Biology subjects

Knapp, S.

Publications and source records attributed to Knapp, S..

At least 19 recordsLinked to original sources

bioRxiv2026

An Atlas of Short Linear Motif-Mediated Human Protein-Protein Interactions

Short linear motifs (SLiMs) within intrinsically disordered protein regions mediate transient interactions crucial for cell physiology1. However, the global interaction landscape of human SLiMs remains largely uncharted. Here we present the Atlas of SLiM-mediated Human protein-protein Interactions (ASHI), which maps more than 20,000 interactions by screening over 800 human protein domains against a library of one million peptides tiling the human disordered proteome. ASHI expands the SLiM interactome, uncovers novel binding modes for known peptide-binding domains, and reveals unexpected peptide-binding activities in enzymes, chaperones, RNA-binding proteins, and modification-reader domains. Furthermore, intrinsically disordered regions emerge as densely encoded interaction platforms where interaction specificity is governed by diverse mechanisms, including key motif determinants, flanking residues, competition, and multivalency. These data provide an unprecedented foundation for modeling dynamic interaction networks, interpreting disease-associated variants, and decoding the dark proteome.

biochemistry↗

bioRxiv2026

The structural basis for LRRK2's activation and autoinhibition

Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) are the second most common cause of autosomal-dominant Parkinsons disease (PD), and increased LRRK2 kinase activity is also observed in idiopathic PD, making LRRK2 a major actionable therapeutic target. LRRK2 is a 286-kDa multidomain enzyme containing a Ras-like GTPase (ROC) and a kinase domain. Using cryo-EM, biochemical reconstitution, and cell-based assays, we show that the ROC GTPase governs switching between autoinhibited and active states: GTP binding promotes activation, whereas GDP binding enforces autoinhibition. Two common PD-linked mutations, G2019S and R1441C/G/H, activate LRRK2 through distinct structural mechanisms, revealing genotype-specific routes to dysregulation. These findings provide a unified framework for understanding LRRK2 regulation with broad therapeutic implications. Stabilizing the GDP-bound state may inhibit LRRK2 by maintaining autoinhibition, whereas promoting the GTP-bound state could be advantageous in specific cellular contexts, such as the lung, where increased LRRK2 kinase activity may play protective or regulatory roles.

neuroscience↗

bioRxiv2026

Kinetic Fingerprints as Mechanistic and Clinical Roadmaps Across KIT Activation States

In cancer therapy, traditional approaches often overlook the dynamic nature of drug-target interactions. We introduce kinetic fingerprints as a mechanistically informative tool to guide kinase inhibitor design and predict clinical performance. Profiling 172 compounds across multiple KIT conformations, including the oncogenic D816V mutation, show that prolonged residence time determines therapeutic success, while mutations accelerating dissociation rates (koff) drive resistance, positioning koff as a robust predictor of clinical failure. Beyond efficacy and resistance, kinetic signatures map molecular behavior: fast-associating scaffolds engage readily populated KIT states, slow binders overcome conformational barriers like juxtamembrane repositioning, and extended residence times highlight ligands stabilizing regulatory elements (G-loop and regulatory spine). Kinetic profiling further unveils mechanisms invisible to conventional methods, such as drug-induced kinase degradation, and exposes selectivity dimensions beyond affinity: avapritinib exhibits durable KIT D816V engagement yet transient off-target binding. Our findings redefine the evaluation of KIT inhibitors, establishing a framework for rational, kinetics-guided drug discovery in KIT-driven cancers. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/726488v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@17f522borg.highwire.dtl.DTLVardef@1ebabc6org.highwire.dtl.DTLVardef@15a5e61org.highwire.dtl.DTLVardef@1ea5e0e_HPS_FORMAT_FIGEXP M_FIG Kinetic profiling of 172 compounds across KIT conformationsincluding D816Vreveals kinetic fingerprints that predict efficacy, selectivity, resistance, and inhibitors ability to stabilize key regulatory elements. Far from a secondary metric, binding kinetics provide mechanistic insights beyond affinity, offering a powerful framework for rational drug design in KIT-driven cancers. C_FIG

