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Protein aggregates are associated with numerous diseases. Here we report a platform for the rapid phenotypic selection of protein aggregation inhibitors from genetically encoded cyclic peptide libraries in Escherichia coli based on phage-assisted continuous evolution (PACE). We developed a new PACE-compatible selection for protein aggregation inhibition and used it to identify cyclic peptides that suppress amyloid-β42 and human islet amyloid polypeptide aggregation. Additionally, we integrated a negative selection that removes false positives and off-target hits, greatly improving cyclic peptide selectivity. We show that selected inhibitors are active when chemically resynthesized in in vitro assays. Our platform provides a powerful approach for the rapid discovery of cyclic peptide inhibitors of protein aggregation and may serve as the basis for the future evolution of cyclic peptides with a broad spectrum of inhibitory activities. A platform for the continuous selection of protein aggregation inhibitors from genetically encoded cyclic peptide libraries in Escherichia coli was developed. This platform was used to discover cyclic peptides that suppress aggregation of amyloid-β42 and human islet amyloid polypeptide.

Raynaud’s phenomenon is a vascular condition characterized by episodic vasoconstriction, and recent reports suggest a potential link between calcitonin gene-related peptide (CGRP) inhibitors, used for migraine treatment, and the onset of this condition. This study evaluated the association between CGRP inhibitors and Raynaud’s phenomenon using data from the FDA Adverse Event Reporting System (FAERS). A retrospective analysis of adverse events from the approval year of each drug through August 2023 was conducted. Disproportionality was assessed using Reporting Odds Ratios (ROR) and Information Components (IC), with significant signals of disproportionate reporting (SDR) identified by a lower 95% confidence interval (CI) for ROR > 1.0 and IC > 0. Intra-class and inter-class analyses were conducted to compare SDRs among CGRP inhibitors and other migraine therapies, including triptans, beta-blockers, and anticonvulsants. CGRP inhibitors demonstrated significant SDRs for Raynaud’s phenomenon (ROR 19.12; 95% CI 15.44–23.69), with rimegepant, ubrogepant, and atogepant showing particularly strong signals. Intra-class analysis revealed a significant SDR only for galcanezumab (ROR 2.01; 95% CI 1.28–3.17). Inter-class analysis indicated significant SDRs for CGRP inhibitors compared to beta-blockers, anticonvulsants, and celecoxib, but not triptans. These findings underscore the importance of ongoing pharmacovigilance and further research to validate these associations and ensure patient safety.

Anti-PD-1 and anti-PD-L1 antibodies have achieved great clinical success in cancer immunotherapy, and peptide and small molecule inhibitors of PD-1/PD-L1 binding also attract much attention. Ascidians are not only seafood, but are also an important source of bioactive substances, including anti-tumor components. In this study, ascidian enzymatic hydrolysates were found to contain PD-1/PD-L1 inhibitory components. Affinity ultrafiltration (AUF) coupled with the nanoLC-MS/MS method was first applied in screening for PD-L1 peptide inhibitors from ascidian enzymatic hydrolysates. Two anti-PD-L1 ascidian peptides, C5 (LDVVIHTVTYGDR) and S2 (VLRDNIQGITKPAIR), were filtered out from the ascidians Ciona intestinalis and Styela clava, respectively. C5 and S2 showed moderate anti-PD-1/PD-L1 effects with the IC50 values of 33.9 µM (C5) and 112.8 μM (S2), respectively, by homogenous time-resolved fluorescence (HTRF) binding assay, and the KD values of 22.9 µM (C5) and 29.1 µM (S2), respectively, by surface plasmon resonance (SPR) assay. The results of this study suggest that ascidian enzymatic hydrolysates may be a potential source of bioactive peptides with anti-PD-1/PD-L1 activity.

IntroductionThe complement system is a key component of the innate immune system, and its aberrant activation underlies the pathophysiology of various diseases. Zilucoplan is a macrocyclic peptide that binds and inhibits the cleavage/activation of human complement component 5 (C5). We present in vitro and ex vivo data on the mechanism of action of zilucoplan for the inhibition of C5 activation, including two clinically relevant C5 polymorphisms at R885.MethodsThe interaction of zilucoplan with C5, including for clinical C5 R885 variants, was investigated using surface plasmon resonance (SPR), hemolysis assays, and ELISA. The interference of C5b6 formation by zilucoplan was investigated by native gel analysis and hemolysis assay. The permeability of zilucoplan in a reconstituted basement membrane was assessed by the partition of zilucoplan on Matrigel-coated transwell chambers.ResultsZilucoplan specifically bound human complement C5 with high affinity, competitively inhibited the binding of C5 to C3b, and blocked C5 cleavage by C5 convertases and the assembly of the cytolytic membrane attack complex (MAC, or C5b9). Zilucoplan fully prevented the in vitro activation of C5 clinical variants at R885 that have been previously reported to respond poorly to eculizumab treatment. Zilucoplan was further demonstrated to interfere with the formation of C5b6 and inhibit red blood cell (RBC) hemolysis induced by plasmin-mediated non-canonical C5 activation. Zilucoplan demonstrated greater permeability than a monoclonal C5 antibody in a reconstituted basement membrane model, providing a rationale for the rapid onset of action of zilucoplan observed in clinical studies.ConclusionOur findings demonstrate that zilucoplan uses a dual mode of action to potently inhibit the activation of C5 and terminal complement pathway including wild-type and clinical R885 variants that do not respond to eculizumab treatment. These data may be relevant to the clinically demonstrated benefits of zilucoplan.

