Biology subjects

Bullock, A. N.

Publications and source records attributed to Bullock, A. N..

14 recordsLinked to original sources

bioRxiv2024

Structural mimicry of UM171 and neomorphic cancer mutants co-opts E3 ligase KBTBD4 for HDAC1/2 recruitment

Neomorphic mutations and drugs can elicit unanticipated effects that hinder mechanistic understanding for clinical practice. Recurrent indel mutations in the Kelch domain of the KBTBD4 E3 ligase rewire epigenetic programs for stemness in medulloblastoma by recruiting LSD1-CoREST-HDAC1/2 complexes as neo-substrates for ubiquitination and degradation. Remarkably, UM171, an investigational drug for haematopoietic stem cell transplantation, was found to degrade LSD1-CoREST-HDAC1/2 complexes in a wild-type KBTBD4-dependent manner, suggesting a potential common mode of action. We identified that these neomorphic interactions were mediated by the HDAC deacetylase domain. Cryo-EM studies of both wild-type and mutant KBTBD4 captured 2:1 and 2:2 KBTBD4-HDAC2 complexes at resolutions spanning 2.7 [A] to 3.1 [A]. The mutant and drug-induced complexes adopted similar structural assemblies requiring both Kelch domains in the KBTBD4 dimer for each HDAC2 interaction. UM171 was identified as a bona fide molecular glue binding across the ternary interface. Most strikingly, the indel mutation reshaped the same surface of KBTBD4 providing the first example of a natural mimic of a molecular glue. Together, the structures provide mechanistic understanding of neomorphic KBTBD4 and help to explain the structure-activity relationships of UM171 derivatives for future drug design.

molecular biology↗

bioRxiv2024

A BTB extension and ion-binding domain contribute to the pentameric structure and TFAP2A binding of KCTD1

KCTD family proteins typically assemble into Cullin-RING E3 ligases. KCTD1 is an atypical member that functions instead as a transcriptional repressor. Mutations in KCTD1 cause developmental abnormalities and kidney fibrosis in scalp-ear-nipple syndrome. Here, we present unexpected mechanistic insights from the structure of full-length KCTD1. Disease-causing mutation P20S maps to an unrecognized extension of the BTB domain that contributes both to its pentameric structure and TFAP2A binding. The C-terminal domain (CTD) shares its fold and pentameric assembly with the protein GFRP despite lacking discernible sequence similarity. Most surprisingly, the KCTD1 CTD establishes a central channel occupied by alternating sodium and iodide ions that restrict TFAP2A dissociation. The elucidation of the structure redefines the KCTD family BTB domain fold and identifies an unexpected ion pore for future study of KCTD1s function in the ectoderm, neural crest and kidney.

molecular biology↗

bioRxiv2024

Discovery and Characterization of a Chemical Probe for Cyclin-Dependent Kinase-Like 2

Acylaminoindazole-based inhibitors of CDKL2 were identified via analyses of cell-free binding and selectivity data. Compound 9 was selected as a CDKL2 chemical probe based on its potent inhibition of CDKL2 enzymatic activity, engagement of CDKL2 in cells, and excellent kinome-wide selectivity, especially when used in cells. Compound 16 was designed as a negative control to be used alongside compound 9 in experiments to interrogate CDKL2-mediated biology. A solved co-crystal structure of compound 9 bound to CDKL2 highlighted key interactions it makes within its ATP-binding site. Inhibition of downstream phosphorylation of EB2, a CDKL2 substrate, in rat primary neurons provided evidence that engagement of CDKL2 by compound 9 in cells resulted in inhibition of its activity. When used at relevant concentrations, compound 9 does not impact the viability of rat primary neurons or certain breast cancer cells nor elicit consistent changes in the expression of proteins involved in epithelial-mesenchymal transition.

cell biology↗

bioRxiv2023

Discovery and characterization of a specific inhibitor of serine-threonine kinase cyclin dependent kinase-like 5 (CDKL5) demonstrates role in hippocampal CA1 physiology

