Biology subjects

Knapp, S.

Publications and source records attributed to Knapp, S..

At least 91 records · Page 5Linked to original sources

bioRxiv2021

Structure and inhibitor binding characterization of oncogenic MLLT1 mutants

Dysfunction of YEATS-domain-containing MLLT1, an acetyl/acyl-lysine dependent epigenetic reader domain, has been implicated in the development of aggressive cancers. Mutations in the YEATS domain have been recently reported as a cause of MLLT1 aberrant reader function. However, structural basis for the reported alterations in affinity for acetyled/acylated histone has remained elusive. Here, we report the crystal structures of both insertion and substitution present in cancer, revealing significant conformational changes of the YEATS-domain loop 8. Structural comparison demonstrates that such alteration not only altered the binding interface for acetylated/acylated histones, but the sequence alterations in the T1 loop may enable dimeric assembly consistent inducing self-association behavior. Nevertheless, we show that also the MLLT1 mutants can be targeted by developed acetyllysine mimetic inhibitors with affinities similarly to wild type. Our report provides a structural basis for the altered behaviors and potential strategy for targeting oncogenic MLLT1 mutants.

biochemistry↗

bioRxiv2020

Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms

Much attention has focused on LRRK2, as autosomal dominant missense mutations that enhance its kinase activity cause inherited Parkinsons disease. LRRK2 regulates biology by phosphorylating a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. In this study we explore whether LRRK1, a less studied homologue of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that the endogenous Rab7A protein, phosphorylated at Ser72 was most impacted by LRRK1 knock-out. This residue is not phosphorylated by LRRK2 but lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinsons mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous Type-2 tyrosine kinase inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. Finally, we show that interaction of Rab7A with its effector RILP is not affected by high stoichiometry LRRK1 phosphorylation. Altogether, these finding reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology.

biochemistry↗

bioRxiv2020

Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2

In a multi-tiered approach, we explored how Parkinsons Disease-related mutations hijack the finely tuned activation process of Leucine-Rich Repeat Kinase 2 (LRRK2) using a construct containing the ROC, Cor, Kinase and WD40 domains (LRRK2RCKW). We hypothesized that the N-terminal domains shield the catalytic domains in an inactive state. PD mutations, type-I LRRK2 inhibitors, or physiological Rab GTPases can unleash the catalytic domains while the active kinase conformation, but not kinase activity, is essential for docking onto microtubules. Mapping solvent accessible regions of LRRK2RCKW employing hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed how inhibitor binding is sensed by the entire protein. Molecular Dynamics simulations of the kinase domain elucidated differences in conformational dynamics between wt and mutants of the DYG{psi} motif. While all domains contribute to regulating kinase activity and spatial distribution, the kinase domain, driven by the DYG{psi} motif, coordinates domain crosstalk and serves as an intrinsic hub for LRRK2 regulation.

biochemistry↗

bioRxiv2020

DFG-1 residue controls inhibitor binding mode and affinity providing a basis for rational design of kinase inhibitor selectivity

ABSTRACTSelectivity remains a challenge for ATP-mimetic kinase inhibitors, an issue that may be overcome by targeting unique residues or binding pockets. However, to date only few strategies have been developed. Here we identify that bulky residues located N-terminal to the DFG motif (DFG-1) represent an opportunity for designing highly selective inhibitors with unexpected binding modes. We demonstrate that several diverse inhibitors exerted selective, non-canonical binding modes that exclusively target large hydrophobic DFG-1 residues present in many kinases including PIM, CK1, DAPK and CLK. Using the CLK family as a model, structural and biochemical data revealed that the DFG-1 valine controlled a non-canonical binding mode in CLK1, providing a rational for selectivity over the closely-related CLK3 which harbors a smaller DFG-1 alanine. Our data suggests that targeting the restricted back pocket in the small fraction of kinases that harbor bulky DFG-1 residues offers a versatile selectivity filter for inhibitor design.Competing Interest StatementThe authors have declared no competing interest.ABBREVIATIONSCLKCdc2 like kinasePIMProviral insertion in murine kinaseACVR1Activin receptor type IDAPKdeath-associated protein kinaseDYRKdual specificity tyrosine-phosphorylation-regulated kinasesDRAKDAP kinase-related apoptosis-inducing protein kinasesCKcasein kinaseRETProto-oncogene tyrosine-protein kinase receptor RetCDK2Cyclin-dependent kinase 2DFG motiftripeptide Asp-Phe-Gly motifVIAK motifvaline-isoleucine-alanine-lysine motif.View Full Text

biochemistry↗

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↗

bioRxiv2020

Nucleotide binding, evolutionary insights and interaction partners of the pseudokinase Unc-51-like kinase 4

