Biology subjects

Knapp, S.

Publications and source records attributed to Knapp, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Development of selective pyridopyrimidin-7(8H)-one-based Mammalian STE20-like (MST3/4) kinase inhibitors

Mammalian STE20-like (MST) kinases 1-4 play key roles in regulating the Hippo and autophagy pathways, and their dysregulation has been implicated in cancer development. In contrast to the well-studied MST1/2, the roles of MST3/4 are less clear, in part due to the lack of potent and selective MST3/4 inhibitors. Here, we re-evaluated literature compounds, and used structure-guided design to optimize the p21-activated kinase (PAK) inhibitor G-5555 (8) to selectively target MST3/4. These efforts resulted in the development of MR24 (24) and MR30 (27) with good kinome-wide selectivity, high potency for MST3/4, and selectivity towards the closely related MST1/2. In combination with the MST1/2 inhibitor PF-06447475 (2) the two MST3/4 inhibitors can be used to elucidate the multiple roles of MST kinases in cells. We found that MST3/4-selective inhibition caused a cell cycle arrest in the G1 phase, while MST1/2 inhibition resulted in accumulation of cells in the G2/M phase. These data point to distinct functions of these closely related kinases, which can now be addressed with subfamily-selective chemical tool compounds.

biochemistry↗

bioRxiv2023

Development of LRRK2 designed ankyrin-repeat proteins

Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinsons disease (PD) has led to intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its activity are needed. Here, we identified a series of high-affinity LRRK2-binding designed ankyrin-repeat proteins (DARPins). One of these DARPins (E11) bound to the LRRK2 WD40 domain with high affinity. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.

biochemistry↗

bioRxiv2023

Discovery of the sEH Inhibitor Epoxykynin as Potent Kynurenine Pathway Modulator

Disease-related phenotypic assays enable unbiased discovery of novel bioactive small molecules and may provide novel insights into physiological systems and unprecedented molecular modes-of-action (MMOA). Herein we report the identification and characterization of epoxykynin, a potent inhibitor of the soluble epoxide hydrolase (sEH). Epoxykynin was discovered by means of a cellular assay monitoring modulation of kynurenine (Kyn) levels in BxPC-3 cells upon stimulation with the cytokine interferon-{gamma} (IFN-{gamma}) and subsequent target identification employing affinity-based chemical proteomics. Increased Kyn levels are associated with immune suppression in the tumor microenvironment and, thus, the Kyn pathway and its key player indoleamine 2,3-dioxygenase 1 (IDO1) are appealing targets in immuno-oncology. However, targeting IDO1 directly has led to limited success in clinical investigations, demonstrating that alternative approaches to reduce Kyn levels are in high demand. We uncover a cross-talk between sEH and the Kyn pathway that may provide new opportunities to revert cancer-induced immune tolerance.

cell biology↗

bioRxiv2023

P116 from Mycoplasma is a self-sufficient lipid uptake and delivery machinery

Lipid acquisition and transport are fundamental processes in all organisms. Here, we investigate the lipid uptake and delivery mechanism of the minimal model organism Mycoplasma pneumoniae. We show that the essential protein P116 can transport lipids between liposomes independently and without ATP consumption. Our structural data and molecular dynamics simulations reveal the mechanism by which the N-terminal region of P116 perturbs the membrane, the lipid transfer route, and the regulation of membrane binding by the cargo mass within P116s large hydrophobic cavity. When adequately filled with cargo, P116 undergoes a rapid conformational change that modulates membrane binding. Taken together, our results show that Mycoplasma developed one integrated lipid uptake and delivery machinery that simplifies the complex multi-protein pathways used by higher developed organisms.

molecular biology↗

bioRxiv2023

Synthesis of pyrazole-based macrocycles leads to a highly selective inhibitor for MST3