Pathological loss-of-function mutations in cyclin-dependent kinase-like 5 (CDKL5) cause CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental disorder associated with severe and medically refractory early-life epilepsy, motor, cognitive, visual and autonomic disturbances in the absence of any structural brain pathology. Analysis of genetic variants in CDD have indicated that CDKL5 kinase function is central to disease pathology. CDKL5 encodes a serine-threonine kinase with significant homology to GSK3{beta}, which has also been linked to synaptic function. Further, Cdkl5 knock-out rodents have increased GSK3{beta} activity and often increased long-term potentiation (LTP). Thus, development of a specific CDKL5 inhibitor must be careful to exclude cross-talk with GSK3{beta} activity. We synthesized and characterized specific, high-affinity inhibitors of CDKL5 that do not have detectable activity for GSK3{beta}. These compounds are very soluble in water but blood-brain barrier penetration is low. In rat hippocampal brain slices, acute inhibition of CDKL5 selectively reduces post-synaptic function of AMPA-type glutamate receptors in a dose-dependent manner. Acute inhibition of CDKL5 reduces hippocampal LTP. These studies provide new tools and insights into the role of CDKL5 as a newly appreciated, key kinase necessary for synaptic plasticity. Comparisons to rodent knock-out studies suggest that compensatory changes have limited the understanding of the roles of CDKL5 in synaptic physiology, plasticity and human neuropathology.

neuroscience↗

bioRxiv2023

Structural and biochemical characterization establishes a detailed understanding of KEAP1-CUL3 complex assembly

KEAP1 promotes the ubiquitin-dependent degradation of NRF2 by assembling into a CUL3-dependent ubiquitin ligase complex. Oxidative and electrophilic stress inhibit KEAP1 allowing NRF2 to accumulate for transactivation of stress response genes. To date there are no structures of the KEAP1-CUL3 interaction nor binding data to show the contributions of different domains to their binding affinity. We determined a crystal structure of the BTB and 3-box domains of human KEAP1 in complex with the CUL3 N-terminal domain that showed a heterotetrameric assembly with 2:2 stoichiometry. To support the structural data, we developed a versatile TR-FRET-based assay system to profile the binding of BTB-domain-containing proteins to CUL3 and determine the contribution of distinct protein features, revealing the importance of the CUL3 N-terminal extension for high affinity binding. We further provide direct evidence that the investigational drug CDDO does not disrupt the KEAP1-CUL3 interaction, even at high concentrations, but reduces the affinity of KEAP1-CUL3 binding. The TR-FRET-based assay system offers a generalizable platform for profiling this protein class and may form a suitable screening platform for ligands that disrupt these interactions by targeting the BTB or 3-box domains to block E3 ligase function. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/528651v2_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@3db02dorg.highwire.dtl.DTLVardef@1f97940org.highwire.dtl.DTLVardef@1879542org.highwire.dtl.DTLVardef@5847d2_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA new crystal structure defines KEAP1 BTB and 3-box domain interactions with CUL3 C_LIO_LIKEAP1 and CUL3 form a heteromeric 2:2 complex with a KD value of 0.2 {micro}M C_LIO_LIA generalizable TR-FRET platform enables multimodal profiling of BTB proteins C_LIO_LIThe investigational drug CDDO is a partial antagonist of the KEAP1-CUL3 interaction C_LI

molecular biology↗

bioRxiv2023

A Potent and Selective CDKL5/GSK3 Chemical Probe is Neuroprotective

Despite mediating several essential processes in the brain, including during development, cyclin-dependent kinase-like 5 (CDKL5) remains a poorly characterized human protein kinase. Accordingly, its substrates, functions, and regulatory mechanisms have not been fully described. We realized that availability of a potent and selective small molecule probe targeting CDKL5 could enable illumination of its roles in normal development as well as in diseases where it has become aberrant due to mutation. We prepared analogs of AT-7519, a known inhibitor of several cyclin dependent and cyclin-dependent kinase-like kinases that has been advanced into Phase II clinical trials. We identified analog 2 as a highly potent and cell-active chemical probe for CDKL5/GSK3 (glycogen synthase kinase 3). Evaluation of its kinome-wide selectivity confirmed that analog 2 demonstrates excellent selectivity and only retains GSK3/{beta} affinity. As confirmation that our chemical probe is a high-quality tool to use in directed biological studies, we demonstrated inhibition of downstream CDKL5 and GSK3/{beta} signaling and solved a co-crystal structure of analog 2 bound to CDKL5. A structurally similar analog (4) proved to lack CDKL5 affinity and maintain potent and selective inhibition of GSK3/{beta}. Finally, we used our chemical probe pair (2 and 4) to demonstrate that inhibition of CDKL5 and/or GSK3/{beta} promotes the survival of human motor neurons exposed to endoplasmic reticulum (ER) stress. We have demonstrated a neuroprotective phenotype elicited by our chemical probe pair and exemplified the utility of our compounds to characterize the role of CDKL5/GSK3 in neurons and beyond.