Unc-51-like kinase 4 (ULK4) is a pseudokinase that has been linked to the development of several diseases. Even though sequence motifs required for ATP binding in kinases are lacking, ULK4 still tightly binds ATP and the presence of the cofactor is required for structural stability of ULK4. Here we present a high-resolution structure of a ULK4-ATP{gamma}S complex revealing a highly unusual ATP binding mode in which the lack of the canonical VAIK motif lysine is compensated by K39, located N-terminal to C. Evolutionary analysis suggests that degradation of active site motifs in metazoan ULK4 has co-occurred with an ULK4 specific activation loop, which stabilizes the C-helix. In addition, cellular interaction studies using BioID and biochemical validation data revealed high confidence interactors of the pseudokinase and armadillo repeat domains. Many of the identified ULK4 interaction partners were centrosomal and tubulin associated proteins and several active kinases suggesting new roles for ULK4. HighlightsStructure of the ULK4 ATP complex reveals a unique ATP binding mode. Disease associated mutations modulate ATP binding and ULK4 stability Degradation of active site motifs co-occurred in evolution with an ULK4 specific activation loop BioID suggests a role of ULK4 regulating centrosomal and cytoskeletal functions

biochemistry↗

bioRxiv2020

Catalytic domain plasticity of MKK7 reveals structural mechanisms of allosteric activation and new targeting opportunities

MKK7 (MEK7) is a key regulator of the JNK stress signaling pathway and targeting MKK7 has been proposed as a chemotherapeutic strategy. Detailed understanding of the MKK7 structure and factors that impact its activity is therefore of critical importance. Here, we present a comprehensive set of MKK7 crystal structures revealing insights into catalytic domain plasticity and the role of the N-terminal regulatory helix, conserved in all MAP2Ks, mediating kinase activation. Crystal structures harboring this regulatory helix revealed typical structural features of active kinase, providing exclusively a first model of the MAP2K active state. A small molecule screening campaign yielded multiple scaffolds, including type-II irreversible inhibitors a binding mode that has not been reported previously. We also observed an unprecedented allosteric pocket located in the N-terminal lobe for the approved drug ibrutinib. Collectively, our structural and functional data expand and provide alternative targeting strategies for this important MAP2K kinase.

biochemistry↗

bioRxiv2020

STK32A is a dual-specificity AGC kinase with a preference for acidic substrates

The STK32 kinases are a small subfamily of three uncharacterised serine/threonine kinases from the AGC kinase family whose functional role is so far unknown. Here, we analyse the consensus peptide sequence for STK32A phosphorylation, showing that STK32A is directed towards acidic substrate sequences and exhibits dual-specificity for serine/threonine and tyrosine residues. A crystal structure of STK32A reveals an overall structure typical of the AGC protein kinase family but with significant and unique features including an altered binding mode of the hydrophobic motif to the N-terminal lobe of the kinase domain, and a novel alpha-helix in between the turn motif and the hydrophobic motif. The crystal structure combined with phylogenetic analysis reveals the evolutionary conservation of the acidic substrate preference. In vitro binding assays demonstrated that the STK32 kinases bind significant numbers of clinically used kinase inhibitors.

biochemistry↗

bioRxiv2020

A Highly Selective Chemical Probe for Activin Receptor-like Kinases ALK4 and ALK5