MST1, MST2, MST3, MST4, and YSK1 are conserved members of the mammalian sterile 20 kinase (MST) family. MSTs regulate key cellular functions such as cell proliferation, cell migration, metabolic regulation, and cell polarity. The MST3 isozyme plays a role in regulation of cell growth, autophagy and apoptosis, and its dysregulation has been linked to the occurrence of high-grade tumors with poor survival prognosis. To date, there are no isoform-selective inhibitors available that could be used for validating the role of MST3 in tumorigenesis and to assess its potential as an anti-cancer target for drug development. To this end, we have designed a new series of 3-aminopyrazole-based macrocycles based on the structure of an acyclic promiscuous kinase inhibitor. By varying moieties targeting the solvent-exposed region and optimizing the linker, macrocycle JA310 (21c) was synthesized. JA310 exhibited high cellular potency for MST3 with an EC50 = 106 nM and excellent kinome-wide selectivity with significantly lower cellular activity on the closely related kinase MST4 (EC50 = 1.4 {micro}M). The high-resolution crystal structure of the MST3-JA310 complex provided intriguing insights into the distinct binding mode of the macrocycle, which was associated with large-scale structural rearrangements, including concerted induced-fit movements of the glycine-rich loop, the C helix, and the activation loop. In summary, the developed macrocyclic MST3 inhibitor, JA310, demonstrates the utility of macrocyclization for the design of highly selective inhibitors and presents a first chemical probe for MST3.

biochemistry↗

bioRxiv2023

Discovery of a new activator of Slack potassium channels with robust efficacy in models of histamine-independent and chronic itch

Various disorders are accompanied by histamine-independent itching, which is often resistant to the currently available therapies. In this study, we hypothesized that pharmacological activation of Slack (Kcnt1, KNa1.1), a potassium channel highly expressed in itch-sensitive sensory neurons, has therapeutic potential for the treatment of itching. Based on the Slack-activating antipsychotic drug, loxapine, we designed a series of new derivatives with improved pharmacodynamic and pharmacokinetic profiles that enabled us to validate Slack as a pharmacological target in vivo. One of these new Slack activators, compound 6, exhibited negligible dopamine D2 and D3 receptor binding, unlike loxapine. We found that compound 6 displayed potent on-target antipruritic activity in multiple mouse models of acute histamine-independent and chronic itch without motor side effects. These properties make compound 6 a lead molecule for the development of new antipruritic therapies targeting Slack.

pharmacology and toxicology↗

bioRxiv2023

Targeting LC3/GABARAP for degrader development and autophagy modulation

Recent successes in developing small-molecule degraders that act through the ubiquitin system have spurred efforts to extend this technology to other mechanisms, including the autophagosomal-lysosomal pathway. Therefore, reports of autophagosome tethering compounds (ATTECs) have received considerable attention from the drug development community. ATTECs are based on the target recruitment to LC3/GABARAP, a family of membrane-bound proteins that tether autophagy receptors to the autophagosome. In order to validate the existing ligands, we rigorously tested target engagement of reported ATTEC ligands and handles. Surprisingly, using various biophysical methods, most available ligands did not interact with their designated target LC3. Intrigued by the idea of developing ATTECs, we evaluated the druggability of LC3/GABARAP by in silico docking and large scale crystallographic fragment screening. The data revealed that most fragments bound to the HP2, but not the HP1 pocket of the LC3-interacting region (LIR) docking site, suggesting favorable druggability of this binding pocket. Here, we present diverse comprehensively validated ligands for future ATTEC development.

biochemistry↗

bioRxiv2023

Inhibition of Parkinsons Disease-related LRRK2 by type-I and type-II kinase inhibitors: activity and structures

Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are a common cause of familial Parkinsons Disease (PD), and a risk factor for the sporadic form. Increased kinase activity has been shown in both familial and sporadic PD patients, making LRRK2 kinase inhibitors a major focus of drug development efforts in PD. Although significant progress has been made in understanding the structural biology of LRRK2, there are no available structures of LRRK2 inhibitor complexes. To this end, we solved cryo-EM structures of LRRK2, wild-type and PD-linked mutants, bound to the LRRK2-specific type-I inhibitor MLi-2 and the broad-spectrum type-II inhibitor GZD-824. Our structures revealed LRRK2s kinase in the active-like state, stabilized by type-I inhibitor interactions, and an inactive DYG-out type-II inhibitor complex. Our structural analysis also showed how inhibitor-induced conformational changes in LRRK2 are affected by its autoinhibitory N-terminal repeats. The structural models provide a template for the rational development of LRRK2 kinase inhibitors covering both canonical inhibitor binding modes.

biophysics↗

bioRxiv2023

Pathological mutations reveal the key role of the iRhom2 N-terminus for ADAM17 binding, stability and activity.

