Biology subjects

Forli, S.

Publications and source records attributed to Forli, S..

16 recordsLinked to original sources

bioRxiv2026

Autophagy activator AA-20 engages CDK5 to promote lipophagy and improve neurodegeneration-associated phenotypes

Lipophagy, the selective degradation of lipid droplets (LDs) by autophagy, has the potential to ameliorate neurodegenerative diseases such as Parkinson's (PD) and Alzheimer's (AD), wherein LD accumulation has been linked to the exacerbation of the proteinopathy component of neurodegeneration. Pharmacological lipophagy inducers remain scarce. We previously identified AA-20 as a novel autophagy activator that acts independently of mTORC1 inhibition, thereby providing a means of stimulating autophagy while avoiding the immunosuppressive side effects associated with direct mTORC1 inhibition. Prior work also showed that AA-20 treatment reduces PolyQ aggregate levels in several C. elegans tissues. Here, we demonstrate that AA-20 enhances LD recruitment to autophagosomes and promotes LD degradation via lysosomal acid lipase activation in murine hippocampal neuronal cells, consistent with lipophagy activation. In human Swedish Amyloid Precursor Protein mutated cerebral organoids, two weeks of AA-20 treatment normalized aberrant lipidomic profiles by selectively reducing various disease-associated lipids, while also correcting the AD-associated electrical hyperexcitability phenotype. Moreover, we identified cyclin-dependent kinase 5 (CDK5), a neuronal kinase linked to AD hyperexcitability and PD pathology, as a target of AA-20. AA-20-induced autophagy and lipid clearance in HT22 cells require CDK5. In a neuronal seeding model of -synuclein pathology, AA-20 reduced pS129--synuclein levels. Collectively, these findings indicate AA-20 can promote lipophagic clearance of LDs and mitigate multiple cellular phenotypes associated with lipidopathy and proteinopathy in neurodegenerative disease models.

molecular biology↗

bioRxiv2026

The SLC15A4-LAMTOR1 interaction licenses endolysosomal TLR-mediated mTOR signaling and inflammatory cytokine production

Endolysosomal Toll-like receptors (TLRs) 7-9 are critical mediators of antimicrobial immunity, but their dysregulation drives pathogenic inflammation in autoimmune diseases such as systemic lupus erythematosus (SLE). The endolysosomal solute carrier SLC15A4 is essential for TLR7-9-mediated cytokine production and is strongly linked to SLE susceptibility, yet our understanding of the precise mechanisms by which it coordinates innate immune signaling remains incomplete. Here, we identify a direct interaction between SLC15A4 and LAMTOR1, a core scaffold subunit of the lysosomal Ragulator complex, and show that is required for coupling endolysosomal TLR activation to mTORC1 and downstream inflammatory responses. Structural modeling and site-directed mutagenesis suggest that the 1 helix of LAMTOR1 engages the substrate-binding pocket of SLC15A4, and disruption of this interface broadly suppresses TLR7-9-mediated cytokine production. Notably, this interaction regulates mTOR activation, IRF5/7 signaling, and downstream transcriptional responses through stimulus-dependent mechanisms. These findings define a previously uncharacterized SLC15A4-LAMTOR1 signaling interface that coordinates endolysosomal TLR-mTOR coupling and shapes downstream cytokine output. Significance StatementDysregulated activation of endolysosomal Toll-like receptors (TLRs) 7-9 drives pathogenic inflammatory cytokine responses that underlie autoimmune diseases such as systemic lupus erythematosus. Despite the established role of the endolysosomal transporter SLC15A4 in this process, how it coordinates downstream immune signaling has remained incompletely understood. We show that SLC15A4 directly interacts with LAMTOR1, a core component of the lysosomal Ragulator complex, and that this interaction is required to activate mTOR and engage IRF5/7-dependent inflammatory programs downstream of TLR7-9. Disrupting this interface suppresses type I interferon and cytokine production, identifying the SLC15A4-LAMTOR1 interaction as a central node in endolysosomal innate immune signaling and validating it as a compelling target for therapeutic intervention in interferon-driven autoimmune disease.

immunology↗

bioRxiv2026

Pharmacological Inhibition of SLC33A1 Promotes Endoplasmic Reticulum Hyperoxidation and Induces Adaptive IRE1/XBP1s Signaling

