Biology subjects

Kraemer, A.

Publications and source records attributed to Kraemer, A..

18 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

Comprehensive ctDNA Profiling Enables Tissue-of-Origin Prediction and Actionable Biomarker Detection in Cancer of Unknown Primary

Most patients with cancer of unknown primary (CUP) still receive platinum-based chemotherapy and have a poor prognosis, with overall survival of less than one year. Recent studies suggest improved outcomes with molecularly guided or site-specific therapies informed by molecular tissue profiling. Here, we analyzed ctDNA from 190 CUP patients using an integrated genomic and epigenomic assay to identify actionable alterations and predict tissue-of-origin (ToO). Integration of actionable biomarkers, ToO prediction and clinical data yielded diagnostic, prognostic or therapeutic information in 90% of unfavorable CUP cases and 88% of patients analyzed at first diagnosis. High ctDNA tumor fraction was associated with poorer prognosis in both favorable and unfavorable CUP. These findings highlight the clinical utility of ctDNA analysis for therapeutic decision-making in CUP and support its incorporation into the diagnostic work-up, particularly when tissue samples are unavailable or insufficient for molecular testing.

Cancer Biology↗

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↗

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

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

Covalent targeting leads to the development of LIMK1 isoform-selective inhibitors

Selectivity for closely related isoforms of protein kinases is a major challenge in the design of drugs and chemical probes. Covalent targeting of unique cysteines is a potential strategy to achieve selectivity for highly conserved binding sites. Here, we used a pan-LIMK inhibitor to selectively probe LIMK1 over LIMK2 by targeting the LIMK1-specific cysteine C349 located in the glycine-rich loop region. Binding kinetics of both non-covalent and covalent LIMK inhibitors were investigated, and the fast on-rate and small size of type-I inhibitors were used in the design of a covalent LIMK1 inhibitor. The developed cell-active, isoform-selective LIMK1 inhibitor showed excellent proteome-wide selectivity in pull-down assays, enabling studies of LIMK1 isoform-selective functions in cellular model systems and providing a versatile chemical tool for studies of the LIMK signalling pathway.

biochemistry↗

bioRxiv2025

Repurposing of the RIPK1 selective benzooxazepin-4-one scaffold for the development of a type-III LIMK1/2 inhibitor

Benzoxazepinones have been extensively studied as exclusively selective RIP kinase 1 inhibitors. This scaffold binds as a type-III inhibitor targeting the C-out/DFG-out conformation. This inactive conformation results in a large expansion of the kinase back pocket, a conformation that has also been reported for LIM kinases. Scaffold hopping is common in the design of orthosteric kinase inhibitors, but has not been explored in the design of allosteric inhibitors, mainly due to the typically exclusive selectivity of type III inhibitors. Here, we hypothesized that the shared structural properties of LIMKs and RIPKs could lead to novel type III LIMK inhibitors using the benzoxazepinone scaffold. We report the discovery of a novel LIMK1/2 inhibitor that relies on this scaffold-based approach. The discovered compound 10 showed low nanomolar potency on LIMK1/2 and exceptional selectivity, as confirmed by a comprehensive selectivity panel with residual RIPK activity as the only off-target. The study provides one of the few examples for scaffold hopping for type-III inhibitors which are usually associated with exclusive target selectivity. Table of contents (TOC) graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/636296v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@19bad48org.highwire.dtl.DTLVardef@18d8929org.highwire.dtl.DTLVardef@1280043org.highwire.dtl.DTLVardef@1296ddf_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Synthesis and evaluation of chemical linchpins for highly selective CK2α targeting

Casein kinase-2 (CK2) are serine/threonine kinases with dual co-factor (ATP and GTP) specificity, that are involved in the regulation of a wide variety of cellular functions. Small molecules targeting CK2 have been described in the literature targeting different binding pockets of the kinase with a focus on type I inhibitors such as the recently published chemical probe SGC-CK2-1. In this study, we investigated whether known allosteric inhibitors binding to a pocket adjacent to helix D could be combined with ATP mimetic moieties defining a novel class of ATP competitive compounds with a unique binding mode. Linking both binding sites requires a chemical linking moiety that would introduce a 90-degree angle between the ATP mimetic ring system and the D targeting moiety, which was realized using a sulfonamide. The synthesized inhibitors were highly selective for CK2 with binding constants in the nM range and low micromolar activity. While these inhibitors need to be further improved, the present work provides a structure-based design strategy for highly selective CK2 inhibitors.