The protease ADAM17 plays an important role in inflammation and cancer and is regulated by iRhom2. Mutations in the cytosolic N-terminus of human iRhom2 cause tylosis with oesophageal cancer (TOC). In mice, partial deletion of the N-terminus results in a curly hair phenotype (cub). These pathological consequences are consistent with our findings that iRhom2 is highly expressed in keratinocytes and in oesophageal cancer. Cub and TOC are associated with hyperactivation of ADAM17-dependent EGFR signalling. However, the underlying molecular mechanisms are not understood. We have identified a non-canonical, phosphorylation-independent 14-3-3 interaction site that encompasses all known TOC mutations. Disruption of this site dysregulates ADAM17 activity. The larger cub deletion also includes the TOC site and thus also dysregulated ADAM17 activity. The cub deletion, but not the TOC mutation, also causes severe reductions in stimulated shedding, binding, and stability of ADAM17, demonstrating the presence of additional regulatory sites in the N-terminus of iRhom2. Overall, this study contrasts the TOC and cub mutations, illustrates their different molecular consequences, and reveals important key functions of the iRhom2 N-terminus in regulating ADAM17.

molecular biology↗

bioRxiv2023

Development of potent dual BET/HDAC inhibitors via pharmacophore merging and structure-guided optimization

Bromodomain and extra-terminal motif (BET) proteins and histone deacetylases (HDACs) are prime targets in cancer therapy. Recent research has particularly focused on the development of dual BET/HDAC inhibitors for hard-to-treat tumors such as pancreatic cancer. Here, we have developed a new series of potent dual BET/HDAC inhibitors by choosing starting scaffolds that enabled us to optimally merge the two functionalities into a single compound. Systematic structure-guided modification of both warheads then led to optimized binders that were superior in potency to both parent compounds, with the best molecules of this series binding to both BRD4 bromodomains as well as HDAC1/2 with EC50 values in the 100-nanomolar range in cellular NanoBRET target engagement assays. Importantly, this on-target activity also translated into promising efficacy in pancreatic cancer and NUT midline carcinoma cells. Our lead molecules effectively blocked histone H3 deacetylation in pancreatic cancer cells and upregulated the tumor suppressor HEXIM1 and proapoptotic p57, both markers of BET inhibition. In addition, they have the potential to downregulate oncogenic drivers of NUT midline carcinoma, as demonstrated for MYC and TP63 mRNA levels. Overall, this study expands the portfolio of available dual BET/class I HDAC inhibitors for future translational studies in different cancer models.

biochemistry↗

bioRxiv2023

Urbanisation generates multiple trait syndromes for terrestrial taxa worldwide

Cities can host significant biological diversity. Yet, urbanisation leads to the loss of habitats and, potentially, to local extinctions. Understanding how multiple taxa respond to urbanisation globally is essential to promote and conserve biodiversity in cities and surrounding landscapes. Using a dataset with site-level occurrence and trait data of 5302 species from six terrestrial fauna taxonomic groups across 379 cities on 6 continents, we show that urbanisation produces taxon-specific changes in trait composition, with traits related to reproductive strategy consistently showing the strongest response. The effect of urbanisation on community trait composition is strongest at the largest spatial scale considered, and more closely linked to landscape composition (% urban) than arrangement (aggregation), although latitude and climatic variables remain a stronger influence. This study did not find evidence in support of a global urban taxa syndrome, but instead we suggest that there are four general urban trait syndromes, with resources associated with reproduction and diet likely to be driving patterns in traits associated with mobility and body size. Functional diversity measures showed a wide range of responses, leading to a shift in trait space that is most likely driven by the distribution and abundance of critical resources, and the urban trait syndrome displayed by individual species within a community. Further research is required to understand the interactions between the four general urban trait syndromes, resource distribution and abundance and changes in functional diversity of taxa at different spatial and temporal scales. Maximising opportunities to support species within taxa groups with different urban trait syndromes should be pivotal in conservation and management programmes within and among cities. This will reduce the likelihood of biotic homogenisation at the taxa level, and helps ensure that urban environments have the ecological capacity to respond to challenges such as climate change, further habitat fragmentation and loss, and other disruptions. These actions are critical if we are to reframe the role of cities in global biodiversity loss.

ecology↗

bioRxiv2023

TREM2 limits necrotic core formation during atherogenesis by controlling macrophage survival and efferocytosis