The transforming growth factor beta-receptor I/activin receptor-like kinase 5 (TGFBR1/ALK5) and its close homologue ALK4 are receptor protein kinases associated with the development of diverse diseases, including cancer, fibrosis, heart diseases and dysfunctional immune response. Therefore, ALK4/5 are among the most studied kinases and several inhibitors have been developed. However, current commercially available inhibitors either lack selectivity or have not been comprehensively characterized, limiting their value for studying ALK4/5 function in cellular systems. To this end, we report the characterization of the 2-oxo-imidazopyridine, TP-008, a potent chemical probe with dual activity for ALK4 and ALK5 as well as the development of a matching negative control compound. TP-008 has excellent cellular potency and strongly abrogates phosphorylation of the substrate SMAD2 (mothers against decapentaplegic homolog 2). Thus, this chemical probe offers an excellent tool for mechanistic studies on the ALK4/5 signaling pathway and the contribution of these targets to disease.

biochemistry↗

bioRxiv2020

Parkinson's Disease-linked LRRK2 structure and model for microtubule interaction

Leucine Rich Repeat Kinase 2 (LRRK2) is the most commonly mutated gene in familial Parkinsons disease. LRRK2 is proposed to function in membrane trafficking and co-localizes with microtubules. We report the 3.5[A] structure of the catalytic half of LRRK2, and an atomic model of microtubule-associated LRRK2 built using a reported 14[A] cryo-electron tomography in situ structure. We propose that the conformation of LRRK2s kinase domain regulates its microtubule interaction, with a closed conformation favoring binding. We show that the catalytic half of LRRK2 is sufficient for microtubule binding and blocks the motility of the microtubule-based motors kinesin and dynein in vitro. Kinase inhibitors that stabilize an open conformation relieve this interference and reduce LRRK2 filament formation in cells, while those that stabilize a closed conformation do not. Our findings suggest that LRRK2 is a roadblock for microtubule-based motors and have implications for the design of therapeutic LRRK2 kinase inhibitors.

biophysics↗

bioRxiv2019

The Kinase Chemogenomic Set (KCGS): An open science resource for kinase vulnerability identification

We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS) is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.

cell biology↗

bioRxiv2019

Structural insights into interaction mechanisms of alternative piperazine-urea YEATS domain binders in MLLT1

YEATS-domain-containing MLLT1 is an acetyl/acyl-lysine reader domain, which is structurally distinct from well-studied bromodomains and has been strongly associated in development of cancer. Here, we characterized piperazine-urea derivatives as an acetyl/acyl-lysine mimetic moiety for MLLT1. Crystal structures revealed distinct interaction mechanisms of this chemotype compared to the recently described benzimidazole-amide based inhibitors, exploiting different binding pockets within the protein. Thus, the piperazine-urea scaffold offers an alternative strategy for targeting the YEATS domain family.

biochemistry↗

bioRxiv2019

p63 sets the threshold for induction of apoptosis using a kinetically encoded ‘doorbell-like’ mechanism

Cell fate decisions such as apoptosis require cells to translate signaling input into a binary yes/no response. A tight control of the process is required to avoid loss of cells by accidental activation of cell death pathways. One particularly critical situation exists in primary oocytes because their finite number determines the reproductive capacity of females. On the one hand a stringent genetic quality control is necessary to maintain the genetic integrity of the entire species; on the other hand an overly stringent mechanism that kills oocytes with even minor DNA damage can deplete the whole primary oocyte pool leading to infertility. The p53 homolog TAp63 is the key regulator of genome integrity in oocytes. After DNA damage TAp63 is activated by multistep phosphorylation involving multiple phosphorylation events by the kinase CK1, which triggers the transition from a dimeric and inactive conformation to an open and active tetramer. By measuring activation kinetics in ovaries and single site phosphorylation kinetics in vitro with peptides and full length protein we show that TAp63 phosphorylation follows a biphasic behavior. While the first two CK1 phosphorylation events are fast, the third one that constitutes the decisive step to form the active conformation is slow. We reveal the structural mechanism for the difference in the kinetic behavior based on an unusual CK1/TAp63 substrate interaction and demonstrate by quantitative simulation that the slow phosphorylation phase determines the threshold of DNA damage required for induction of apoptosis.

biochemistry↗

bioRxiv2019

Natural selection towards wild-type in composite cross populations of winter wheat