The endoplasmic reticulum (ER) transporter solute carrier family 33 member 1 (SLC33A1) has emerged as an attractive therapeutic target in etiologically diverse diseases, ranging from lung cancer to neurodegenerative disorders. Yet, no pharmacologic SLC33A1 modulators have been described. Here, we show that the small molecule IXA4, a highly selective activator of the adaptive IRE1/XBP1s signaling arm of the unfolded protein response (UPR), binds to SLC33A1 and inhibits its activity. Genetic depletion of SLC33A1 phenocopies the selective induction of IRE1/XBP1s signaling brought about by IXA4 treatment. Chemoproteomic analyses and cryo-electron microscopy show that IXA4 binds SLC33A1 within the central channel to inhibit transport of its substrate metabolite(s). Binding of IXA4 to SLC33A1 leads to the accumulation of oxidized glutathione within the ER, hyperoxidizing the ER lumen and inducing activation of adaptive IRE1/XBP1s signaling. Consistent with this function, we find that pharmacologic inhibition of SLC33A1 with IXA4 selectively reduces viability of KEAP1-deficient lung adenocarcinoma cells that have elevated levels of glutathione, mimicking the sensitivity of these cells to genetic deletion of SLC33A1. Our work demonstrates a new physiologic role of SLC33A1 in regulation of ER redox homeostasis and designates IXA4 as a pharmacologic inhibitor of SLC33A1 that can be used to evaluate the biological impact and therapeutic utility of SLC33A1 inhibition in homeostasis and in disease.

cell biology↗

bioRxiv2025

Inactivation of peptidoglycan remodeling promotes antibiotic susceptibility in vancomycin-resistant Enterococcus faecium

Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of healthcare-associated infections globally and demands new approaches for treatment. Here we show that genetic and pharmacological inactivation of a highly conserved NlpC/P60 peptidoglycan hydrolase, secreted antigen A (SagA), enhanced vancomycin susceptibility of VREfm ex vivo and in vivo. Notably, genetic deletion of sagA impaired VREfm peptidoglycan remodeling, growth and increased the activity of vancomycin. We then identified first-in-class covalent NlpC/P60 peptidoglycan hydrolase inhibitors and demonstrated that pharmacological inactivation of SagA activity also impaired peptidoglycan remodeling and increased the efficacy of vancomycin across genetically distinct VREfm clinical isolates. Our study reveals peptidoglycan hydrolases are druggable targets whose inactivation improves the efficacy of vancomycin against VREfm.

microbiology↗

bioRxiv2025

Post-Translational Modifications Remodel Proteome-Wide Ligandability

Post-translational modifications (PTMs) vastly expand the diversity of human proteome, dynamically reshaping protein activity, interactions, and localization in response to environmental, pharmacologic, and disease-associated cues. While it is well established that PTMs modulate protein function, structure, and biomolecular interactions, their proteome-wide impact on small-molecule recognition--and thus druggability--remains largely unexplored. Here, we introduce a chemical proteomic strategy to delineate how PTM states remodel protein ligandability in human cells. By deploying broad profiling photoaffinity probes, we identified over 400 functionally diverse proteins whose ability to engage small molecules is impacted by phosphorylation or N-linked glycosylation status. Integration of binding site mapping with structural analyses revealed a diverse array of PTM-dependent pockets. Among these targets, we discovered that the phosphorylation status of common oncogenic KRAS mutants impact the action of small molecules, including clinically approved inhibitors. These findings illuminate an underappreciated, PTM-governed layer of proteome plasticity and uncover opportunities for the development of chemical probes to selectively target proteins in defined modification states.

biochemistry↗

bioRxiv2025

Design principles of the common Gly-X6-Gly membrane protein building block

Protein behavior in lipid is poorly understood and inadequately represented in current computational models. Design and prediction abilities for bilayer-embedded molecular structures may be improved by characterizing membrane proteins most frequent, favored structural features to glean both context-specific and general principles. We used protein design to proactively interrogate the sequence-structure relationship and stabilizing atomic details of two highly prevalent antiparallel transmembrane (TM) motifs with Small-X6-Small consensus sequences. A fragment-based data-mining and sequence statistical inference method including cross-evolutionary structure-aligned covariance enabled engineering of de novo multi-span TM protein assemblies by successfully encoding Gly-X6-Gly and Ala-X6-Ala building blocks. A highly stable glycine-based designs X-ray structure hosts C-H{middle dot}{middle dot}{middle dot}O=C H-bonding alongside extensive backbone-directed van der Waals packing, idealizing features of this motif in Nature. Data-driven design navigates sequence space to directly inquiry upon how to encode and stabilize vital membrane protein structural elements, facilitating efficacious construction of lipid-embedded architectures of increasing complexity. SignificanceMembrane proteins comprised of -helices pack together within lipid bilayers, establishing stabilities and architectures that brace function and guide evolution. De novo design was used to clarify the consensus sequences and molecular features encoding of one exceedingly common TM helix packing architecture ([~]10% of those in membrane folds). Small-X6-Small residue patterns were proven to reliably drive minimal proteins into these antiparallel TM geometries, revealing glycine mainchain hydrogen bonding or via fully apolar interfaces can encode the motif. Optimizing steric packing was the most decisive feature amongst the synthetic proteins. New membrane-specific design methods and model molecules are validated in route to outlining this important sequence-structure relationship broadly impacting the membrane proteome and revealing generalized structure-energetic imperatives governing interactions in lipid.

