Biology subjects

Miclot, T.

Publications and source records attributed to Miclot, T..

12 recordsLinked to original sources

bioRxiv2026

Targeting the Myeloid Immune Checkpoint ILT3 (LILRB4) with Small Molecules Enables Reprogramming of Suppressive Tumor Immunity

Cancer immunotherapy has transformed cancer treatment; however, durable responses remain limited by suppressive myeloid populations within the tumor microenvironment. Leukocyte immunoglobulin-like receptor B4 (LILRB4/ILT3) is an emerging myeloid immune checkpoint implicated in immune evasion and resistance to immunotherapy, yet small molecule targeting of ILT3 remains largely unexplored. Here, we report the discovery of small molecule ILT3 modulators identified using a Dianthus-based temperature-related intensity change (TRIC) screening platform. Screening of an 8,961-member Enamine Library identified multiple direct ILT3 binders, with lead compound ICB-7 demonstrating high-affinity binding to recombinant human ILT3 by microscale thermophoresis and robust cellular target engagement in CETSA assays. Molecular docking and molecular dynamics simulations revealed a stable hydrophobic binding pocket within the D2 domain of ILT3. Functionally, ICB-7 disrupted the ILT3-SCG2 interaction and inhibited downstream SHP1, SHP2, and STAT3 signaling. In patient-derived colorectal cancer and acute myeloid leukemia co-culture models, ICB-7 enhanced cytotoxic T-cell activity, and reduced tumor-cell viability. The compound also demonstrated favorable pharmacokinetic and safety properties together with significant anti-tumor efficacy in the CT26 syngeneic colorectal carcinoma model. Collectively, these findings establish ILT3 as a tractable target for small-molecule immunomodulation and support pharmacological targeting of suppressive myeloid checkpoints as a promising cancer immunotherapy strategy.

pharmacology and toxicology↗

bioRxiv2025

Beyond contacts: the important role of the support region to distinguish stable and transient protein interfaces

Protein-protein interactions (PPIs) are fundamental to virtually all cellular processes; however, elucidating the principles that govern protein association into complexes remains a significant challenge. In this study, we present a comparative analysis of stable and transient protein interfaces, offering a detailed perspective on the interactions that form them. Moving beyond the traditional focus on pairs of neighboring residues, we examine interacting pairs by identifying their distinct non-bonded interactions. Additionally, we account for the contextual dependence of these interactions by analyzing the regions of the interface where they occur. Our approach quantifies the diversity of pairs in each region, considering the type of interface. Furthermore, we introduce an innovative strategy to analyze pair co-occurrence, enabling a comparison of the inner local organization of stable and transient interfaces. Our findings reveal that stable and transient interfaces differ not in overall residue composition, but in the residue- and interaction-partitioning patterns across the variably hydrated regions of the interface. These results underscore the importance of considering the contextual environment in which pairs interact and identify the support region as a key determinant for distinguishing transient and stable protein complexes. The software package developed for this analysis is available as open source.

bioinformatics↗

bioRxiv2023

Mechanism of the Covalent Inhibition of Human Transmembrane Protease Serine 2 as an Original Antiviral Strategy

The Transmembrane Protease Serine 2 (TMPRSS2) is a human enzyme which is involved in the maturation and post-translation of different proteins. In addition of being overexpressed in cancer cells, TMPRSS2 plays a further fundamental role in favoring viral infections by allowing the fusion of the virus envelope and the cellular membrane, notably in SARS-CoV-2. In this contribution we resort to multiscale molecular modeling to unravel the structural and dynamical features of TMPRSS2 and its interaction with a model lipid bilayer. Furthermore, we shed light into the mechanism of action of a potential inhibitor (Nafamostat), determining the free-energy profile associated with the inhibition reaction, and showing the facile poisoning of the enzyme. Our study, while providing the first atomistically resolved mechanism of TMPRSS2 inhibition, is also fundamental in furnishing a solid framework for further rational design targeting transmembrane proteases in a host-directed antiviral strategy. TOC GRAPHICS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/537985v1_ufig1.gif" ALT="Figure 1"> View larger version (108K): org.highwire.dtl.DTLVardef@19dd8c9org.highwire.dtl.DTLVardef@394c0org.highwire.dtl.DTLVardef@11ad383org.highwire.dtl.DTLVardef@347a8c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Resolving a Guanine-Quadruplex Structure in the SARS-CoV-2 Genome through Circular Dichroism and Multiscale Molecular Modeling.

