Accessible drug modalities have continued to increase in number in recent years. Peptides play a central role as pharmaceuticals and biomaterials in these new drug modalities. Although traditional peptide synthesis using chain-elongation from C- to N-terminus is reliable, it produces large quantities of chemical waste derived from protecting groups and condensation reagents, which place a heavy burden on the environment. Here we report an alternative N-to-C elongation strategy utilizing catalytic peptide thioacid formation and oxidative peptide bond formation with main chain-unprotected amino acids under aerobic conditions. This method is applicable to both iterative peptide couplings and convergent fragment couplings without requiring elaborate condensation reagents and protecting group manipulations. A recyclable N-hydroxy pyridone additive effectively suppresses epimerization at the elongating chain. We demonstrate the practicality of this method by showcasing a straightforward synthesis of the nonapeptide DSIP. This method further opens the door to clean and atom-efficient peptide synthesis.
","authors":["Kanai M","Tatsumi T","Sasamoto K","Matsumoto T","Hirano R","Oikawa K","Nakano M","Yoshida M","Oisaki K."],"year":2023,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.1101/2025.02.14.638354","title":"Discovery of first-in-class inhibitors of the TRF1-TIN2 protein-protein interaction by fragment screening","abstract":"TRF1 is a subunit of the shelterin complex that binds to and protects the linear ends of chromosomes known as telomeres. Both genetic deletion and chemical inhibition of TRF1 have been shown to block the growth of lung carcinoma, glioblastoma, and renal cell carcinoma in mice without affecting mouse survival or tissue function, making TRF1 a potential therapeutic target in cancer 1–3 . Here, we report the discovery of a series of fragment hits that bind at the interface between the TRFH domain of TRF1 (TRF1 TRFH ) and a peptide of TIN2 (TIN2 TBM ), an interaction essential for the recruitment of TRF1 to shelterin, using X-ray crystallography (XChem) and ligand-observed NMR (LO-NMR) fragment screening. We discovered a first-in-class inhibitor of the TRF1-TIN2 interaction (compound 40 ) that binds to TRF1 TRFH with a K D of 29 μM (95% CI: 20 – 41 μM), displaces a TIN2 probe with an IC 50 of 67 ± 28 μM, and expels TRF1 from purified shelterin. Aided by a novel crystal system of TRF1 TRFH , we characterised fragments binding in a hotspot at the TRF1-TIN2 interface which will serve as a starting point for the structure-guided development of potent inhibitors of TRF1 protein-protein interactions to disrupt shelterin complex assembly.","authors":["Casale G","Liu M","Le Bihan Y","Inian O","Stammers E","Caldwell J","van Montfort RLM","Collins I","Guettler S."],"year":2025,"journal":"PPR","source":"PPR","preprint":true}],"preprints":[{"pmid":"","doi":"10.1101/2024.02.12.579891","title":"Sensing the bactericidal and bacteriostatic antimicrobial mode of action using Raman-Deuterium stable isotope probing (DSIP)","abstract":"The mode of actions of antibiotics can be broadly classified as bacteriostatic and bactericidal. The bacteriostatic mode leads to the arrested growth of the cells while the bactericidal mode causes cell death. In this work, we report the applicability of Deuterium stable isotope probing (DSIP) in combination with Raman spectroscopy (Raman DSIP) for discrimination among antibiotics on the basis of their mode of action at community level. We optimized the concentration of deuterium oxide required for metabolic activity monitoring without compromising the microbial growth. We also identified a novel carbon-deuterium Raman metabolic qualitative spectral marker in the biofingerprint region. This can be used for early identification of the antibiotic’s mode of action. Our results explores the new perspective which supports the utility of Deuterium based vibrational tags in the field of clinical spectroscopy. Understanding the antibiotic’s mode of action on bacterial cells in a short and objective manner can significantly enhance the clinical management abilities of infectious diseases and may also help in personalised antimicrobial therapy.Accessible drug modalities have continued to increase in number in recent years. Peptides play a central role as pharmaceuticals and biomaterials in these new drug modalities. Although traditional peptide synthesis using chain-elongation from C- to N-terminus is reliable, it produces large quantities of chemical waste derived from protecting groups and condensation reagents, which place a heavy burden on the environment. Here we report an alternative N-to-C elongation strategy utilizing catalytic peptide thioacid formation and oxidative peptide bond formation with main chain-unprotected amino acids under aerobic conditions. This method is applicable to both iterative peptide couplings and convergent fragment couplings without requiring elaborate condensation reagents and protecting group manipulations. A recyclable N-hydroxy pyridone additive effectively suppresses epimerization at the elongating chain. We demonstrate the practicality of this method by showcasing a straightforward synthesis of the nonapeptide DSIP. This method further opens the door to clean and atom-efficient peptide synthesis.