CDK9 (cyclin-dependent kinase 9) plays a significant role in numerous pathological conditions, such as HIV-1 infection and cancer. The interaction between CDK9 and cyclin T1 is crucial for maintaining the kinase’s active state. Therefore, targeting this protein-protein interaction offers a promising strategy for inhibiting CDK9. In this study, we aimed to design and characterize a library of mutant peptides based on the binding region of cyclin T1 to CDK9. Using Osprey software, a total of 7,776 mutant peptides were generated. After conducting a comprehensive analysis, three peptides, namely, mp3 (RAADVEGQRKRRE), mp20 (RAATVEGQRKRRE), and mp29 (RAADVEGQDKRRE), were identified as promising inhibitors that possess the ability to bind to CDK9 with high affinity and exhibit low free binding energy. These peptides exhibited favorable safety profiles and displayed promising dynamic behaviors. Notably, our findings revealed that the mp3 and mp29 peptides interacted with a conserved sequence in CDK9 (residues 60–66). In addition, by designing the structure of potential peptides in the plasmid vector pET28a (+), we have been able to pave the way for facilitating the process of their recombinant production in an Escherichia coli expression system in future studies. Predictions indicated good solubility upon overexpression, further supporting their potential for downstream applications. While these results demonstrate the promise of the designed peptides as blockers of CDK9 with high affinity, additional experimental studies are required to validate their biological activity and assess their selectivity. Such investigations will provide valuable insights into their therapeutic potential and pave the way for the future development of peptide-based inhibitors targeting the CDK9-cyclin T1 complex.

TGF‐β (transforming growth factor β) is a pleiotropic cytokine found in three isoforms in humans. It regulates cell proliferation, wound healing, immune cell recruitment, contributes to epithelial‐to‐mesenchymal transition (EMT) and to the conversion of fibroblasts to myofibroblasts. TGF‐β signalling pathway hyperactivity underlies many human disorders. The aim of this study was to evaluate a series of novel, in silico–designed peptide inhibitors (PIs) of the TGFβ/TGFβRI/TGFβRII complex. Luciferase‐based luminescence assays on HEK293T cells were used to comparatively assess PI biological activity and calculate IC50 values. Flow cytometry was used to assess PI cytotoxicity on HEK293T cells. The PIs caused significant luminescence level reductions compared to controls. Additionally, three of the PIs caused luminescence reductions that did not differ significantly from the effects of SD‐208, a small molecule TGFβ inhibitor. None of the PIs exhibited cytotoxicity. Our TGFBR PIs have demonstrated activity in vitro, with no observed cytotoxicity. Our results suggest the PIs may be of interest in the treatment of fibrotic disorders, chronic inflammatory diseases, or certain neoplastic cancers. The PIs will be further refined in silico and tested via assays carried out on cancer cell lines and CD4+/CD8+ T cells.

Formation of the transient protein complexes in response to activation of cellular receptors is a common mechanism by which cells respond to external stimuli. This article presents the concept of blocking interactions of signaling proteins by the peptide inhibitors, and describes the progress achieved to date in the development of signaling inhibitors that act by blocking the signal-dependent protein interactions.

