Biology subjects

Roche, P.

Publications and source records attributed to Roche, P..

7 recordsLinked to original sources

bioRxiv2025

Engineering SIRPα conformational plasticity to reveal a cryptic pocket suitable for structure-based drug design

The protein-protein interaction between Signal Regulatory Protein alpha (SIRP) and CD47 is a critical immune checkpoint that enables tumor immune escape, making it a key target for cancer immunotherapy. While antibody-based therapies exist, the development of small-molecule inhibitors has been hindered by the flat, featureless binding interface. Here, we report the discovery of a novel, druggable cryptic pocket within the SIRP D1 domain (the WYF pocket), revealed through a structure-based fragment screening campaign using x-ray crystallography. This pocket, defined by residues Trp38, Tyr50, and Phe74, is only accessible in a conformation that is incompatible with CD47 binding, making it a candidate for structure-based drug design and immune checkpoint inhibitor development. Through a combination of NMR spectroscopy, molecular dynamics simulations, and biophysical assays, we demonstrate that access to this cryptic site is dynamically controlled by a single "gatekeeper" residue, Gln52. The rotameric state of Gln52 dictates a conformational equilibrium between a "closed," state and a ligand-accessible "open" state. We validated this mechanism by engineering SIRP mutants to bias this equilibrium. A Q52F mutation locked the pocket in a closed state, abolishing both CD47 and fragment binding, while Q52A and Q52R mutations biased the protein toward an open state. These "open-biased" mutants not only exhibited decreased affinity for CD47 but also significantly improved binding to small-molecule fragments that inhibit the SIRP-CD47 interaction. This work reveals the intrinsic conformational plasticity of SIRP and establishes a validated structure-based roadmap for a new class of allosteric inhibitors. This flexibility-for-inhibition strategy functions by trapping a non-binding conformation and represents a broadly applicable framework for targeting this and other challenging immune checkpoints.

biochemistry↗

bioRxiv2024

A new nanoDSF approach to anti-tubulin compounds screening revealed novel MTAs among approved drugs.

Microtubule Targeting Agents (MTAs) constitute a vital category of tubulin-binding compounds, deployed across anticancer therapies. Despite the array of MTA drugs developed by pharmaceutical entities, the quest for novel efficacious molecules continues unabated. We unveil an innovative in vitro MTA screening methodology employing nano differential scanning fluorimetry (nanoDSF), presenting distinct advantages over known assays. This novel approach not only assesses compound-tubulin binding but also quantitatively analyzes their impact on tubulin polymerization. Proposed nanoDSF assay was rigorously validated using the Prestwick Chemical Library, which encompasses 1,520 approved compounds, successfully identifying all previously known MTAs. Furthermore, this screening has unearthed potential anti-tubulin agents among drugs currently utilized for non-related medical conditions, offering insights into their mechanisms of action in inhibiting cancer cell proliferation and/or inducing cytotoxicity. These discoveries herald new opportunities for drug repositioning involving the newly identified MTAs and substantially streamline the process of screening extensive chemical libraries for MTAs featuring novel chemical structures.

cancer biology↗

bioRxiv2021

CRCM5484: A BET- BDII Selective Compound With Differential Anti-Leukemic Drug Potentiation

Differentially screening the Fr-PPIChem chemical library on the BET BRD4-BDII versus -BDI bromodomains led to the discovery of a BDII selective tetrahydropyridothienopyrimidinone (THPTP)-based compound. Structure-activity relationship (SAR) and hit-to-lead approaches allowed us to develop CRCM5484, a potent inhibitor of BET proteins with a preferential and 475-fold selectivity for the second bromodomain of the BRD3 protein (BRD3-BDII) over its first bromodomain (BRD3-BDI). Its very low activity was demonstrated in various cell-based assays, corresponding with recent data describing other selective BDII compounds. However, screening on a drug sensitivity and resistance-profiling platform revealed its ability to modulate the anti-leukemic activity in combination with various FDA-approved and/or in-development drugs in a cell- and context-dependent differential manner. Altogether, the results confirm the originality of the THPTP molecular mode of action in the BD cavity and its potential as starting scaffold for the development of potent and selective bromodomain inhibitors.

cancer biology↗

bioRxiv2021

Discovery of small molecule inhibitors of the PTK7/β-catenin interaction targeting the Wnt signaling pathway in colorectal cancer