biochemistry↗

bioRxiv2026

Small-molecule modulators of HIPK4 activity and proteostasis

Homeodomain-interacting protein kinase 4 (HIPK4) is a dual-specificity kinase that is predominantly expressed in differentiating spermatids, required for sperm development, and a promising target for nonhormonal male contraception. Genetic and functional studies have established an essential role for HIPK4 in spermiogenesis, where it acts at least in part through regulation of the F-actin-scaffolded acroplaxome during spermatid head shaping. The direct molecular targets of HIPK4 and their downstream effectors remain poorly defined, and small-molecule probes would be versatile tools for further investigating HIPK4 functions. Synthetic HIPK4 ligands could also be valuable leads for the development of nonhormonal male contraceptives. Here, we report the discovery of a cyanoquinoline-based series of HIPK4 inhibitors with nanomolar potency. Our lead compounds are selective for HIPK4, both within the HIPK family and across the broader kinome, establishing this scaffold as a useful starting point for probe and lead development. Unexpectedly, we found that a subset of these cyanoquinolines also perturbs HIPK4 proteostasis in a cell type-specific manner. In spermatids, these compounds induce the formation of detergent-insoluble HIPK4 aggregates and promote interactions between this kinase and the autophagy receptor Tax1-binding protein 1 (TAX1BP1). Together, our findings establish cyanoquinoline ligands as a new chemotype for probing HIPK4 biology and advancing male contraceptive discovery.

biochemistry↗

bioRxiv2026

Macrocyclization of Broad-Spectrum Kinase Inhibitor Bosutinib leads to Potent and Selective Quinoline-based HIPK4 Inhibitor AZ137

Homeodomain-interacting protein kinase 4 (HIPK4) remains an understudied member of the dark kinome. While genetic knockout studies suggest roles for HIPK4 in spermiogenesis and cutaneous squamous cell carcinoma, whether these cellular functions can be recapitulated by pharmacological inhibition remains to be determined. However, such investigations have been hampered by a lack of high-quality chemical tools. To address this, we employed a rational design strategy utilizing macrocyclization of a bosutinib-based scaffold. Systematic optimization led to the discovery of AZ137 (28e), a potent and selective HIPK4 inhibitor (IC50 = 11 nM; cellular EC50 = 76 nM). AZ137 exhibits exceptional selectivity across three comprehensive orthogonal panels, high solubility, and no detectable cytotoxicity. Its cellular activity was confirmed in cell-based assays of HIPK4-dependent F-actin remodeling. Together with a negative control compound, this probe set provides a foundational framework for the validating HIPK4 as a therapeutic target and a high-quality resource to elucidate its roles in normal physiology and disease.

biochemistry↗

bioRxiv2026

A targetable dependency on nonsense-mediated decay for proteostasis and immune control in small cell lung cancer

Small cell lung cancer (SCLC) is among the deadliest cancers with excessive somatic alterations, expectedly resulting in immunogenic epitopes. However, patient benefit from immunotherapy is limited. We found abundant frameshift mutations in SCLC, regarded as highly immunogenic, counterbalanced by a hyperactive nonsense-mediated decay (NMD) pathway, responsible for frameshift-mRNA degradation. NMD activity correlated with tumor mutational burden (TMB) across cancers, suggesting that SCLC depends on NMD for cellular homeostasis and immune evasion. NMD inhibition impaired SCLC proliferation and induced ER stress-dependent apoptosis in a TMB-dependent manner, thereby enabling pharmacological inhibition in vivo which effectively controlled tumor growth. By integrating genome and transcriptome sequencing with MHC-I immunopeptidomics and functional in vitro and in vivo assays, we identified that NMD inhibition boosted neoantigen expression and presentation by tumor cells and increased T cell recognition, thus enhancing overall tumor immunogenicity and further improving immunotherapy efficacy in vivo. Our work shows that SCLC - as a TMBhigh cancer - relies on NMD for survival and immune escape, uncovering a novel TMB-dependent tractable vulnerability for this devastating disease.