biophysics↗

bioRxiv2025

Microbiota-derived secondary bile acids promote STING activation and antitumor activity

Microbiota metabolism generates diverse bile acids that are associated with health and disease, but the molecular targets and mechanisms of action for these metabolites have not been fully elucidated. Using deoxycholic acid (DCA) photoaffinity probes, with cholic acid (CA) probe as a comparator, we found many protein targets of microbiota-derived DCA in mammalian cells. Of note, we discovered that DCA binds the transmembrane domain of stimulator of interferon genes (STING), and enhances agonist-induced accumulation of higher-order STING species, type I interferon signaling and antitumor immunity. Moreover, DCA-producing microbiota species enhanced STING agonist-induced antitumor immunity primarily through the activation of natural killer (NK) cells. This reverse chemical microbiology approach revealed an unpredicted mechanism of action for DCA on STING activation and suggests that specific secondary bile acids and their associated microbiota species may promote NK activation and impact the efficacy of STING-targeted therapeutics.

immunology↗

bioRxiv2024

De novo designed pMHC binders facilitate T cell induced killing of cancer cells

The recognition of intracellular antigens by CD8+ T cells through T-cell receptors (TCRs) is central to adaptive immunity, enabling responses against infections and cancer. The recent approval of TCR-gene-edited T cells for cancer therapy demonstrates the therapeutic advantage of using pMHC recognition to eliminate cancer. However, identification and selection of TCRs from patient material is complex and influenced by the TCR repertoire of the donors used. To overcome these limitations, we here present a rapid and robust de novo binder design platform leveraging state-of-the-art generative models, including RFdiffusion, ProteinMPNN, and AlphaFold2, to engineer minibinders (miBds) targeting the cancer-associated pMHC complex, NY-ESO-1(157-165)/HLA-A*02:01. By incorporating in silico cross-panning and molecular dynamics simulations, we enhanced specificity screening to minimise off-target interactions. We identified a miBd that exhibited high specificity for the NY-ESO-1-derived peptide SLLMWITQC in complex with HLA-A*02:01 and minimal cross-reactivity in mammalian display assays. We further demonstrate the therapeutic potential of this miBd by integrating it into a chimeric antigen receptor, as de novo Binders for Immune-mediated Killing Engagers (BIKEs). BIKE-transduced T cells selectively and effectively killed NY-ESO-1+ melanoma cells compared to non-transduced controls, demonstrating the promise of this approach in precision cancer immunotherapy. Our findings underscore the transformative potential of generative protein design for accelerating the discovery of high-specificity pMHC-targeting therapeutics. Beyond CAR-T applications, our workflow establishes a foundation for developing miBds as versatile tools, heralding a new era of precision immunotherapy.

immunology↗

bioRxiv2024

An mRNA Display Approach for Covalent Targeting of a Staphylococcus aureus Virulence Factor

Staphylococcus aureus (S. aureus) is an opportunistic human pathogen that causes over one million deaths around the world each year. We recently identified a family of serine hydrolases termed fluorophosphonate binding hydrolases (Fphs) that play important roles in lipid metabolism and colonization of a host. Because many of these enzymes are only expressed in Staphylococcus bacteria, they are valuable targets for diagnostics and therapeutics. Here we developed and screened highly diverse cyclic peptide libraries using mRNA display with a genetically encoded oxadiazolone (Ox) electrophile that was previously shown to potently and covalently inhibit multiple Fph enzymes. By performing multiple rounds of counter selections with WT and catalytic dead FphB, we were able to tune the selectivity of the resulting selected cyclic peptides containing the Ox residue towards the desired target. From our mRNA display hits, we developed potent and selective fluorescent probes that label the active site of FphB at single digit nanomolar concentrations in live S. aureus bacteria. Taken together, this work demonstrates the potential of using direct genetically encoded electrophiles for mRNA display of covalent binding ligands and identifies potent new probes for FphB that have the potential to be used for diagnostic and therapeutic applications.