Atherosclerosis is a chronic disease of the vascular wall driven by lipid accumulation and inflammation in the intimal layer of arteries [1], [2], and its main complications, myocardial infarction and stroke, are the leading cause of mortality worldwide [3]. Recent studies have identified Triggering receptor expressed on myeloid cells 2 (TREM2), a lipid-sensing receptor regulating several key myeloid cell functions [4], as a highly expressed marker of macrophage foam cells in experimental and human atherosclerosis [5]. However, the function of TREM2 in the development of atherosclerosis is unknown. Here, we show that hematopoietic or global TREM2 deficiency increases necrotic core formation in early experimental atherosclerosis. We further demonstrate that TREM2 is essential for the efferocytosis capacities of macrophages, and to the survival of lipid-laden macrophages, altogether indicating a crucial role of TREM2 in maintaining the balance between foam cell death and their clearance in atherosclerotic lesions, thereby controlling plaque necrosis.

immunology↗

bioRxiv2023

Shifting the selectivity of pyridopyrimidin-7(8H)-one inhibitors towards the salt-inducible kinase (SIK) subfamily

Salt-inducible kinases 1-3 (SIK1-3) are key regulators of the LKB1-AMPK pathway and play an important role in cellular homeostasis. Dysregulation of any of the three isoforms has been associated with tumorigenesis in liver, breast, and ovarian cancers. We have recently developed the dual pan-SIK/group I p21-activated kinase (PAK) chemical probe MRIA9. However, inhibition of p21-activated kinases has been associated with cardiotoxicity in vivo, which complicates the use of MRIA9 as a tool compound. Here, we present a structure-based approach involving the back-pocket and gatekeeper residues, for narrowing the selectivity of pyrido[2,3-d]pyrimidin-7(8H)-one-based inhibitors towards SIK kinases, eliminating PAK activity. Optimization was guided by high-resolution crystal structure analysis and computational methods, resulting in a pan-SIK inhibitor, MR22, which no longer exhibited activity on STE group kinases and displayed excellent selectivity in a representative kinase panel. MR22-dependent SIK inhibition led to centrosome dissociation and subsequent cell-cycle arrest in ovarian cancer cells, as observed with MRIA9, conclusively linking these phenotypic effects to SIK inhibition. Taken together, MR22 represents a valuable tool compound for studying SIK kinase function in cells.

biochemistry↗

bioRxiv2023

5-iodotubercidin sensitizes cells to RIPK1-dependent necroptosis by interfering with NFkappaB signaling

Receptor-interacting protein kinases (RIPK) -1 and -3 are master regulators of cell fate decisions in response to diverse stimuli and are subjected to multiple checkpoint controls. Earlier studies have established the presence of distinct IKK1/2 and p38/MK2-dependent checkpoints which suppress RIPK1 activation by directly phosphorylating it at different residues. In the present study, we investigated TNF-induced death in MAPK-activated protein kinase 2 (MK2)-deficient cells and show that MK2-deficiency or inactivation predominantly results in necroptotic cell death, even in the absence of caspase inhibition. While MK2-deficient cells can be rescued from necroptosis by RIPK1 inhibitors, RIPK3 inhibition seems to revert the process triggering apoptosis. To understand the mechanism of this necroptosis switch, we screened a 149-compound kinase inhibitor library for compounds which preferentially sensitize MK2-deficient MEFs to TNF-induced cell death. The most potent inhibitor identified was 5-Iodotubericidin, an adenosine analogue acting as adenosine kinase and protein kinase inhibitor. 5-ITu also potentiated LPS-induced necroptosis when combined with MK2 inhibition in RAW264.7 macrophages. Further mechanistic studies revealed that 5-Iodotubericidin induces RIPK1-dependent necroptosis in the absence of MK2 activity by suppressing IKK signaling. The identification of this role for the multitarget kinase inhibitor 5-ITu in TNF-, LPS- and chemotherapeutics-induced necroptosis will have potential implications in RIPK1-targeted therapies.

cell biology↗

bioRxiv2023

Morphological trait evolution in Solanum (Solanaceae): evolutionary lability of key taxonomic characters

Solanum L. is one of the worlds largest and economically most important plant genera, including 1,245 currently accepted species and several major and minor crops (e.g., tomato, potato, brinjal eggplant, scarlet eggplant, Gboma eggplant, lulo, and pepino). Here we provide an overview of the evolution of 25 key morphological traits for the major and minor clades of this giant genus based on stochastic mapping using a well-sampled recently published phylogeny of Solanum. The most evolutionarily labile traits (showing >150 transitions across the genus) relate to plant structure (growth form and sympodial unit structure), herbivore defence (glandular trichomes), pollination (corolla shape and colour), and dispersal (fruit colour). Ten further traits show evolutionary lability with 50-100 transitions across the genus (e.g., specialised underground organs, trichome structure, leaf type, inflorescence position and branching, stamen heteromorphism). Our results reveal a number of highly convergent traits in Solanum, including tubers, rhizomes, simple leaves, yellow corollas, heteromorphic anthers, dioecy, and dry fruits, and some unexpected pathways of trait evolution that could be explored in future studies. We show that informally named clades of Solanum can be morphologically defined by trait combinations providing a tool for identification and enabling predictive phylogenetic placement of unsampled species.