biochemistry↗

bioRxiv2024

Back-pocket optimization of 2-aminopyrimidine-based macrocycles leads to potent dual EPHA2/GAK kinase inhibitors with antiviral activity

Macrocyclization of acyclic compounds is a powerful strategy for improving inhibitor potency and selectivity. Here, we developed a 2-aminopyrimidine-based macrocyclic dual EPHA2/GAK kinase inhibitor as a chemical tool to study the role of these two kinases in viral entry and assembly. Starting with a promiscuous macrocyclic inhibitor, 6, we performed a structure-guided activity relationship and selectivity study using a panel of over 100 kinases. The crystal structure of EPHA2 in complex with the developed macrocycle 23 provided a basis for further optimization by specifically targeting the back pocket, resulting in compound 55 as a potent dual EPHA2/GAK inhibitor. Subsequent front-pocket derivatization resulted in an interesting in cellulo selectivity profile, favoring EPHA4 over the other ephrin receptor kinase family members. The dual EPHA2/GAK inhibitor 55 prevented dengue virus infection of Huh7 liver cells, mainly via its EPHA2 activity, and is therefore a promising candidate for further optimization of its activity against dengue virus.

biochemistry↗

bioRxiv2023

STIL overexpression shortens lifespan and reduces tumor formation in mice

Centrosomes are the major microtubule organizing centers of animal cells. Supernumerary centrosomes are a common feature of human tumors and associated with karyotype abnormalities and aggressive disease, but whether they are cause or consequence of cancer remains controversial. Here, we analyzed the consequences of centrosome amplification by generating transgenic mice in which centrosome numbers can be increased by overexpression of the structural centrosome protein STIL. We show that STIL overexpression induces centrosome amplification and aneuploidy, leading to senescence, apoptosis, and impaired proliferation in mouse embryonic fibroblasts, and microcephaly with increased perinatal lethality and shortened lifespan in mice. Importantly, both overall tumor formation in mice with constitutive, global STIL overexpression and chemical skin carcinogenesis in animals with inducible, skin-specific STIL overexpression were reduced, an effect that was not rescued by concomitant p53 inactivation. These results suggest that supernumerary centrosomes impair proliferation in vitro as well as in vivo, resulting in reduced lifespan and spontaneous as well as carcinogen-induced tumor formation.

cancer biology↗

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

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

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

Tracking the PROTAC degradation pathway in living cells highlights the importance of ternary complex measurement for PROTAC optimization

The multistep PROTAC (PROteolysis TArgeting Chimeras) degradation process poses challenges for their rational development, as rate limiting steps determining PROTAC efficiency remain largely unknown. Moreover, the slow throughput of currently used endpoint assays does not allow the comprehensive analysis of larger series of PROTACs. Here we developed cell-based assays using NanoLuciferase and HaloTags, that allow measuring PROTAC induced degradation and ternary complex formation kinetics and stability in cells. Using PROTACs developed for degradation of WDR5, the characterization of the mode of action of these PROTACs in the early degradation cascade revealed a key role of ternary complex formation and stability. Comparing a series of ternary complex crystal structures highlighted the importance of an efficient E3-target interface for ternary complex stability. The developed assays outline a strategy for the rational optimization of PROTACs using a series of live cell assays monitoring key steps of the early PROTAC induced degradation pathway. SignificanceThe multistep PROTAC induced degradation process of a POI poses a significant challenge for the rational design of these bifunctional small molecules as critical steps that limit PROTAC efficacy cannot be easily assayed at required throughput. In addition, the cellular location of the POI may pose additional challenges as some cellular compartments, such as the nucleus, may not be easily reached by PROTAC molecules and the targeted E3 ligases may not be present in this cellular compartment. We propose therefore a comprehensive assay panel for PROTACs evaluation in cellular environments using a sensor system that allows continuous monitoring of the protein levels of the endogenous POI. We developed a cell line expressing WDR5 from its endogenous locus in fusion with a small sequence tag (HiBIT) that can be reconstituted to functional NanoLuciferase (NLuc). This system allowed continuous monitoring of endogenous WDR5 levels in cells and together with HaloTag system also the continuous monitoring of ternary complex (E3, WDR5 and PROTAC) formation. As this assay can be run at high throughput, we used this versatile system monitoring three diverse chemical series of WDR5 PROTACs that markedly differ in their degradation properties. Monitoring cell penetration, binary complex formation (PROTAC-WDR5 and PROTAC-VHL) as well as ternary complex formation we found that PROTAC efficiency highly correlated with synergy of ternary complex formation in cells. This study represents a first data set on diverse PROTACs studying this property in cellulo and it outlines a strategy for the rational optimization of PROTACs. It also provided kinetic data on ternary complex assembly and dissociation that may serve as a benchmark for future studies utilizing also kinetic properties for PROTAC development. Comparative structural studies revealed larger PROTAC mediated interaction surfaces for PROTACs that efficiently formed ternary complexes highlighting the utility of structure based optimization of PROTAC induced ternary complexes in the development process.