The genome of SARS-CoV-2 coronavirus is made up of a single-stranded RNA fragment that can assume a specific secondary structure, whose stability can influence the virus ability to reproduce. Recent studies have identified putative guanine quadruplex sequences in SARS-CoV-2 genome fragments that are involved in coding for both structural and non-structural proteins. In this contribution, we focus on a specific G-rich sequence referred as RG-2, which codes for the non-structural protein 10 (Nsp10) and assumes a guanine-quadruplex (G4) arrangement. We provide the secondary structure of the RG-2 G4 at atomistic resolution by molecular modeling and simulation, validated by the superposition of experimental and calculated electronic circular dichroism spectrum. Through both experimental and simulation approaches, we have demonstrated that pyridostatin (PDS), a widely recognized G4 binder, can bind to and stabilize RG-2 G4 more strongly than RG-1, another G4 forming sequence that was previously proposed as a potential target for antiviral drug candidates. Overall, this study highlights RG-2 as a valuable target to inhibit the translation and replication of SARS-CoV-2 paving the way towards original therapeutic approaches against emerging RNA viruses.

biophysics↗

bioRxiv2023

Predicting the Three-dimensional Structure of the c-KIT Oncogene Promoter and the Dynamics of Strongly Coupled Guanine-Quadruplexes

Guanine-quadruplexes (G4s) are non-canonical DNA structures that play important protective and regulatory roles within cells, influencing, for instance, gene expression. Although the secondary structure of many human G4s is well characterized, in several gene-promoter regions multiple G4s are located in close proximity and may form three-dimensional structures which could ultimately influence their biological roles. In this contribution, we analyze the interplay between the three neighboring G4s present in the c-KIT proto-oncogene promoter, namely WK1, WSP and WK2. In particular, we highlight how these three G4s are structurally linked and how their crosstalk favors the formation of a parallel structure for WSP, differently from what observed for this isolated G4 in solution. Relying on all-atom molecular dynamic simulations exceeding the s time-scale and using enhanced sampling methods, we provide the first computationally-resolved structure of a well-organized G4 cluster in the promoter of a crucial gene involved in cancer development. Our results indicate that neighboring G4s influence their mutual three-dimensional arrangement and provide a powerful tool to predict and interpret complex DNA structures that ultimately can be used as starting point for drug discovery purposes.

biophysics↗

bioRxiv2022

G-quadruplex recognition by DARPIns through epitope/paratope analogy

We investigated the mechanisms leading to the specific recognition of Guanine Guadruplex (G4) by DARPins peptides, which can lead to the design of G4s specific sensors. To this end we carried out all-atom molecular dynamic simulations to unravel the interactions between specific nucleic acids, including human-telomeric (h-telo), Bcl-2, and c-Myc, with different peptides, forming a DARPin/G4 complex. By comparing the sequences of DARPin with that of a peptide known for its high affinity for c-Myc, we show that the recognition cannot be ascribed to sequence similarity but, instead, depends on the complementarity between the three-dimensional arrangement of the molecular fragments involved: the -helix/loops domain of DARPin and the G4 backbone. Our results reveal that DARPins tertiary structure presents a charged hollow region in which G4 can be hosted, thus the more complementary the structural shapes, the more stable the interaction.

biophysics↗

bioRxiv2021

Structure of the 5' untranslated region in SARS-CoV-2 genome and its specific recognition by innate immune system via the human oligoadenylate synthase 1

2-5-Oligoadenylate synthetase 1 (OAS1) is one of the key enzymes driving the innate immune system response to SARS-CoV-2 infection whose activity has been related to COVID-19 severity. In particular, OAS1 is a sensor of endogenous RNA that triggers the 2-5 oligoadenylate/RNase L pathway in response to viral infections, ultimately activating the RNA-Lyase which cleaves endogenous and exogenous RNA impeding the viral maturation. Upon SARS-CoV-2 infection, OAS1 is responsible for the recognition of viral RNA and has been shown to possess a particularly high sensitivity for the 5-untranslated (5-UTR) RNA region, which is organized in a double-strand stem loop motif (SLA). Since the structure of the nucleic acid/protein complex has not been resolved, here we report its structure obtained by molecular modeling, including enhanced sampling approaches. We also pinpoint that the SL1 region enhances the interaction network with the enzyme, promoting specific hydrogen bonds, absent in normal double strand RNA fragments, hence rationalizing the high affinity for OAS1.

biophysics↗

bioRxiv2021

Never cared for what they do. High structural stability of Guanine-quadruplexes in presence of strand-break damages