Background Migraine is a prevalent and disabling condition affecting one billion people worldwide. calcitonin gene-related peptide (CGRP) inhibitors have transformed migraine management. We describe real-world trends in CGRP inhibitor treatment, switching, discontinuation, and migraine-related health care use (including emergency department, ED, and ambulatory care visits). Methods We used the Merative™ MarketScan ® Research data, providing data on inpatient and outpatient healthcare use and drug dispensation from a large sample of individuals with employer-sponsored health insurance in the United States. We identified adults using CGRP inhibitors (comprising both monoclonal antibodies, mAbs, and small-molecule CGRP receptor antagonists, gepants) as preventive treatment for migraine between May 2018 and December 2022. We evaluated treatment patterns, including switching between CGRP agents or other prophylactic migraine treatments, and therapy discontinuation. Healthcare and migraine-related medication use were compared one year pre- and post-CGRP inhibitor initiation. Migraine-related medications included preventive migraine-specific treatments (e.g., onabotulinumtoxinA), non-specific preventives (e.g., antidepressants), and acute treatments—both migraine-specific (e.g., triptans) and non-specific (e.g., opioids). Results We studied 148,100 adults with at least one CGRP inhibitor dispensation. CGRP inhibitor use increased over time, with newer agents being more commonly dispensed in recent years. Within the first year, 10.3% of individuals switched between CGRP inhibitors, and an additional 13.5% discontinued their first CGRP inhibitor without switching to another. Post-initiation, there was a 4.5% reduction in migraine-related medication use (95% confidence interval, CI: -4.9% to -4.0%, and 12.8% reduction in healthcare use (95% CI: -13.2% to -12.3%). Conclusions CGRP inhibitors are increasingly used over time. About 10% switch between CGRP agents within the first year of initiation. There was reduction in other migraine-related medications and healthcare visits following CGRP initiation. Trial registration N/A.

Acute liver failure (ALF) is a fatal syndrome associated with massive hepatocyte death. Previous studies have found that Farnesyltransferase (FTase) inhibitors improve disease progression in mouse models of endotoxemia, sepsis, and autoimmune hepatitis. PANoptosis is a novel type of programmed cell death (PCD), including pyroptosis, apoptosis, and necrosis, that plays an important role in ALF. This study was designed and investigated whether the FTase inhibitor PD083176 (d2,d3,d5) could attenuate ALF progression by modulating PANoptosis. Combining the technical tools of computational biology, structural biology and pharmacology, we designed and obtained three high-affinity human FTase inhibitors of PD083176(d2,d3,d5). Then, these FTase inhibitors were investigated by animal experiments by administering PD083176(d2,d3,d5) (10 mg/kg) before modeling with LPS (100 μg/kg)/D-GalN (300 mg/kg) or TAA (800 mg/kg). We found that ALF induced by LPS/D-GaIN or TAA were associated with increased farnesylated protein in the liver. PD083176(d2,d3,d5) not only inhibited hepatic farnesylated proteins but also significantly attenuated liver injury and mortality in ALF mice. Importantly, PD083176(d2,d3,d5) treatment effectively inhibited hepatocyte apoptosis (Bax, Bcl-xL and TUNEL cell counts), pyroptosis (Caspase-1 and GSDMD), and necrotic apoptosis (RIPK1 and RIPK3). Collectively, these findings demonstrate that PD081376(d2,d3,d5) could alleviate LPS/D-GaIN or TAA-induced ALF by regulating apoptosis, pyroptosis, and necrotizing apoptosis, which might provide a new therapeutic strategy and scalability challenge for ALF.

Previous studies reported the critical role of the brefeldin A–inhibited guanine nucleotide exchange protein 3–prohibitin 2 (BIG3‐PHB2) complex in modulating estrogen signaling activation in breast cancer cells, yet its pathophysiological roles in osteosarcoma (OS) cells remain elusive. Here, we report a novel function of BIG3‐PHB2 in OS malignancy. BIG3‐PHB2 complexes were localized mainly in mitochondria in OS cells, unlike in estrogen‐dependent breast cancer cells. Depletion of endogenous BIG3 expression by small interfering RNA (siRNA) treatment led to significant inhibition of OS cell growth. Disruption of BIG3‐PHB2 complex formation by treatment with specific peptide inhibitor also resulted in significant dose‐dependent suppression of OS cell growth, migration, and invasion resulting from G2/M‐phase arrest and in PARP cleavage, ultimately leading to PARP‐1/apoptosis‐inducing factor (AIF) pathway activation–dependent apoptosis in OS cells. Subsequent proteomic and bioinformatic pathway analyses revealed that disruption of the BIG3‐PHB2 complex might lead to downregulation of inner mitochondrial membrane protein complex activity. Our findings indicate that the mitochondrial BIG3‐PHB2 complex might regulate PARP‐1/AIF pathway–dependent apoptosis during OS cell proliferation and progression and that disruption of this complex may be a promising therapeutic strategy for OS. In this study, we focused on understanding the critical role of the mitochondrial BIG3‐PHB2 complex in osteosarcoma (OS) cell proliferation and survival. Its complex disruption by the specific dominant‐peptide inhibitor causes mitochondrial dysfunction, resulting in apoptotic cell death and decreases in the migration and invasion abilities of OS cells. These findings suggest that inhibiting the BIG3‐PHB2 complex formation may be a new therapeutic strategy for the treatment of OS.