molecular biology↗

bioRxiv2021

Design, Synthesis and Evaluation of WD-repeat containing protein 5 (WDR5) degraders

Histone H3K4 methylation serves as post-translational hallmark of actively transcribed genes and is introduced by histone methyltransferases (HMT) and its regulatory scaffolding proteins. One of these is the WD-repeat containing protein 5 (WDR5) that has also been associated with controlling long non-coding RNAs and transcription factors including MYC. The wide influence of dysfunctional HMTs complexes and the typically upregulated MYC levels in diverse tumor types suggested WDR5 as an attractive drug target. Indeed, protein-protein interface inhibitors for two protein interaction interfaces on WDR5 have been developed. While such compounds only inhibit a subset of WDR5 interactions, chemically induced proteasomal degradation of WDR5 might represent an elegant way to target all oncogenic function. This study presents the design, synthesis and evaluation of two diverse WDR5 degrader series based on two WIN site binding scaffolds and shows that linker nature and length strongly influence degradation efficacy.

cancer biology↗

bioRxiv2020

The evolution of trait variance creates a tension between species diversity and functional diversity

It seems intuitive that species diversity promotes functional diversity. For example, more plant species imply more diverse leaf chemistry and thus more kinds of food for herbivores. Here we argue that the evolution of functional trait variance challenges this view. We show that trait-based eco-evolutionary processes force species to evolve narrower trait breadths in tightly packed communities, in their effort to avoid competition with neighboring species. This effect is so strong as to reduce overall trait space coverage, overhauling the expected positive relationship between species- and functional diversity. Empirical data from Galápagos land snail communities proved consistent with this claim. As a consequence, trait data from species-poor communities may misjudge functional diversity in species-rich ones, and vice versa.Competing Interest StatementThe authors have declared no competing interest.View Full Text

ecology↗

bioRxiv2020

Optimization of pyrazolopyrimidines lead to the identification of a highly selective casein kinase 2 inhibitor

ABSTRACTCasein kinase 2 (CK2) is a constitutively expressed serine/threonine kinase that has a large diversity of cellular substrates. Thus, CK2 has been associated with a plethora of regulatory functions and dysregulation of CK2 has been linked to disease development in particular to cancer. The broad implications in disease pathology makes CK2 an attractive target. To date, the most advanced CK2 inhibitor is silmitasertib, which has been investigated in clinical trials for treatment of various cancers, albeit several off-targets for silmitasertib have been described. To ascertain the role of CK2 inhibition in cancer, other disease and normal physiology the development of a selective CK2 inhibitor would be highly desirable. In this study we explored the pyrazolo[1,5-a]pyrimidine hinge-binding moiety for the development of selective CK2 inhibitors. Optimization of this scaffold, which included macrocyclization, led to IC20 (31) a compound that displayed high in vitro potency for CK2 (KD = 12 nM) and exclusive selectivity for CK2. X-ray analysis revealed a canonical type-I binding mode for IC20. However, the polar carboxylic acid moiety that is shared by many CK2 inhibitors including silmitasertib was required for potency and reduced somewhat cellular activity. In summary, IC20 represents a highly selective and potent inhibitor of CK2, which can be used as a tool compound to study CK2 biology and potential new applications for the treatment of diseases.Notes The authors declare no conflict of interest.Competing Interest StatementThe authors have declared no competing interest.Abbreviations2-MTHF2-methyltetrahydrofuranATPAdenosine triphosphateB2pin2Bis(pinacolato)diboronCK2Casein kinase 2DIADdiisopropyl azodicarboxylateDIPEAN,N-diisopropylethylamineHEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidIPTGIsopropyl-thio-galactopyranosideMES2-(N-morpholino)ethanesulfonic acidMGCmammalian gene collectionPEG MMEpolyethylene glycol monomethyl etherP(Cy)3TricyclohexylphosphineTCEPTris(2-carboxyethyl)phosphineTEVTobacco Etch VirusTf2Otrifluoromethanesulfonic anhydrideTPP triphenylphosphine;XPhos2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenylView Full Text

biochemistry↗