cell biology↗

bioRxiv2021

Selection and structural characterisation of anti-TREM2 scFvs that reduce levels of shed ectodomain

Single point mutations in TREM2, a receptor expressed by microglia in the brain, are associated with an increased risk of neurodegeneration including Alzheimers disease. Numerous studies support a role for TREM2 in sensing damaging stimuli and triggering signalling cascades necessary for neuroprotection. Despite its significant role, ligands and regulators of TREM2 activation, and the mechanisms governing TREM2-dependent responses and its cleavage from the membrane, remain poorly characterised. Here, we present phage display generated scFv antibody binders to human TREM2 ectodomain. Cocrystal structures revealed the binding of two scFvs to an epitope on the globular TREM2 domain distal to the putative ligand-binding site. Enhanced functional activity was observed for oligomeric scFv species which inhibited the production of soluble TREM2 in a HEK293 cell model. We hope that detailed characterisation of their epitopes and properties will facilitate the use of these renewable binders as structural and functional biology tools for TREM2 research.

molecular biology↗

bioRxiv2020

Saracatinib is an efficacious clinical candidate for fibrodysplasia ossificans progressiva

Currently, no effective therapies exist for fibrodysplasia ossificans progressiva (FOP), a rare congenital syndrome in which heterotopic bone is formed in soft tissues due to dysregulated activity of the bone morphogenetic protein (BMP) receptor kinase ALK2/ACVR1. From a screen of known biologically active compounds, we identified saracatinib as a potent ALK2 kinase inhibitor. In enzymatic and cell-based assays, saracatinib preferentially inhibited ALK2 compared with other receptors of the BMP/TGF{beta} signaling pathway, and induced dorsalization in zebrafish embryos consistent with BMP antagonism. We further tested the efficacy of saracatinib using an inducible ACVR1Q207D transgenic mouse line, which provides a model of heterotopic ossification, as well as an inducible ACVR1R206H knock-in, which serves as a genetically and physiologically faithful model of FOP. In both models, saracatinib was well tolerated and potently inhibited the development of heterotopic ossification even when administered transiently following soft tissue injury. Together, these data suggest that saracatinib is an efficacious clinical candidate for repositioning in the treatment of FOP, offering an accelerated path to clinical proof of efficacy studies and potentially significant benefits to individuals with this devastating condition.

molecular biology↗

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

Sequence and structural variations determining the recruitment of WNK kinases to the KLHL3 E3 ligase

The BTB-Kelch protein KLHL3 is a Cullin3-dependent E3 ligase that mediates the ubiquitin-dependent degradation of kinases WNK1-4 to control blood pressure and cell volume. A crystal structure of KLHL3 has defined its binding to an acidic degron motif containing a PXXP sequence that is strictly conserved in WNK1, WNK2 and WNK4. Mutations in the second proline abrograte the interaction causing the hypertension syndrome pseudohypoaldosteronism type II. WNK3 shows a diverged degron motif containing 4 amino acid substitutions that remove the PXXP motif raising questions as to the mechanism of its binding. To understand this atypical interaction, we determined the crystal structure of the KLHL3 Kelch domain in complex with a WNK3 peptide. The electron density enabled the complete 11-mer WNK-family degron motif to be traced for the first time revealing several conserved features not captured in previous work, including additional salt bridge and hydrogen bond interactions. Overall, the WNK3 peptide adopted a conserved binding pose except for a subtle shift to accommodate bulkier amino acid substitutions at the binding interface. At the centre, the second proline was substituted by WNK3 Thr541, providing a unique phosphorylatable residue among the WNK-family degrons. Fluorescence polarisation and structural modelling experiments revealed that its phosphorylation would abrogate the KLHL3 interaction similarly to hypertension-causing mutations. Together, these data reveal how the KLHL3 Kelch domain can accommodate the binding of multiple WNK isoforms and highlight a potential regulatory mechanism for the recruitment of WNK3.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsBTBBric-a-brac, Tramtrack, and Broad complexCCTConserved C-terminalCRL3Cullin3-dependent RING E3 ligaseDMEMDulbecco’s modified Eagle’s mediumDTTDithiothreitolFBSfetal bovine serumGSTGlutathione S-transferaseHCDHigher-energy collisional dissociationHEK293Thuman embryonic kidney 293 cell line expressing the SV40 large T antigenKCCK+/Cl− co-transportersIPTGIsopropyl β-D-1-thiogalactopyranosideKLHL3Kelch-like protein 3LC/MSLiquid chromatography-mass spectrometryN[K]CCNa+/Cl− ion co-transportersNKCC2Na+/K+/2Cl− co-transporter 2OSR1oxidative stress-responsive kinase 1PDBProtein DatabankPHAIIpseudohypoaldosteronism type IIRINGreally interesting new geneSPAKSPS1-related proline/alanine-rich kinaseTCEPtris(2-carboxyethyl)phosphineTEVtobacco etch virusWNKwith no lysine (K) kinaseView Full Text

molecular biology↗

bioRxiv2020

Identification of a PGXPP degron motif in dishevelled and structural basis for its binding to the E3 ligase KLHL12