Second cause of death due to cancer worldwide, colorectal cancer (CRC) is a major public health issue. The discovery of new therapeutic targets is thus essential. The pseudokinase PTK7 intervenes in the regulation of the Wnt/{beta}-catenin pathway signaling, in part, through a kinase-domain dependent interaction with the {beta}-catenin protein. PTK7 is overexpressed in CRC; an event associated with metastatic development and reduced survival of non-metastatic patient. In addition, numerous alterations have been identified in CRC inducing constitutive activation of Wnt/{beta}-catenin pathway signaling through {beta}-catenin accumulation. Thus, we thought that targeting PTK7/{beta}-catenin interaction could be of interest for future drug development. In this study, we have developed a NanoBRET screening assay recapitulating the interaction between PTK7 and {beta}-catenin to identify compounds able to disrupt this protein-protein interaction. A high-throughput screening allowed us to identify small molecule inhibitors targeting the Wnt pathway signaling and inducing anti-proliferative effect in vitro in CRC cells harboring {beta}-catenin or APC mutations downstream of PTK7. Thus, inhibition of the PTK7/{beta}-catenin interaction could represent, in the future, a new therapeutic strategy to inhibit cell growth dependent on Wnt signaling pathway. Moreover, despite a lack of enzymatic activity of its tyrosine kinase domain, targeting the PTK7 kinase domain-dependent functions appears to be of interest for further therapeutic development.

pharmacology and toxicology↗

bioRxiv2021

Systemic inflammation recruits fast-acting anti-inflammatory innate myeloid progenitors from BM into lymphatics

Innate immune cellular effectors are actively consumed during systemic inflammation but the systemic traffic and the mechanisms that support their replenishment remain unknown. Here we demonstrate that acute systemic inflammation induces the emergent activation of a previously unrecognized system of rapid migration of granulocyte-macrophage progenitors and committed macrophage-dendritic progenitors, but not other progenitors or stem cells, from bone marrow (BM) to lymphatic capillaries. The progenitor traffic to the systemic lymphatic circulation is mediated by Ccl19/Ccr7 and is NF{kappa}B independent, Traf6/I{kappa}B-kinase/SNAP23 activation which is responsible for the secretion of pre-stored Ccl19 by a subpopulation of CD205+/CD172a+ conventional dendritic cells type 2 (cDC2) and upregulation of BM myeloid progenitor Ccr7 signaling. The consequence of this progenitor traffic is anti-inflammatory with promotion of early survival and initiation of replenishment of lymph node cDC.

immunology↗

bioRxiv2018

Receptor Mediated Delivery of Cas9-Nanobody Induces Cisplatin Synthetic Dose Sensitivity

The CRISPR/Cas9 system has shown great potential for precisely editing genomic DNA sequences by introducing site-specific DNA cuts that are subsequently repaired by the cell. However, delivery of the CRISPR ribonucleoprotein remains an understudied area and hinders realizing the full potential of the system. We prepared Cas9 ribonucleoprotein complexes chemically conjugated to the 7D12 nanobody and demonstrate receptor-mediated transfection of Cas9 into A549 non-small-cell lung cancer cells via binding to the epithelial growth factor receptor for subsequent cell internalization. We further show that transfection with a Cas9 ribonucleoprotein targeting the BRCA2 gene results in an enhanced sensitivity to the chemotherapeutic drug Cisplatin, and thereby induces a synthetic dose lethality in A549 cells.

biochemistry↗

bioRxiv2017

A divergent CheW stabilizes nucleoid-associated chemosensory arrays

Chemosensory systems are highly organized signaling pathways that allow bacteria to adapt to environmental changes. The Frz chemosensory system from M. xanthus possesses two CheW-like proteins, FrzA (the core CheW) and FrzB. We found that FrzB does not interact with FrzE (the cognate CheA) as it lacks the amino acid region responsible for this interaction. FrzB, instead, acts upstream of FrzCD in the regulation of M. xanthus chemotaxis behaviors and activates the Frz pathway by allowing the formation and distribution of multiple chemosensory clusters on the nucleoid. These results, together, show that the lack of the CheA-interacting region in FrzB confers new functions to this small protein.\n\nAUTHOR SUMMARYChemosensory systems are signaling complexes that are widespread in bacteria and allow the modulation of different cellular functions, such as taxis and development, in response to the environment. We show that the Myxococcus xanthus FrzB is a divergent CheW lacking the region involved in the interaction with the histidine kinase FrzE. Instead, it acts upstream of FrzCD to allow the formation of multiple distributed Frz chemosensory arrays at the nucleoid. The loss of the CheA-interacting region in FrzB might have been selected to confer plasticity to nucleoid-associated chemosensory systems. By unraveling a new accessory protein and its function, this work opens new insights into the knowledge of the regulatory potentials of bacterial chemosensory systems.

microbiology↗