Biology subjects

Marto, J. A.

Publications and source records attributed to Marto, J. A..

14 recordsLinked to original sources

bioRxiv2025

Overcoming EGFR resistance by monovalent and bident inhibitors targeting Cys775

Covalent targeting of EGFR cysteine 797 by osimertinib is one of the most successful breakthroughs in targeted therapy, fundamentally transforming the treatment landscape for non-small cell lung cancer (NSCLC) patients. However, resistance driven by mutation of C797 remains a major clinical challenge. Developing novel covalent strategies beyond C797 targeting presents a compelling opportunity for next-generation EGFR inhibitors. We first demonstrated that cysteine 775, located deep within the ATP-binding pocket, is accessible by a rationally designed covalent molecule ZNL-3, which as the first-in-class covalent cysteine 775 inhibitor exhibited strong efficacy in osimertinib-resistant mouse models. To further enhance resilience to resistance-causing mutations, we developed a dual-warhead, bident compound--YNW-1--which covalently targets both cysteine 775 and 797 simultaneously. YNW-1 is the first intramolecular lock to exhibit balanced reactive efficiency on both cysteines, rendering single-site mutations ineffective to confer resistance. The discovery of ZNL-3 and YNW-1 represents significant advancements in EGFR-targeted drug development, and further optimization toward clinical translation is a worthwhile strategy. SIGNIFICANCE: This study establishes the therapeutic potential of an EGFR covalent inhibitor through unprecedented targeting of cysteine 775 and provides the first demonstration that dual cysteine engagement offers superior efficacy over conventional covalent inhibitors by delaying resistance.

cancer biology↗

bioRxiv2024

Leveraging HILIC/ERLIC Separations for Online Nanoscale LC- MS/MS Analysis of Phosphopeptide Isoforms from RNA Polymerase II C-terminal Domain

The eukaryotic RNA polymerase II (Pol II) multi-protein complex transcribes mRNA and coordinates several steps of co-transcriptional mRNA processing and chromatin modification. The largest Pol II subunit, Rpb1, has a C-terminal domain (CTD) comprising dozens of repeated heptad sequences (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), each containing five phospho-accepting amino acids. The CTD heptads are dynamically phosphorylated, creating specific patterns correlated with steps of transcription initiation, elongation, and termination. This CTD phosphorylation code choreographs dynamic recruitment of important co-regulatory proteins during gene transcription. Genetic tools were used to engineer protease cleavage sites across the CTD (msCTD), creating tryptic peptides with unique sequences amenable to mass spectrometry analysis. However, phosphorylation isoforms within each msCTD sequence are difficult to resolve by standard reversed phase chromatography typically used for LC-MS/MS applications. Here, we use a panel of synthetic CTD phosphopeptides to explore the potential of hydrophilic interaction and electrostatic repulsion hydrophilic interaction (HILIC and ERLIC) chromatography as alternatives to reversed phase separation for CTD phosphopeptide analysis. Our results demonstrate that ERLIC provides improved performance for separation of singly- and doubly-phosphorylated CTD peptides for sequence analysis by LC-MS/MS. Analysis of native yeast msCTD confirms that phosphorylation on Ser5 and Ser2 represents the major endogenous phosphoisoforms. We expect this methodology will be especially useful in the investigation of pathways where multiple protein phosphorylation events converge in close proximity.

biochemistry↗

bioRxiv2023

Template-assisted covalent modification of DCAF16 underlies activity of BRD4 molecular glue degraders