Wnt signaling is critically dependent on dishevelled proteins (DVL1-3), which are required to assemble an intracellular Wnt signalosome at the plasma membrane. The levels of dishevelled proteins are strictly regulated by multiple E3 ubiquitin ligases that target DVL1-3 for ubiquitination and proteasomal degradation. The BTB-Kelch family protein KLHL12 is a substrate-specific adaptor for a Cullin-RING E3 ligase complex that contains Cullin3 and Rbx1. KLHL12 was the first E3 ligase to be identified for DVL1-3, but the molecular mechanisms determining its substrate interactions have remained unknown. Here, we mapped the interaction of DVL1-3 to a PGXPP motif that is conserved in other known partners and substrates of KLHL12, including PLEXHA4, PEF1 and SEC31. A 20-mer peptide containing the DVL1 motif bound to the Kelch domain of KLHL12 with low micromolar affinity. In cells, the mutation or deletion of this motif caused a striking reduction in the binding, ubiquitination and stability of DVL1 confirming this sequence as a degron motif for KLHL12 recruitment. To determine the binding mechanism we solved a 2.4 [A] crystal structure of the Kelch domain of KLHL12 in complex with a DVL1 peptide. The structure revealed that the DVL1 substrate adopted a U-shaped turn conformation that enabled hydrophobic interactions with all six blades of the Kelch domain {beta}-propeller. Overall, these results define the molecular mechanisms determining DVL regulation by KLHL12 and establish the KLHL12 Kelch domain as a new protein interaction module for a novel proline-rich motif.

molecular biology↗

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↗

bioRxiv2019

Structural consequences of BMPR2 kinase domain mutations causing pulmonary arterial hypertension

Bone morphogenetic proteins (BMPs) are secreted ligands of the transforming growth factor-{beta} (TGF-{beta}) family that control embryonic patterning, as well as tissue development and homeostasis. Loss of function mutations in the type II BMP receptor BMPR2 are the leading cause of pulmonary arterial hypertension (PAH), a rare disease of vascular occlusion and heart hypertrophy. To understand the structural consequences of these mutations, we determined the crystal structure of the human BMPR2 kinase domain at 2.35 [A] resolution. The structure revealed an activate conformation of the catalytic domain that formed canonical interactions with the bound ligand Mg-ADP. Disease-associated missense mutations were mapped throughout the protein structure, but clustered predominantly in the larger kinase C-lobe. Modelling revealed that the mutations will destabilize the protein structure by varying extents consistent with their previously reported functional heterogeneity. The most severe mutations introduced steric clashes in the hydrophobic protein core, whereas those found on the protein surface were less destabilizing and potentially most favorable for therapeutic rescue strategies currently under clinical investigation.

molecular biology↗

bioRxiv2018

Structural basis for recruitment of DAPK1 to the KLHL20 E3 ligase

BTB-Kelch proteins form the largest subfamily of Cullin-RING E3 ligases, yet their substrate complexes are mapped and structurally characterized only for KEAP1 and KLHL3. KLHL20 is a related CUL3-dependent ubiquitin ligase linked to autophagy, cancer and Alzheimers disease that promotes the ubiquitination and degradation of substrates including DAPK1, PML and ULK1. We identified a LPDLV-containing recruitment site in the DAPK1 death domain and determined the 1.1 [A] crystal structure of a KLHL20-DAPK1 complex. DAPK1 binds to KLHL20 as a loose helical turn that inserts deeply into the central pocket of the Kelch domain to contact all six blades of the {beta}-propeller. Here, KLHL20 forms a salt bridge as well as hydrophobic interactions that include a tryptophan and cysteine residue ideally positioned for covalent inhibitor development. The structure highlights the diverse binding modes of circular substrate pockets versus linear grooves and suggests a novel E3 ligase for protac-based drug design.

molecular biology↗