Most of our crops are grown in monoculture with single genotypes grown over wide acreage. An alternative approach, where segregating populations are used as crops is an exciting possibility, but outcomes of natural selection upon this type of crop are not well understood. We tracked allelic frequency changes in evolving composite cross populations (CCPs) of wheat grown over ten generations under organic and conventional farming. At three generations, each population was genotyped with 19 SSR and 8 SNP markers. The latter were diagnostic for major functional genes. Gene diversity was constant at SSR markers but decreased over time for genic markers. Population differentiation between the four locations could not be detected, suggesting that organic vs. non-organic crop management did not drive allele frequency changes. However, we did see changes for genes controlling plant height and phenology in all populations independently and consistently. We interpret these changes as the result of a consistent natural selection towards wild-type. Independent selection for alleles that are associated with plant height suggests that competition for light was central, resulting in the predominance of stronger intraspecific competitors, and highlighting a potential trade-off between individual and population performance.

genetics↗

bioRxiv2019

Conservation of structure, function and inhibitor binding in UNC-51-like kinase 1 and 2 (ULK1/2)

Autophagy is essential for cellular homeostasis and when deregulated this survival mechanism has been associated with disease development. Inhibition of autophagy initiation by inhibiting the kinase ULK1 has been proposed as a potential cancer therapy. While inhibitors and crystal structures of ULK1 have been reported, little is known about the other closely related kinase ULK2. Here we present the crystal structure of ULK2 in complex with ATP competitive inhibitors. Surprisingly, the ULK2 structure revealed a dimeric assembly reminiscent of dimeric arrangements of auto-activating kinases suggesting a role for this association in ULK activation. Screening of a kinase focused library of pre-clinical and clinical compounds revealed several potent ULK1/2 inhibitors and good correlation of inhibitor binding behavior with both ULK kinases. Aurora A was identified as a major off-target of currently used ULK1 inhibitors. Autophagic flux assays demonstrated that this off-target activity by strongly inducing autophagy in different cellular systems conferred an additional layer of complexity in the interpretation of cellular data. The data presented here provides structural models and chemical starting points for the development of ULK1/2 dual inhibitors with improved selectivity for future exploitation of autophagy inhibition.

biochemistry↗

bioRxiv2018

A structure-based approach towards identification of inhibitory fragments for eleven-nineteen-leukemia protein (ENL) YEATS domain

Lysine acetylation is an epigenetic mark that is principally recognized by bromodomains and recently structurally diverse YEATS domains also emerged as readers of lysine acetyl/acylations. Here we present a crystallography-based strategy and the discovery of fragments binding to the ENL YEATS domain, a potential drug target. Crystal structures combined with synthetic efforts led to the identification of a sub-micromolar binder, providing first starting points for the development of chemical probes for this reader domain family.

biochemistry↗

bioRxiv2018

A Chemical Toolbox for the Study of Bromodomains and Epigenetic Signaling

Bromodomains (BRDs) are evolutionary conserved epigenetic protein interaction modules which recognize (\"read\") acetyl-lysine, however their role(s) in regulating cellular states and their potential as targets for the development of targeted treatment strategies is poorly understood. Here we present a set of 25 chemical probes, selective tool small molecule inhibitors, covering 29 human bromodomain targets. We comprehensively evaluate the selectivity of this probe-set using BROMOscan(R) and demonstrate the utility of the set using studies of muscle cell differentiation and triple negative breast cancer (TNBC). We identified cross talk between histone acetylation and the glycolytic pathway resulting in a vulnerability of TNBC cell lines to inhibition of BRPF2/3 BRDs under conditions of glucose deprivation or GLUT1 inhibition. This chemical probe set will serve as a resource for future applications in the discovery of new physiological roles of bromodomain proteins in normal and disease states, and as a toolset for bromodomain target validation.

cell biology↗

bioRxiv2018

SGC-GAK-1: a chemical probe for cyclin G associated kinase (GAK)

We describe SGC-GAK-1 (11), a potent, selective, and cell-active inhibitor of cyclin G associated kinase (GAK), together with a structurally-related negative control SGC-GAK-1N (14). SGC-GAK-1 is highly selective in a kinome-wide screen, but cellular engagement assays defined RIPK2 as a collateral target. We identified 18 as a potent inhibitor of RIPK2 lacking GAK activity. Together, the chemical probe set of 11, 14, and 18 can be used to interrogate the cellular biology of GAK inhibition.

cell biology↗