Small molecules that induce protein-protein interactions to exert proximity-driven pharmacology such as targeted protein degradation are a powerful class of therapeutics1-3. Molecular glues are of particular interest given their favorable size and chemical properties and represent the only clinically approved degrader drugs4-6. The discovery and development of molecular glues for novel targets, however, remains challenging. Covalent strategies could in principle facilitate molecular glue discovery by stabilizing the neo-protein interfaces. Here, we present structural and mechanistic studies that define a trans-labeling covalent molecular glue mechanism, which we term "template-assisted covalent modification". We found that a novel series of BRD4 molecular glue degraders act by recruiting the CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). BRD4BD2, in complex with DCAF16, serves as a structural template to facilitate covalent modification of DCAF16, which stabilizes the BRD4-degrader-DCAF16 ternary complex formation and facilitates BRD4 degradation. A 2.2 [A] cryo-electron microscopy structure of the ternary complex demonstrates that DCAF16 and BRD4BD2 have pre-existing structural complementarity which optimally orients the reactive moiety of the degrader for DCAF16Cys58 covalent modification. Systematic mutagenesis of both DCAF16 and BRD4BD2 revealed that the loop conformation around BRD4His437, rather than specific side chains, is critical for stable interaction with DCAF16 and BD2 selectivity. Together our work establishes "template-assisted covalent modification" as a mechanism for covalent molecular glues, which opens a new path to proximity driven pharmacology.

biochemistry↗

bioRxiv2022

Covalent Disruptor of YAP-TEAD Association Suppresses Defective Hippo Signaling

The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03-69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03-69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03-69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03-69 led to an in vivo compatible compound MYF-03-176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration.

cancer biology↗

bioRxiv2021

A non-canonical convergence of carbohydrate and glutamine metabolism is required after metabolic rewiring in 3D environment

The fate of pyruvate, which is modulated by the activity of the mitochondrial pyruvate carrier (MPC), is a defining metabolic feature in many cancers. Diffuse large B-cell lymphomas (DLBCLs) are a genetically and metabolically heterogeneous cancer. Although MPC expression and activity differed between DLBCL subgroups, mitochondrial pyruvate was uniformly consumed by glutamate pyruvate transaminase 2 (GPT2) to support -ketoglutarate production as part of glutaminolysis. This led us to discover that glutamine exceeds pyruvate as a carbon source for the tricarboxylic acid (TCA) cycle in DLBCLs. Furthermore, we found that MPC inhibition unexpectedly leads to decreased glutaminolysis, which is contrary to previous observations in other cell types. We also discovered that MPC inhibition and depletion only decreased DLBCL proliferation in an extracellular matrix (ECM) environment and in vivo xenografts, but not in the typical DLBCL suspension environment. We also have found that the metabolic profile of DLBCL cells in ECM is markedly different from cells in suspension environment. Thus, we report that besides the canonical glutamate dehydrogenase (GDH)-mediated glutaminolysis, the non-canonical GPT2 mediated consumption and assimilation of glutamine and pyruvate in DLBCLs enables their proliferation in an extracellular environment-dependent manner. HIGHLIGHTSO_LIGlutamine, but not glucose, is a major carbon source for the tricarboxylic acid cycle in diffuse large B-cell lymphomas. C_LIO_LIMitochondrial pyruvate supports glutaminolysis in diffuse large B-cell lymphomas by supplying pyruvate for glutamate pyruvate transaminase 2-mediated -ketoglutarate production. C_LIO_LIMitochondrial pyruvate carrier inhibition leads to decreased glutaminolysis in diffuse large B-cell lymphomas. C_LIO_LI -ketoglutarate production is important for diffuse large B-cell lymphoma proliferation in a solid extracellular matrix environment. C_LIO_LIMitochondrial pyruvate carrier activity supports diffuse large B-cell lymphoma proliferation in a solid extracellular matrix environment and in mouse xenografts. C_LI

cancer biology↗

bioRxiv2020

IER5, a DNA-damage response gene, is required for Notch-mediated induction of squamous cell differentiation