microbiology↗

bioRxiv2024

Assessing AF2's ability to predict structural ensembles of proteins

Recent breakthroughs in protein structure prediction have enhanced the precision and speed at which protein configurations can be determined, setting new benchmarks for accuracy and efficiency in the field. However, the fundamental mechanisms of biological processes at a molecular level are often connected to conformational changes of proteins. Molecular dynamics (MD) simulations serve as a crucial tool for capturing the conformational space of proteins, providing valuable insights into their structural fluctuations. However, the scope of MD simulations is often limited by the accessible timescales and the computational resources available, posing challenges to comprehensively exploring protein behaviors. Recently emerging approaches have focused on expanding the capability of AlphaFold2 (AF2) to predict conformational substates of protein structures by manipulating the input multiple sequence alignment (MSA). These approaches operate under the assumption that the MSA also contains information about the heterogeneity of protein structures. Here, we benchmark the performance of various workflows that have adapted AF2 for ensemble prediction focusing on the subsampling of the MSA as implemented in ColabFold and compare the obtained structures with ensembles obtained from MD simulations and NMR. As test cases, we chose four proteins namely the bovine pancreatic inhibitor protein (BPTI), thrombin and two antigen binding fragments (antibody Fv and nanobody), for which reliable experimentally validated structural information (X-ray and/or NMR) was available. Thus, we provide an overview of the levels of performance and accessible timescales that can currently be achieved with machine learning (ML) based ensemble generation. In three out of the four test cases, we find structural variations fall within the predicted ensembles. Nevertheless, significant minima of the free energy surfaces remain undetected. This study highlights the possibilities and pitfalls when generating ensembles with AF2 and thus may guide the development of future tools while informing upon the results of currently available applications.

biophysics↗

bioRxiv2024

IP6 and PF74 affect HIV-1 Capsid Stability through Modulation of Hexamer-Hexamer Tilt Angle Preference

The HIV-1 capsid is an irregularly shaped complex of about 1200 protein chains containing the viral genome and several viral proteins. Together, these components are the key to unlocking passage into the nucleus, allowing for permanent integration of the viral genome into the host cell genome. Recent interest into the role of the capsid in viral replication has been driven by the approval of the first-in-class drug lenacapavir, which marks the first drug approved to target a non-enzymatic HIV-1 viral protein. In addition to lenacapavir, other small molecules such as the drug-like compound PF74, and the anionic sugar inositolhexakisphosphate (IP6), are known to impact capsid stability, and although this is widely accepted as a therapeutic effect, the mechanisms through which they do so remain unknown. In this study, we employed a systematic atomistic simulation approach to study the impact of molecules bound to hexamers at the central pore (IP6) and the FG-binding site (PF74) on capsid oligomer dynamics, compared to apo hexamers and pentamers. We found that neither small molecule had a sizeable impact on the free energy of binding of the interface between neighboring hexamers but that both had impacts on the free energy profiles of performing angular deformations to the pair of oligomers akin to the variations in curvature along the irregular surface of the capsid. The IP6 cofactor, on one hand, stabilizes a pair of neighboring hexamers in their flattest configurations, whereas without IP6, the hexamers prefer a high tilt angle between them. On the other hand, having PF74 bound introduces a strong preference for intermediate tilt angles. These results suggest that structural instability is a natural feature of the HIV-1 capsid which is modulated by molecules bound in either the central pore or the FG-binding site. Such modulators, despite sharing many of the same effects on non-bonded interactions at the various protein-protein interfaces, have decidedly different effects on the flexibility of the complex. This study provides a detailed model of the HIV-1 capsid and its interactions with small molecules, informing structure-based drug design, as well as experimental design and interpretation.