cancer biology↗

bioRxiv2026

Leveraging the BAF chromatin remodeling complex for targeted transcriptional rewiring in cancer

Chromatin accessibility is essential for maintaining the fidelity of gene regulation and is dynamically regulated by epigenetic enzymes that are often dysregulated in cancer. The most commonly mutated regulator is the modular, multi-subunit Brahma-associated factor (BAF) chromatin remodeling complex. Previous attempts to exploit BAF complex mutations as potential tumor vulnerabilities using loss-of-function approaches have shown limited clinical success. Here, we instead propose a gain-of-function (GOF) strategy and establish Transcriptional/Remodeling chemical Inducers of Proximity (TRIPs), a class of neomorphic molecules that recruit active BAF complexes to rewire an oncogenic repressor, B-cell lymphoma 6 (BCL6). TRIPs potently induce transcriptional de-repression and apoptosis in Diffuse Large B-cell Lymphoma (DLBCL), enabled by ternary complex formation between BCL6 and BAF. CRISPR knockout screening identifies the PBAF complex as an essential contributor to cellular TRIP efficacy. Finally, we demonstrate that TRIP induces chromatin enrichment of BAF at BCL6-bound sites, resulting in ATPase-dependent eviction of BCL6, and de-repression of pro-apoptotic BCL6 target genes. We establish BAF recruitment for targeted chromatin remodeling as a viable GOF pharmacological strategy for tackling diseases driven by aberrant gene repression.

cancer biology↗

bioRxiv2026

Cytonuclear Conflict and Reticulate Evolution in the Morelloid Clade (Solanum, Solanaceae): Insights from Genome Skimming and Network Phylogenomics

The Morelloid clade (black nightshades) is one of the most strongly supported clades within the megadiverse Solanum genus. It comprises 76 globally distributed, non-spiny herbaceous and suffrutescent species. While often erroneously considered poisonous weeds, several species are economically important as orphan crops. The clade is closely related to tomato and potato but, due to a lack of focused breeding efforts, remains a reservoir of genetic diversity for crop improvement. Despite this potential, we lack fundamental knowledge on the evolution of the Morelloid clade. The group includes polyploid species with unknown parental origins--likely reflecting reticulate processes such as hybridization, introgression, and associated backcrossing events. Prior analyses have been unable to disentangle these processes, leaving the mechanisms underlying reticulate evolution in the Morelloid clade poorly understood. Here, we use genome skimming to produce a well-supported maximum likelihood plastid phylogeny from complete circularized plastomes and a coalescent-based species tree from combined Angiosperms353 and conserved ortholog set nuclear markers. Our dataset, composed of previously published data and deep genome skimming from herbarium samples, spans 26 Morelloid species. To investigate patterns of non-treelike evolution, we used a nuclear phylogenetic network, multispecies coalescent simulations, a fused rooted nuclear chloroplast tree, and quantification of nuclear gene tree concordance. We show that incongruence between nuclear and plastid trees is pervasive and cannot be explained by incomplete lineage sorting alone. Instead, our results demonstrate that events consistent with repeated chloroplast capture have shaped the reticulate evolutionary history of the clade, especially among African polyploid and Pan-American diploid lineages.

evolutionary biology↗

bioRxiv2025

Evaluation of peptidomimetic inhibitors

Scientific research in drug discovery relies on robust, versatile, and well-established methodologies. One effective strategy for targeting disease-relevant proteins, such as E3 ligases, involves rational drug design using degron peptides as starting points for compound development. Here, we present a comprehensive set of complementary assays for the evaluation of degron-based binders, enabling their integration into an efficient drug discovery pipeline. We introduce screening strategies such as Differential Scanning Fluorimetry (DSF) and Fluorescence Polarization (FP), alongside both solution-based and immobilization-based biophysical techniques, including Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR) for reliable affinity determination. To assess intracellular interactions with full-length target proteins, we also employ Nano Bioluminescence Resonance Energy Transfer (NanoBRET). Together, these methods establish a robust framework for the discovery and characterization of degron-based peptidomimetic compounds.