Notch signaling regulates normal squamous cell proliferation and differentiation and is frequently disrupted in squamous cell carcinomas, in which Notch is a key tumor suppressive pathway. To identify the direct targets of Notch that produce these phenotypes, we introduced a conditional Notch transgene into squamous cell carcinoma cell lines, which respond to Notch activation in 2D culture and in organoid cultures by undergoing differentiation. RNA-seq and ChIP-seq analyses show that in squamous cells Notch activates a context-specific program of gene expression that depends on lineage-specific regulatory elements, most of which lie in long- range enhancers. Among the direct Notch target genes are multiple DNA damage response genes, including IER5, which is regulated by Notch through several enhancer elements. We show that IER5 is required for Notch-induced differentiation in squamous carcinoma cells and in TERT-immortalized keratinocytes. Its function is epistatic to PPP2R2A, which encodes the B55 subunit of PP2A, and IER5 interacts with B55 in cells and in purified systems. These results show that Notch and DNA-damage response pathways converge in squamous cells and that some components of these pathways promote differentiation, which may serve to eliminate DNA-damaged cells from the proliferative pool in squamous epithelia. Crosstalk involving Notch and PP2A may enable Notch signaling to be tuned and integrated with other pathways that regulate squamous differentiation. Our work also suggests that squamous cell carcinomas remain responsive to Notch signaling, providing a rationale for reactivation of Notch as a therapeutic strategy in these cancers. ImpactOur findings highlight context-specific crosstalk between Notch, DNA damage response genes, and PP2A, and provide a roadmap for understanding how Notch induces the differentiation of squamous cells.

cancer biology↗

bioRxiv2020

Sulfopin, a selective covalent inhibitor of Pin1, blocks Myc-driven tumor initiation and growth in vivo

The peptidyl-prolyl cis-trans isomerase, Pin1, acts as a unified signaling hub that is exploited in cancer to activate oncogenes and inactivate tumor suppressors, in particular through up-regulation of c-Myc target genes. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to discover covalent inhibitors targeting Pin1s active site nucleophile - Cys113, leading to the development of Sulfopin, a double-digit nanomolar Pin1 inhibitor. Sulfopin is highly selective for Pin1, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement, and phenocopies genetic knockout of Pin1. Although Pin1 inhibition had a modest effect on viability in cancer cell cultures, Sulfopin induced downregulation of c-Myc target genes and reduced tumor initiation and tumor progression in murine and zebrafish models of MYCN-driven neuroblastoma. Our results suggest that Sulfopin is a suitable chemical probe for assessing Pin1-dependent pharmacology in cells and in vivo. Moreover, these studies indicate that Pin1 should be further investigated as a potential cancer target.

cancer biology↗

bioRxiv2020

Fast transport of RNA granules by direct interactions with KIF5A/KLC1 motors prevents axon degeneration

Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long distance transport of non-membrane enclosed organelles such as RNA granules is not known. Here we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts directly with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that binding of SFPQ to KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie-Tooth (CMT) Disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations show that non-membrane enclosed organelles can move autonomously and that replacing axonally translated proteins provides a therapeutic approach to axonal degenerative disorders.

cell biology↗

bioRxiv2019

STRIPAK directs PP2A activity toward MAP4K4 to promote oncogenic transformation

Alterations involving serine-threonine phosphatase PP2A subunits occur in a range of human cancers and partial loss of PP2A function contributes to cell transformation. Displacement of regulatory B subunits by the SV40 Small T antigen (ST) or mutation/deletion of PP2A subunits alters the abundance and types of PP2A complexes in cells, leading to transformation. Here we show that ST not only displaces common PP2A B subunits but also promotes A-C subunit interactions with alternative B subunits (B, striatins) that are components of the Striatin-interacting phosphatase and kinase (STRIPAK) complex. We found that STRN4, a member of STRIPAK, is associated with ST and is required for ST-PP2A-induced cell transformation. ST recruitment of STRIPAK facilitates PP2A-mediated dephosphorylation of MAP4K4 and induces cell transformation through the activation of the Hippo pathway effector YAP1. These observations identify an unanticipated role of MAP4K4 in transformation and show that the STRIPAK complex regulates PP2A specificity and activity.

cancer biology↗

bioRxiv2019

Targeting the PI5P4K lipid kinase family in cancer using novel covalent inhibitors

The PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of potent, specific small molecules available. Here we present the discovery and characterization of a novel pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4K/{beta}/{gamma}. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4Ks reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders.

cancer biology↗

bioRxiv2019

Molecular basis for feedback inhibition of Notch signaling by the Notch regulated ankyrin repeat protein NRARP