biophysics↗

bioRxiv2024

Structural insights into IMP2 dimerization and RNA binding

IGF2BP2 (IMP2) is an RNA-binding protein that contributes to cancer tumorigenesis and metabolic disorders. Structural studies focused on individual IMP2 domains have provided important mechanistic insights into IMP2 function; however, structural information on full-length IMP2 is lacking but necessary to understand how to target IMP2 activity in drug discovery. In this study, we investigated the behavior of full-length IMP2 and the influence of RNA binding using biophysical and structural methods including mass photometry, hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS), and small angle x-ray scattering (SAXS). We found that full-length IMP2 forms multiple oligomeric states but predominantly adopts a dimeric conformation. Molecular models derived from SAXS data suggest the dimer is formed in a head-to-tail orientation by the KH34 and RRM1 domains. Upon RNA binding, IMP2 forms a pseudo-symmetric dimer different from its apo/RNA-free state, with the KH12 domains of each IMP2 molecule forming the dimer interface. We also found that the formation of IMP2 oligomeric species, which includes dimers and higher-order oligomers, is sensitive to ionic strength and RNA binding. Our findings provide the first insight into the structural properties of full-length IMP2, which may lead to novel opportunities for disrupting its function with more effective IMP2 inhibitors.

molecular biology↗

bioRxiv2024

The conformational landscape of human transthyretin revealed by cryo-EM

Transthyretin (TTR) is a natively tetrameric thyroxine transporter found in blood and cerebrospinal fluid whose misfolding and aggregation causes transthyretin amyloidosis. A rational drug design campaign identified the small molecule tafamidis (Vyndaqel/Vyndamax) as an effective stabilizer of the native TTR fold, and this aggregation inhibitor is regulatory agency-approved for the treatment of TTR amyloidosis. Despite 50 years of structural studies on TTR and this triumph of structure-based drug design, there remains a notable dearth of structural information available to understand ligand binding allostery and amyloidogenic TTR unfolding intermediates. We used single-particle cryo-electron microscopy (cryo-EM) to investigate the conformational landscape of this 55 kiloDalton tetramer in the absence and presence of one or two ligands, revealing inherent asymmetries in the tetrameric architecture and previously unobserved conformational states. These findings provide critical mechanistic insights into negatively cooperative ligand binding and the structural pathways responsible for TTR amyloidogenesis. This study underscores the capacity of cryo-EM to provide new insights into protein structures that have been historically considered too small to visualize and to identify pharmacological targets suppressed by the confines of the crystal lattice, opening uncharted territory in structure-based drug design.

molecular biology↗

bioRxiv2020

A strain-specific inhibitor of receptor-bound HIV-1 targets a pocket near the fusion peptide and offers a template for drug design

Disruption of viral fusion represents a viable, albeit under-explored, target for HIV therapeutics. While studying the receptor-bound envelope glycoprotein conformation by cryo-EM, we identified a pocket near the base of the trimer containing a bound detergent molecule and performed in silico drug screening using a library of drug-like and commercially available molecules. After down-selection, we solved cryo-EM structures that validated binding of two small molecule hits in very similar manners to the predicted binding poses, including interactions with aromatic residues within the fusion peptide. One of the molecules demonstrated low micromolar inhibition of the autologous virus by utilizing a very rare phenylalanine in the fusion peptide and stabilizing the surrounding region. This work demonstrates that small molecules can target the fusion process, providing a new target for anti-HIV therapeutics, and highlights the need to explore how fusion peptide sequence variations affect receptor-mediated conformational states across diverse HIV strains.

biophysics↗

bioRxiv2019

An activity-guided map of electrophile-cysteine interactions in primary human immune cells

Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving direct functional perturbation and/or ligand-induced degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells, underscoring the potential of electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders.

immunology↗

bioRxiv2019

Integrative x-ray structure and molecular modeling for the rationalization of procaspase-8 inhibitor potency and selectivity

Caspases are a critical class of proteases involved in regulating programmed cell death and other biological processes. Selective inhibitors of individual caspases, however, are lacking, due in large part to the high structural similarity found in the active sites of these enzymes. We recently discovered a small-molecule inhibitor, 63-R, that covalently binds the zymogen, or inactive precursor (pro-form), of caspase-8, but not other caspases, pointing to an untapped potential of procaspases as targets for chemical probes. Realizing this goal would benefit from a structural understanding of how small molecules bind to and inhibit caspase zymogens. There have, however, been very few reported procaspase structures. Here, we employ x-ray crystallography to elucidate a procaspase-8 crystal structure in complex with 63-R, which reveals large conformational changes in active-site loops that accommodate the intramolecular cleavage events required for protease activation. Combining these structural insights with molecular modeling and mutagenesis-based biochemical assays, we elucidate key interactions required for 63-R inhibition of procaspase-8. Our findings inform the mechanism of caspase activation and its disruption by small molecules, and, more generally, have implications for the development of small molecule inhibitors and/or activators that target alternative (e.g., inactive precursor) protein states to ultimately expand the druggable proteome.

biochemistry↗