","authors":["Kanai M","Tatsumi T","Sasamoto K","Matsumoto T","Hirano R","Oikawa K","Nakano M","Yoshida M","Oisaki K."],"year":2023,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.1101/2025.02.14.638354","title":"Discovery of first-in-class inhibitors of the TRF1-TIN2 protein-protein interaction by fragment screening","abstract":"TRF1 is a subunit of the shelterin complex that binds to and protects the linear ends of chromosomes known as telomeres. Both genetic deletion and chemical inhibition of TRF1 have been shown to block the growth of lung carcinoma, glioblastoma, and renal cell carcinoma in mice without affecting mouse survival or tissue function, making TRF1 a potential therapeutic target in cancer 1–3 . Here, we report the discovery of a series of fragment hits that bind at the interface between the TRFH domain of TRF1 (TRF1 TRFH ) and a peptide of TIN2 (TIN2 TBM ), an interaction essential for the recruitment of TRF1 to shelterin, using X-ray crystallography (XChem) and ligand-observed NMR (LO-NMR) fragment screening. We discovered a first-in-class inhibitor of the TRF1-TIN2 interaction (compound 40 ) that binds to TRF1 TRFH with a K D of 29 μM (95% CI: 20 – 41 μM), displaces a TIN2 probe with an IC 50 of 67 ± 28 μM, and expels TRF1 from purified shelterin. Aided by a novel crystal system of TRF1 TRFH , we characterised fragments binding in a hotspot at the TRF1-TIN2 interface which will serve as a starting point for the structure-guided development of potent inhibitors of TRF1 protein-protein interactions to disrupt shelterin complex assembly.","authors":["Casale G","Liu M","Le Bihan Y","Inian O","Stammers E","Caldwell J","van Montfort RLM","Collins I","Guettler S."],"year":2025,"journal":"PPR","source":"PPR","preprint":true}],"consensus_view":"The literature consensus is that DSIP has genuine but poorly understood biological activity, particularly in sleep regulation and broad CNS neuromodulation. No consensus exists on its receptor target — indeed, no DSIP-specific receptor has ever been cloned or definitively identified after nearly 50 years of research. The hypothesis that DSIP engages GABA-A receptor α1 subunit is speculative and lacks direct published support; it is a reasonable but unproven mechanistic inference from DSIP's sleep-promoting pharmacological profile. The consensus also supports that native DSIP is conformationally flexible and that structural analogues may surpass it in bioactivity, providing indirect rationale for the proposed backbone rigidification strategy.","knowledge_gaps":"Critical gaps include: (1) No structural or biophysical data on DSIP-receptor interactions at GABA-A or any other receptor — binding affinity, co-crystal structures, and docking models are entirely absent from the published literature. (2) No SAR studies specifically mapping DSIP's Gly-3/Gly-4 hinge to bioactivity; the analogues noted by Kovalzon & Strekalova as more potent than native DSIP are uncharacterized in the retrieved literature. (3) No NMR or circular dichroism (CD) conformational studies of DSIP in solution that define the native β-turn population or quantify the conformational ensemble. (4) No published D-amino acid substitution studies on DSIP. (5) No electrophysiology (patch-clamp) or radioligand displacement assays with DSIP at GABA-A receptor subtypes. These gaps mean the team's computational or experimental predictions would constitute genuinely novel contributions.","supporting_evidence":"The strongest supporting evidence is the Kovalzon & Strekalova (2006) observation that artificial DSIP structural analogues — not the native peptide — show significant SWS-promoting activity, implying that conformational or steric optimization of DSIP enhances CNS potency and that the native sequence is suboptimal, consistent with the backbone rigidification rationale. DSIP's sedative-hypnotic clinical profile (Schneider-Helmert, 1984) is mechanistically consistent with positive GABA-A modulation (the predominant mechanism of benzodiazepine-class drugs). The small steric footprint of glycine in positions 3-4 is chemically logical as a flexible hinge, and D-Ala substitution is a well-established medicinal chemistry strategy for introducing backbone constraint without major steric penalty. The synthetic preprint (Kanai et al., 2023) confirms that DSIP analogues are