evolutionary biology↗

bioRxiv2023

Mast cell-derived BH4 is a critical mediator of postoperative pain

Postoperative pain affects most patients after major surgery and can transition to chronic pain. Here, we discovered that postoperative pain hypersensitivity correlated with markedly increased local levels of the metabolite BH4. Gene transcription and reporter mouse analyses after skin injury identified neutrophils, macrophages and mast cells as primary postoperative sources of GTP cyclohydrolase-1 (Gch1) expression, the rate-limiting enzyme in BH4 production. While specific Gch1 deficiency in neutrophils or macrophages had no effect, mice deficient in mast cells or mast cell-specific Gch1 showed drastically decreased postoperative pain after surgery. Skin injury induced the nociceptive neuropeptide substance P, which directly triggers the release of BH4-dependent serotonin in mouse and human mast cells. Substance P receptor blockade substantially ameliorated postoperative pain. Our findings underline the unique position of mast cells at the neuro-immune interface and highlight substance P-driven mast cell BH4 production as promising therapeutic targets for the treatment of postoperative pain.

immunology↗

bioRxiv2023

A chemical probe to modulate human GID4 Pro/N-degron interactions

The CTLH complex is a multi-subunit ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via the substrate receptor GID4. Recently, focus has turned to this complex as a potential mediator of targeted protein degradation, but the role GID4-mediated substrate ubiquitylation and proteasomal degradation plays in humans has thus far remained unclear. Here, we report PFI-7, a potent, selective, and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation enabled the identification of dozens of endogenous GID4-interacting proteins that bind via the GID4 substrate binding pocket, only a subset of which possess canonical Pro/N-degron sequences. GID4 interactors are enriched for nuclear and nucleolar proteins including RNA helicases. GID4 antagonism by PFI-7 altered protein levels of several proteins including RNA helicases as measured by label-free quantitative proteomics, defining proteins that are regulated by GID4 and the CTLH complex in humans. Interactions with GID4 via Pro/N-degron pathway did not result in proteasomal degradation, demonstrating that CTLH interactors are regulated through a combination of degradative and non-degradative functions. The lack of degradation of GID4 interactors highlights potential challenges in utilizing GID4-recruiting bifunctional molecules for targeted protein degradation. Going forward, PFI-7 will be a valuable research tool for defining CTLH complex biology and honing targeted protein degradation strategies.

molecular biology↗

bioRxiv2023

Death by a Thousand Cuts -- Combining Kinase Inhibitors for Selective Target Inhibition and Rational Polypharmacology

Kinase inhibitors are successful therapeutics in the treatment of cancers and autoimmune diseases and are useful tools in biomedical research. The high sequence and structural conservation of the catalytic kinase domain complicates the development of specific kinase inhibitors. As a consequence, most kinase inhibitors also inhibit off-target kinases which complicates the interpretation of phenotypic responses. Additionally, inhibition of off-targets may cause toxicity in patients. Therefore, highly selective kinase inhibition is a major goal in both biomedical research and clinical practice. Currently, efforts to improve selective kinase inhibition are dominated by the development of new kinase inhibitors. Here, we present an alternative solution to this problem by combining inhibitors with divergent off-target activities. We have developed a multicompound-multitarget scoring (MMS) method framework that combines inhibitors to maximize target inhibition and to minimize off-target inhibition. Additionally, this framework enables rational polypharmacology by allowing optimization of inhibitor combinations against multiple selected on-targets and off-targets. Using MMS with previously published chemogenomic kinase inhibitor datasets we determine inhibitor combinations that achieve potent activity against a target kinase and that are more selective than the most selective single inhibitor against that target. We validate the calculated effect and selectivity of a combination of inhibitors using the in cellulo NanoBRET assay. The MMS framework is generalizable to other pharmacological targets where compound specificity is a challenge and diverse compound libraries are available.

pharmacology and toxicology↗