The Notch regulated ankyrin repeat protein (NRARP) is a negative feedback regulator of Notch signaling in higher organisms. The molecular basis for NRARP function, however, has remained elusive. Mass spectrometry-based proteomic studies show that human NRARP associates with the core Notch transcriptional activation complex (NTC), containing the RBPJ transcription factor, the Notch intracellular domain (NICD), and a Mastermind-like co-activator. Binding of NRARP is direct, requires both RBPJ and NICD, and is independent of Mastermind-like proteins or DNA. The X-ray structure of an NRARP/RBPJ/NOTCH1/DNA complex, determined to 3.75 [A] resolution, reveals a non-canonical mode of binding in which NRARP extends the NOTCH1 ankyrin-repeat stack by three additional repeats. Mutations of NRARP residues at the binding interface disrupt entry into complexes and suppress feedback inhibition in signaling assays. These studies establish the structural basis for NTC engagement by NRARP and provide insight into its mechanism of feedback inhibition of Notch signaling.

biochemistry↗

bioRxiv2019

Development of a Covalent Inhibitor of Gut Bacterial Bile Salt Hydrolases

Bile salt hydrolase (BSH) enzymes are widely expressed by human gut bacteria and catalyze the gateway reaction leading to secondary bile acid formation. Bile acids regulate key metabolic and immune processes by binding to host receptors. There is an unmet need for a potent tool to inhibit BSHs across all gut bacteria in order to study the effects of bile acids on host physiology. Here, we report the development of a covalent pan-inhibitor of gut bacterial BSH. From a rationally designed candidate library, we identified a lead compound bearing an alpha-fluoromethyl ketone warhead that modifies BSH at the catalytic cysteine residue. Strikingly, this inhibitor abolished BSH activity in conventional mouse feces. Mice gavaged with a single dose of this compound displayed decreased BSH activity and decreased deconjugated bile acid levels in feces. Our studies demonstrate the potential of a covalent BSH inhibitor to modulate bile acid composition in vivo.

microbiology↗

bioRxiv2019

In vitro analysis of RNA polymerase II elongation complex dynamics

RNA polymerase II elongation complexes (ECs) were assembled from nuclear extract on immobilized DNA templates and analyzed by quantitative mass spectrometry. Time course experiments showed that initiation factor TFIIF can remain bound to early ECs, while levels of core elongation factors Spt4-Spt5, Paf1C, Spt6-Spn1, and Elf1 levels remain steady. Importantly, the dynamic phosphorylation patterns of the Rpb1 C-terminal domain (CTD), and the factors that recognize them, change as a function of post-initiation time, rather than distance elongated. Chemical inhibition of Kin28/Cdk7 blocks both Serine 5 and Serine 2 phosphorylation, affects initiation site choice, and inhibits elongation efficiency. EC components dependent on CTD phosphorylation include capping enzyme, Cap Binding Complex, Set2, and the PAF1 complex. By recapitulating many known features of in vivo elongation, this system reveals new details that clarify how EC-associated core elongation factors, chromatin regulators, and RNA processing factors change at each step of transcription.

biochemistry↗

bioRxiv2017

Downstream promoter interactions of TFIID TAFs facilitate transcription reinitiation

TFIID binds promoter DNA to recruit RNA polymerase II and other basal factors for transcription. Although the TATA-Binding Protein (TBP) subunit of TFIID is necessary and sufficient for in vitro transcription, the TBP-Associated Factor (TAF) subunits recognize downstream promoter elements, act as co-activators, and interact with nucleosomes. Here we show that transcription induces stable TAF binding to downstream promoter DNA, independent of upstream contacts, TBP, or other basal transcription factors. This transcription-dependent TAF complex promotes subsequent activator-independent transcription, and promoter response to TAF mutations in vivo correlates with the level of downstream, rather than overall, Taf1 crosslinking. We propose a new model in which TAFs function as reinitiation factors, accounting for the differential responses of promoters to various transcription factor mutations.

molecular biology↗