synthetically tractable.","challenging_evidence":"Several findings complicate or challenge the hypothesis: (1) No study has ever directly demonstrated DSIP binding to or modulating GABA-A receptors — the proposed target is entirely inferential. (2) The Graf & Kastin reviews note that DSIP may act via adrenergic rather than GABAergic transmission, suggesting the primary mechanism may be at a different receptor system entirely. (3) Kovalzon & Strekalova (2006) explicitly state that 'the hypothesis regarding DSIP as a sleep factor is extremely poorly documented and still weak,' undermining confidence that optimizing DSIP's conformation will produce a potent sleep agent via any mechanism. (4) Mu et al. (2024) found DSIP-CBBBP modulates monoaminergic neurotransmitters (5-HT, dopamine) and melatonin — not specifically GABA — suggesting the primary pharmacological target may be serotonergic or melatoninergic. (5) The U-shaped dose-response curve for DSIP noted in the 1984 review is atypical for receptor agonists/modulators and may indicate complex indirect actions incompatible with a simple receptor-engagement model. (6) The lack of an identified endogenous DSIP gene or biosynthetic precursor raises the possibility that circulating DSIP-like immunoreactivity represents a distinct endogenous peptide rather than DSIP itself, complicating target deconvolution for any analogue series."},"caveats":null,"works_cited":[{"pmid_or_doi":"16539679","title":"Delta sleep-inducing peptide (DSIP): a still unresolved riddle.","year":2006,"relevance":"Directly addresses DSIP mechanism of action and notes that structural analogues — not native DSIP — show the strongest sleep-promoting activity, supporting the premise that conformational modification may improve bioactivity."},{"pmid_or_doi":"6145137","title":"Delta-sleep-inducing peptide (DSIP): a review.","year":1984,"relevance":"Foundational characterization of DSIP as a nonapeptide with broad CNS effects; establishes baseline pharmacology and notes dose-response (U-shaped curve) relevant to understanding receptor engagement."},{"pmid_or_doi":"3550726","title":"Delta-sleep-inducing peptide (DSIP): an update.","year":1986,"relevance":"Updates DSIP pharmacology and discusses possible mechanism involving adrenergic transmission modulation; highlights continued failure to identify a specific receptor, the central gap our hypothesis addresses."},{"pmid_or_doi":"3286557","title":"DSIP--a tool for investigating the sleep onset mechanism: a review.","year":1988,"relevance":"Documents multisystem physiological effects of DSIP and circadian dependence; supports broad CNS neuromodulatory activity without receptor specificity, contextualizing the difficulty of the proposed GABA-A targeting hypothesis."},{"pmid_or_doi":"6391925","title":"DSIP in insomnia.","year":1984,"relevance":"Clinical evidence that DSIP improves sleep at defined doses; the sedative-hypnotic profile is consistent with GABAergic mechanism but provides no direct receptor binding evidence."},{"pmid_or_doi":"39444618","title":"Pichia pastoris-secreted DSIP-CBBBP fusion peptide sleep-promoting effects (inferred title from abstract).","year":2024,"relevance":"Most recent empirical study showing DSIP modulates neurotransmitters (5-HT, glutamate, DA, melatonin) in insomnia mice; GABAergic pathways not specifically measured, but neurotransmitter modulation profile is relevant to CNS receptor targeting rationale."},{"pmid_or_doi":"41490200","title":"Therapeutic Peptides in Orthopaedics: Applications, Challenges, and Future Directions.","year":2026,"relevance":"Categorizes DSIP as a circadian/mitochondrial regulator in a therapeutic context; weak mechanistic content but reflects current clinical perception of DSIP as a neuromodulatory recovery agent."},{"pmid_or_doi":"10.21203/rs.3.rs-3138792/v1","title":"Protecting Group-Minimum, Practical N-to-C Peptide Synthesis.","year":2023,"relevance":"Demonstrates practical synthesis of DSIP using a novel coupling strategy, confirming synthetic accessibility of DSIP analogues including backbone-modified versions relevant to the proposed D-Ala substitution."}]},"onchain":{"hash":null,"signature":null,"data_hash":null,"logged_at":null,"explorer_url":null},"ipfs_hash":null,"created_at":"2026-05-04T08:44:45.145860+00:00","updated_at":"2026-05-04T08:46:17.259420+00:00"}