biochemistry↗

bioRxiv2025

Synthesis and Characterization of ULK1/2 Kinase Inhibitors that Inhibit Autophagy and Upregulate Expression of Major Histocompatibility Complex I for the Treatment of Non-Small Cell Lung Cancer

Autophagy inhibition represents a promising therapeutic approach for the management of various cancers including non-small cell lung cancer (NSCLC). We previously reported SBP-7455, a dual inhibitor of unc-51-like kinase 1 (ULK1) and its homologue ULK2, and described its effects on triple-negative breast cancer (TNBC) cells. Herein we report the design, synthesis, and characterization of SBP-5147 and SBP-7501, two new dual ULK1/2 inhibitors that are cytotoxic against NSCLC cells, inhibit autophagic flux in A549 cells, and present greater oral exposure than SBP-7455 at a lower dose. In addition, SBP-5147 effectively modulates autophagy and increases the expression of major histocompatibility complex (MHC) class I in NSCLC cells, which may support the rationale for ULK1/2 inhibition as a strategy to overcome resistance to immunotherapy. Together these data support the use of ULK inhibitors as part of a cancer treatment strategy, both as a single agent as well as in combination with current therapies.

cancer biology↗

bioRxiv2025

Proteome-wide impact of LRRK1 and 2 inhibitors on protein interactions and phosphorylation

The Leucine-rich repeat kinases 1 and 2 (LRRK1 and 2) are large, multidomain proteins and closely related members of the Roco protein family. They share a high similarity in domain structure and are both phosphorylate members of the Rab GTPase family. However, despite these similarities, there are substantial differences between the two kinases. While mutations to LRRK1 are only implicated in rare cases of osteopetrosis, LRRK2 is associated with multiple diseases, most prominently with familial and sporadic forms of Parkinsons disease, where pathogenic LRRK2 is associated with an increased kinase activity. While LRRK2 has received major attention from the research community, LRRK1 has been largely understudied. In this work, we employ proximity labelling mass spectrometry in combination with quantitative phosphoproteomics in a model cell line to obtain the cellular interactomes of LRRK1 and LRRK2 and corresponding phosphorylation sites. We then use this dataset to characterize the impact of small molecules targeting both LRRK1 and 2. We identify phosphorylation sites across the proteome that are impacted by these inhibitors and identify novel candidate substrates for LRRK2, including MICALL2. Taken together our data provide a powerful resource for future studies on the cellular role and function of LRRK proteins and their potential use as therapeutic targets.

molecular biology↗

bioRxiv2025

Overcoming Ligand Discovery Challenges: Developing Peptide-Based Tracers for SPSB2

Developing new E3 ligase ligands for the design of heterobivalent molecules, such as PROteolysis TArgeting Chimeras (PROTACs), requires careful evaluation of target engagement (TE). Characterizing protein-protein interactions (PPIs) is therefore essential in drug discovery, as it enables the assessment of ligand binding to sites that are often difficult to target. Degrons, peptide motifs recognized by E3 ligases, may serve as valuable starting points for designing E3 ligands. However, many degrons are highly polar and lack intrinsic membrane permeability, requiring alternative strategies for efficient cellular delivery. In this study, we used the SPRY domain-containing SOCS box protein 2 (SPSB2) E3 ligase as a model system to develop TE strategies for in vitro and in cellulo using polar degron-based peptides. By conjugating various polycationic cell-penetrating peptides (CPPs) to the degron sequence, we present a study demonstrating efficient cellular delivery. We obtained a high-resolution crystal structure and used various biophysical techniques to assess the influence of each modification, while confocal microscopy and BRET-based assays confirmed successful cellular delivery as well as potent target engagement. O_FIG O_LINKSMALLFIG WIDTH=183 HEIGHT=200 SRC="FIGDIR/small/673904v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@191848borg.highwire.dtl.DTLVardef@94fae6org.highwire.dtl.DTLVardef@e43016org.highwire.dtl.DTLVardef@110f9f0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