DNA integrity is an important factor to assure genome stability and, more generally, cells and organisms viability. In presence of DNA damage, the normal cell cycle is perturbed while cells activate their repair processes. Although efficient, the repair system is not always able to ensure the complete restoration of gene integrity. In these cases, not only mutations may occur, but the accumulation of lesions can either lead to carcinogenesis or reach a threshold which induces apoptosis and the programmed cell death. Among the different types of DNA lesions, strand breaks produced by ionizing radiations are the most toxic, due to their inherently difficult repair, which may lead to genomic instability. In this article we show, by using classical molecular simulations techniques, that differently from the canonical double-helical B-DNA, guanine-quadruplex (G4) arrangements show a remarkable structural stability, even in presence of two strand breaks. Since G4-DNA are recognized for their regulatory roles in cell senescence and gene expression, also involving oncogene, their stability can be related to an evolutionary cellular response aimed at minimizing the effects of ionizing radiation.

biophysics↗

bioRxiv2021

How fragile we are. Influence of STimulator of INterferon Genes, STING, variants on pathogen recognition and immune response efficiency

The STimulator of INterferon Genes (STING) protein is a cornerstone of the human immune response. Its activation by cGAMP upon the presence of cytosolic DNA stimulates the production of type I interferons and inflammatory cytokines which are crucial for protecting cells from infections. STING signaling pathway can also influence both tumor-suppressive and tumor-promoting mechanisms, rendering it an appealing target for drug design. In the human population, several STING variants exist and exhibit dramatic differences in their activity, impacting the efficiency of the host defense against infections. Understanding the differential molecular mechanisms exhibited by these variants is of utmost importance notably towards personalized medicine treatments against diseases such as viral infections (COVID-19, Dengue...), cancers, or auto-inflammatory diseases. Owing to micro-seconds scale molecular modeling simulations and post-processing by contacts analysis and Machine Learning techniques, we reveal the dynamical behavior of four STING variants (wild type, G230A, R293Q, and G230A-R293Q) and we rationalize the variability of efficiency observed experimentally. Our results show that the decrease of STING activity is linked to a stiffening of key-structural features of the binding cavity, together with changes of the interaction patterns within the protein.

biochemistry↗

bioRxiv2021

Structure and dynamics of RNA guanine quadruplexes in SARS-CoV-2 genome. Original strategies against emerging viruses

Guanine quadruplexes (G4) structures in viral genome have a key role in modulating viruses biological activity. While several DNA G4 structures have been experimentally resolved, RNA G4s are definitely less explored. We report the first calculated G4 structure of the RG-1 RNA sequence of SARS-CoV-2 genome, obtained by using a multiscale approach combining quantum and classical molecular modelling and corroborated by the excellent agreement between the corresponding calculated and experimental circular dichroism spectra. We prove the stability of RG-1 G4 arrangement as well as its interaction with G4 ligands potentially inhibiting viral protein translation.

biophysics↗

bioRxiv2020

Forever Young: Structural Stability of Telomeric Guanine-Quadruplexes in Presence of Oxidative DNA Lesions.

Human telomeric DNA (h-Telo), in G-quadruplex (G4) conformation, is characterized by a remarkable structural stability that confers it the capacity to resist to oxidative stress producing one or even clustered 8-oxoguanine lesions. We present a combined experimental/computational investigation, by using circular dichroism in aqueous solutions, cellular immunofluorescence assays and molecular dynamics (MD) simulations, that identifies the crucial role of the stability of G4s to oxidative lesions, related also to their biological role as inhibitors of telomerase, an enzyme overexpressed in most cancers associated to oxidative stress.

biophysics↗

bioRxiv2020

Role of RNA Guanine Quadruplexes in Favoring the Dimerization of SARS Unique Domain in Coronaviruses

Coronaviruses may produce severe acute respiratory syndrome (SARS). As a matter of fact, a new SARS-type virus, SARS-CoV-2, is responsible of a global pandemic in 2020 with unprecedented sanitary and economic consequences for most countries. In the present contribution we study, by all-atom equilibrium and enhanced sampling molecular dynamics simulations, the interaction between the SARS Unique Domain and RNA guanine quadruplexes, a process involved in eluding the defensive response of the host thus favoring viral infection of human cells. Our results evidence two stable binding modes involving an interaction site spanning either the protein dimer interface or only one monomer. The free energy profile unequivocally points to the dimer mode as the thermodynamically favored one. The effect of these binding modes in stabilizing the protein dimer was also assessed, being related to its biological role in assisting SARS viruses to bypass the host protective response. This work also constitutes a first step of the possible rational design of efficient therapeutic agents aiming at perturbing the interaction between SARS Unique Domain and guanine quadruplexes, hence enhancing the host defenses against the virus. TOC GRAPHICS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/029447v2_ufig1.gif" ALT="Figure 1"> View larger version (99K): org.highwire.dtl.DTLVardef@1497e69org.highwire.dtl.DTLVardef@a4c44org.highwire.dtl.DTLVardef@1511b7org.highwire.dtl.DTLVardef@13e12c1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