toxicity and ability to code for immune evasion. Here, we further examined the ability of stir-4 in ICE (r2) and its encoded STIR-4 protein to mediate immune evasion using comparative genomic analysis. Additionally, the mechanisms underlying the synergistic activities of STIR-1, STIR-2, STIR-3 and STIR-4 in immune evasion were examined. Our results showed that STIR-4 did not contribute to bacterial toxicity, either in vivo nor in vitro, or show the ability to assist in bacterial immune escape. STIR-1, STIR-2, and STIR-3 formed heterotrimers in bacteria while facilitating immune evasion, which we speculate may be essential to maintain their stability. This discovery also partially explains the previous finding that a single gene can mediate immune evasion. Our data provide further knowledge on the distribution of ICE (r2)-like elements in bacteria, validating the prevalence of large-scale gene transfer in pathogens and its potential for enhancing virulence levels. Further studies are necessary to establish the biological significance of the ICE (r2) component.

H/D Isotope Effects on 1H-NMR Chemical Shifts in Cyclic Heterodimers and Heterotrimers of Phosphinic and Phosphoric Acids.

Hydrogen-bonded heterocomplexes formed by POOH-containing acids (diphenylphosphoric 1, dimethylphosphoric 2, diphenylphosphinic 3, and dimethylphosphinic 4) are studied by the low-temperature (100 K) 1H-NMR and 31P-NMR using liquefied gases CDF3/CDF2Cl as a solvent. Formation of cyclic dimers and cyclic trimers consisting of molecules of two different acids is confirmed by the analysis of vicinal H/D isotope effects (changes in the bridging proton chemical shift, δH, after the deuteration of a neighboring H-bond). Acids 1 and 4 (or 1 and 3) form heterotrimers with very strong (short) H-bonds (δH ca. 17 ppm). While in the case of all heterotrimers the H-bonds are cyclically arranged head-to-tail, ···O=P-O-H···O=P-O-H···, and thus their cooperative coupling is expected, the signs of vicinal H/D isotope effects indicate an effective anticooperativity, presumably due to steric factors: when one of the H-bonds is elongated upon deuteration, the structure of the heterotrimer adjusts by shortening the neighboring hydrogen bonds. We also demonstrate the formation of cyclic tetramers: in the case of acids 1 and 4 the structure has alternating molecules of 1 and 4 in the cycle, while in case of acids 1 and 3 the cycle has two molecules of 1 followed by two molecules of 3.

Asperflavipine†A: A Cytochalasan Heterotetramer Uniquely Defined by a Highly Complex Tetradecacyclic Ring System from Aspergillus flavipes QCS12.

Asperflavipines A (1) and B (2), two structurally complex merocytochalasans, were isolated from Aspergillus flavipes. Asperflavipine A (1), which contains two cytochalasan moieties and two epicoccine moieties, is the first cytochalasan heterotetramer to be discovered. It is uniquely defined by 5/6/11/5/6/5/6/5/6/5/5/11/6/5 fused tetradecacyclic rings with three continuous bridged ring systems. Asperflavipine B (2) is a cytochalasan heterotrimer containing a cytochalasan and two epicoccine moieties with a 5/6/11/5/5/6/5/6/5 nonacyclic ring system. The hypothetical biosynthesis of 1 and 2 is proposed to involve Diels-Alder and [3+2] cycloaddition reactions as key steps and reveals unparalleled plasticity in the biosynthesis of merocytochalasans. The existence of 1 adds a new dimension to the diversity of the cytochalasan family. Compound 1 showed moderate cytotoxicity and induced apoptosis in Jurkat, NB4, and HL60 cells through the activation of caspase-3 and degradation of poly(ADP-ribose) polymerase (PARP).

Matrix metalloproteinase inhibition by heterotrimeric triple-helical Peptide transition state analogues.

Matrix metalloproteinases (MMPs) have been implicated in numerous pathologies. An overall lack of selectivity has rendered active-site-targeted MMP inhibitors problematic. The present study describes MMP inhibitors that function by binding both secondary binding sites (exosites) and the active site. Heterotrimeric triple-helical peptide transition-state analogue inhibitors (THPIs) were assembled utilizing click chemistry. Three different heterotrimers were constructed, allowing for the inhibitory phosphinate moiety to be present uniquely in the leading, middle, or trailing strand of the triple helix. All heterotrimeric constructs had sufficient thermally stability to warrant analysis as inhibitors. The heterotrimeric THPIs were effective against MMP-13 and MT1-MMP, with Ki values spanning 100-400 nM. Unlike homotrimeric THPIs, the heterotrimeric THPIs offered complete selectivity between MT1-MMP and MMP-1. Exosite-based approaches such as this provide inhibitors with desired MMP selectivities.