CD8+ T cells regulate the bioenergetic reprogramming of lymphoid organs and the heart during viral infection

The activation of the immune system is a bioenergetically-costly process1. Yet, essential bodily functions require a continuous energy supply, imposing energy constraints and trade-offs between competing processes2. Our understanding of the underlying bioenergetic adaptations reconciling rapid immune activation with other vital processes remains scarce. 3-6 Here, by using experimental models of viral infections, we identified an unexpected CD8+ T cell-driven redistribution of energy substrates between lymphoid organs and the heart. Viral infection promoted systemic hypoglycaemia and ketogenesis, together with systemic reallocation of energy substrates. Across organs analysed, secondary lymphoid organs and the heart showed the most dramatic changes. The former increased glucose uptake and oxidation while the heart showed the opposite, switching to preferential fatty acid utilization. These bioenergetic adaptations were absent in infected mice lacking CD8+ T cells or with T cells lacking the glucose transporter GLUT1. Pharmacological inhibition of fatty acid oxidation forced a systemic switch to glucose oxidation. This was associated with metabolic decompensation, reduced cardiac energetics, left ventricular stress, and mortality in otherwise nonlethal viral infections. Our results reveal how the energetic cost of immune cell activation imposes bioenergetic adaptations on non-lymphoid organs, posing a major challenge for the heart by completely relying on fatty acids.

immunology↗

bioRxiv2025

Click. Screen. Degrade. A Miniaturized D2B Workflow for rapid PROTAC Discovery

Targeted protein degradation is one of the fastest developing fields in medicinal chemistry and chemical biology. Despite significant development in assay technologies and inhibitor discovery, the development of PROTACs remains a challenging endeavor since rational design approaches remain widely elusive. Our workflow eliminates the rate-limiting step of classic synthesis, namely compound purification, and pairs it with high-throughput, semi-automated plate-based synthesis, and direct cellular assay evaluation. We applied this direct-to-biology approach to four diverse targets demonstrating the general applicability of this technology. PROTAC synthesis was realized by using the highly efficient copper-catalyzed azide-alkyne cycloaddition reaction. This simplified reaction setup allowed synthesis in the nanomole scale with reaction volumes as low as 5 L. This high throughput approach enables the synthesis and testing of hundreds of PROTACs within a few days, allowing for a comprehensive assessment of target degradability and the identification of the most suitable E3 ligase for degrader development.

biochemistry↗

bioRxiv2025

Un-LOK-ing a new approach for conformational selective targeting of STK10 (LOK)

STK10 (serine/threonine kinase 10, LOK), is an important regulator of diverse cellular processes, such as cell cycle progression or lymphocyte migration. STK10 has emerged as a potential therapeutic target for diseases associated with impaired cell migration and cell division. Here we present a late-stage optimization of a macrocyclic pyrazolo[1,5-a]pyrimidine scaffold that led to a urea-based lead series targeting the back-pocket of STK10. Co-crystal structure analysis of 23 revealed that the optimized macrocycles adopted a unique binding mode that protrudes deep into the back pocket of STK10. Compound 23 exhibited potent on-target activity in biophysical and activity assays and displayed nanomolar activity for STK10 in cells. In addition, 23 shows good selectivity against the kinome and remarkably also against the closely related kinase SLK (STE20-like kinase). Therefore, we propose that targeting the unique and largely extended pocket in STK10 represents an opportunity to develop highly selective STK10 inhibitors. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/666149v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@36f039org.highwire.dtl.DTLVardef@d55025org.highwire.dtl.DTLVardef@80c007org.highwire.dtl.DTLVardef@bf2471_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

