Biology subjects

Martin, M. P.

Publications and source records attributed to Martin, M. P..

6 recordsLinked to original sources

bioRxiv2026

FragLite mapping to identify the BRD4 recruitment site of P-TEFb

The eukaryotic positive transcription elongation factor b (P-TEFb), composed of CDK9 and cyclin T, plays a central role in regulating RNA polymerase II (RNAPII). Phosphorylation of the RNAPII C-terminal domain (CTD) by P-TEFb promotes promoter proximal pause release and enables productive transcriptional elongation across many genes. Cyclin T mediates protein-protein interactions, several of which have been structurally characterised, that help to recruit and fine-tune P-TEFb activity to ensure a tight regulation of transcription. We have previously reported a set of halogenated chemical fragments termed FragLites that can prospectively identify protein interaction sites. Here, we report the FragLite map of cyclin T2, revealing binding sites corresponding to structurally defined cyclin T partners CDK9, AFF4, and HIV-1 Tat. Furthermore, we demonstrate the utility of FragLites in identifying a previously uncharacterised BRD4 binding site. By integrating FragLite clustering with biophysical analyses and AlphaFold3 modelling, we delineate the cyclin T-BRD4 interface. These analyses provide a comprehensive, chemically enriched fragment map highlighting functionally relevant sites to support future probe and modulator development to selectively target P-TEFb.

biophysics↗

bioRxiv2025

Structural, biophysical and biological analysis and characterisation of IRF4 DNA-binding domain mutations associated with multiple myeloma

IRF4, a transcription factor in the interferon regulatory factor family, is a key regulator in immune cell differentiation indicated to have an essential role in the development of lymphoid malignancies. Genome-wide association studies previously identified a set of overlapping mutations within the IRF4 DNA-binding domain in T-cell lymphoma and multiple myeloma, several of which appeared to be associated with better prognosis. Mapping these mutations to the known crystal structure of the IRF4:PU.1:DNA ternary complex and a new structure of the IRF4 DNA-binding domain in the apo state suggested they might interfere with DNA-binding, directly or via destabilisation of domain structure. We characterised these cancer-associated IRF4 mutants experimentally using the recombinant IRF4 DNA-binding domain (DBD) in vitro and examined the clinically relevant mutant K123R in cellulo. Using fluorescence polarisation, surface plasmon resonance, differential scanning fluorimetry and molecular dynamics, we find that mutation may give rise to significant differences in DNA-binding kinetics and thermal stability without compromising the affinity of IRF4 DNA-binding. The K123R IRF4 mutant showed increased transcriptional activity via a luciferase reporter assay and increased nuclear partitioning, which may be preferentially selected for in multiple myeloma. We discuss our observations in relation to the improved prognosis conferred by this mutation.

biophysics↗

bioRxiv2024

Crystallographic fragment screening of CDK2-cyclin A: FragLites map sites of protein-protein interaction

Protein-protein interaction sites (PPIs) are potentially more selective therapeutic binding sites than protein substrate binding sites. PPIs include distinct regions frequently called "hotspots," sites of key amino acid interactions. Prospective identification of these hotspots through X-ray crystallographic screening could assist in the identification of separation of function mutants for experimental validation, enhance confidence in AI-generated multiprotein complex predictions and accelerate development of selective chemical probes. To explore these applications, we utilize the FragLite library to examine the binding surfaces of CDK2-cyclin A. The many protein- and peptide-CDK2-cyclin A complexes that have been structurally characterised make this complex an appropriate test case. We show that FragLites comprehensively map both known sites of protein-protein interaction on CDK2-cyclin A and identify a possible uncharacterised site, providing a structural method toward directing mechanistic studies and providing starting points for chemical probe design.

biophysics↗

bioRxiv2023

A Comparison of Quantitative Mass Spectrometric Methods for Drug Target Identification by Thermal Proteome Profiling

Thermal proteome profiling (TPP) provides a powerful approach to studying proteome-wide interactions of small therapeutic molecules and their target and off-target proteins, complementing phenotypic-based drug screens. Detecting differences in thermal stability due to target engagement requires high quantitative accuracy and consistent detection. Isobaric tandem mass tags (TMT) are used to multiplex samples and increase quantification precision in TPP analysis by data-dependent acquisition (DDA). However, advances in data-independent acquisition (DIA) can provide higher sensitivity and protein coverage with reduced costs and sample preparation steps. Herein, we explored the performance of different DIA-based label-free quantification (LFQ) approaches compared to TMT-DDA for thermal shift quantitation. Acute myeloid leukaemia (AML) cells were treated with losmapimod, a known inhibitor of MAPK14 (p38). Label-free DIA approaches, and particularly the library-free mode in DIA-NN, were comparable or better than TMT-DDA in their ability to reproducibly detect target engagement of losmapimod with MAPK14 and one of its downstream targets, MAPKAPK3. Using DIA for thermal shift quantitation is a cost-effective alternative to labelled quantitation in the TPP pipeline.

biochemistry↗

bioRxiv2020

Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease

COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments was progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.

biophysics↗

bioRxiv2020

senSCOPE: Modeling radiative transfer and biochemical processes in mixed canopies combining green and senescent leaves with SCOPE

Semi-arid grasslands and other ecosystems combine green and senescent leaves featuring different biochemical and optical properties, as well as functional traits. Knowing how these properties vary is necessary to understand the functioning of these ecosystems. However, differences between green and senescent leaves are not considered in recent models representing radiative transfer, heat, water and CO2 exchange such as the Soil-Canopy Observation of Photosynthesis and Energy fluxes (SCOPE). Neglecting the contribution of senescent leaves to the optical and thermal signal of vegetation limits the possibilities to use remote sensing information for studying these ecosystems; as well as neglecting their lack of photosynthetic activity increases uncertainty in the representation of ecosystem fluxes. In this manuscript we present senSCOPE as a step towards a more realistic representation of mixed green and senescent canopies. senSCOPE is a modified version of SCOPE model that describes a canopy combining green and senescent leaves with different properties and function. The model relies on the same numerical solutions than SCOPE, but exploits the linear nature of the scattering coefficients to combine optical properties of both types of leaf. Photosynthesis and transpiration only take place in green leaves; and different green and senescent leaf temperatures are used to close the energy balance. Radiative transfer of sun-induced fluorescence (SIF) and absorptance changes induced by the xanthophyll cycle action are also simulated. senSCOPE is evaluated against SCOPE both using synthetic simulations, forward simulations based on observations in a Mediterranean tree-grass ecosystem, and inverting dataset of ground measurements of reflectance factors, SIF, thermal radiance and gross primary production on a heterogeneous and partly senescent Mediterranean grassland. Results show that senSCOPE outputs vary quite linearly with the fraction of green leaf area, whereas SCOPE does not respond linearly to the effective leaf properties, calculated as the weighted average of green and senescent leaf parameters. Inversion results and pattern-oriented model evaluation show that senSCOPE improves the estimation of some parameters, especially chlorophyll content, with respect SCOPE retrievals during the dry season. Nonetheless, inaccurate knowledge of the optical properties of senescent matter still complicates model inversion. senSCOPE brings new opportunities for the monitoring of canopies mixing green and senescent leaves, and for improving the characterization of the optical properties of senescent material.

ecology↗