A new phylogeny and phylogenetic classification for Solanaceae

Solanaceae are a globally distributed family of considerable economic importance. Recent phylogenetic work on various clades in the family has resulted in a plethora of names at various taxonomic levels. This results in a confusing set of names applied to clades at different ranks, and to the use of informal names that are often not accepted in online resources. To solve this problem, we present here a phylogenetic classification following the PhyloCode for the Solanaceae. Traditional taxon names previously recognized at the ranks of tribe, genus, and section were maintained when associated with clades. We base our clade definitions on a newly constructed phylogeny of the family using 10 plastid and nuclear markers with a broad sampling of 1,474 species, roughly half of the diversity of the family. We complement this marker-based analysis with a phylogenomic analysis of 110 taxa (including 97 of the 102 genera in the family) using the Angiosperms353 probe set. This pair of analyses produced highly concordant phylogenies and provides strong support for the clades included in the classification. In total, we propose and define rank-free names for 38 major clades, including 16 infrageneric taxa within the mega-diverse Solanum. This rank-free system intends to promote name stability as new data become available, avoiding name changes to well-known groups.

evolutionary biology↗

bioRxiv2025

Seed fossil record of Solanaceae revisited

The fossil record for Solanaceae has a complex taxonomic history, with many species originally described in the family being subsequently shown to belong in other plant families. In this work, we present an in-depth analysis of the nightshade seed fossil record, which corresponds to the largest amount of fossil material for the family despite having relatively few described taxa. We re-examined 110 fossil seeds previously assigned either to one of the six taxa or unplaced within the family. All fossils were imaged and measured for 14 discrete and continuous characters, and a subset of 22 well-preserved specimens was additionally subjected to micro-CT scanning. In parallel, we scored the same set of characters for seeds from 354 extant taxa of Solanaceae. We carried out a multivariate analysis of the combined extinct and extant dataset to visualize the placement of fossil seeds within Solanaceae seed morphospace. This analysis revealed a wide spread of fossils across morphospace, including a variety of shapes, sizes and testal wall features. For one fossil taxon (Hyoscyosperma daturoides Deanna & S.D.Sm. gen. et sp. nov.), the micro-CT images confirmed the presence of an elaiosome, otherwise only known in the family from the genus Datura L. Based on these analyses, we re-circumscribe three species and describe six new taxa (four in new genera), doubling the number of fossil seed species in the family. The broad geographic and stratigraphic distribution of these diverse taxa, from Eocene North America and Europe to Pleistocene Eurasia, support the notion that the Solanaceae is likely a family of a late Cretaceous origin that attained nearly a cosmopolitan distribution by the Eocene.

paleontology↗

bioRxiv2025

Targeted degradation of USP7 in solid cancer cells reveals disparate effects of deubiquitinase inhibition vs. acute protein depletion

Proteolysis-targeting chimeras (PROTACs) co-op the ubiquitin system for targeted protein degradation, creating opportunities to interrogate cellular functions of proteins through "chemical knockdown". However, matched pairs of protein degraders and inhibitors, that possess high specificity and chemical complementarity, for individual components of the ubiquitin system have remained scarce. This includes reagents to modulate activity and abundance of deubiquitinases (DUBs), which critically regulate ubiquitin-mediated signaling. Here, using an integrated chemical biology approach, we explored the cellular function of the DUB USP7 as a case study comparing inhibition and degradation of this DUB in melanoma and pancreatic cancer cells. Through the synthesis of a degrader library, we identified potent USP7 PROTACs for each cancer type, established BRET-based ternary complex formation and quantified degradation efficiency. USP7 degraders and their cognate inhibitor were subsequently employed to characterize treatment-induced phenotypic alterations. Proteomic and cellular analyses revealed that highly specific degradation of USP7 modulated both shared and distinct protein sets across cancer cell types, without impacting cell growth. Notably, cellular responses to USP7 degradation differed markedly from those to USP7 inhibition. Moreover, our data uncovered broad proteomic and metabolic changes induced by prolonged USP7 inhibitor treatment. Collectively, our work provides a chemical toolbox of comprehensively characterized reagents to distinguish on-target phenotypes which will aid the understanding of the role of USP7 in malignant diseases. More broadly, our data emphasize the importance of increased specificity via PROTAC-mediated degradation and the potential of this modality to distinguish catalytic from non-catalytic as well as cell-line specific functions of DUBs.

biochemistry↗