Find research datasets worth reusing

opencc-by-4.0Nov 2020View details →

Dehydrogenase E1 and transketolase domain-containing protein 1 (DHTKD1); A Target Enabling Package

Inherited mutations of the GCDH gene for glutaryl-CoA dehydrogenase, catalysing the sixth enzymatic step in lysine catabolism, lead to the rare neurometabolic disorder Glutaric Aciduria type 1 (GA1). There is a rationale that inhibition of the fifth lysine catabolising step, catalysed by the DHTKD1 enzyme, could provide therapeutic benefit for GA1 by means of substrate reduction. This TEP provides early tools to develop DHTKD1 inhibitors, including recombinant protein, structure, biophysical (activity and stability) assays and fragment hits of human DHTKD1. This work also reports the interaction of DHTKD1 with its functional partner DLST as a binary complex, and an EM reconstruction of the DLST catalytic core.

opencc-by-4.0Feb 2022View details →

Dipeptidyl peptidase 11 (PgDPP11); A Target Enabling Package

Porphyromonas gingivalis (P. gingivalis) is the main causative agent of Periodontitis, the most widespread inflammatory condition world-wide. Recently this organism has been implicated in several systemic conditions, such as Alzheimer’s disease and type 2 diabetes. P. gingivalis does not ferment carbohydrates, instead it uses proteases to generate energy and carbon source. Dipeptidyl peptidase 11 plays a central role in the energy metabolism of this bacterium and has been proposed as an attractive drug target. This TEP provide early tools to develop inhibitors of PgDPP11, including purification protocols of recombinant proteins, a crystal structure of the protein in complex with a dipeptide, crystallisation conditions suitable for crystallography-based fragment screening, an inhibition assay and fragment hits in the active site and an allosteric site. These molecules provide a promising starting point for the development of more specific and potent PgDPP11 inhibitors.

opencc-by-4.0Aug 2018View details →

Human Family With Sequence Similarity 83 Member B (FAM83B); A Target Enabling Package

FAM83A-H are newly identified oncogenes characterised by a conserved DUF1669 domain. FAM83B can substitute for RAS to promote malignant transformation. Ablation of FAM83B or mutation of Lys230 inhibits malignant phenotypes, implicating FAM83B as potential therapeutic target. As part of this TEP, we solved the first crystal structures from the FAM83 family, including FAM83A and FAM83B. The structures of the DUF1669 domain reveal a phospholipase D-like fold lacking conservation of key catalytic residues. We deorphanise the FAM83 DUF1669 domain as a critical docking scaffold for binding of casein kinase 1 isoforms. Finally, using XChem fragment screening we report chemical fragments that bind to Lys230 in the central pocket of the DUF1669 and form starting points for potential drug development.

opencc-by-4.0Sep 2018View details →

Human Poly (ADP-ribose) Polymerase Family Member 14 (PARP14); A Target Enabling Package

This work provides reagents to develop specific inhibitors of the macrodomains of PARP14, for potential use for cancer and/or inflammation. Targeting the macrodomains of PARP14 is an alternative targeting strategy to PARP catalytic domain inhibitors that may allow greater inhibitor selectivity, and an alternative cellular effect. This package includes protein purification protocols, crystal structures of the 2nd and 3rd macrodomain of PARP14 in complex with small molecule chemical starting points, in vitro assays to measure ligand binding to the macrodomains, as well as validation of a PARP14 antibody and reagents to generate PARP14 knock-out cell lines (CRISPR-Cas9).

Human RECQL5 helicase; A Target Enabling Package

RECQL5 is a member of the RecQ family of helicase which have important functions in DNA repair pathways and maintenance of genome integrity. RECQL5 has recently been identified as a synthetic lethal candidate in various haematological malignancies and has been verified by knockdown to sensitize myeloproliferative neoplasms (MPN) to DNA damaging agents. In this TEP we have expressed purified and determined the first ever crystal structures of RECQL5, in both APO and ADP/Mg2+ bound forms which crystallize in two distinctly different conformations. In vitro DNA stimulated ATPase assays suitable for high throughput screening have been developed as well as lower throughput orthogonal assays to verify potential hits. A fragment screening campaign has been initiated and single fragment hit has identified a potential allosteric site that may be targeted to block the transition between conformations that it thought to be part of the helicase mechanism. Finally included as part of the package we present 3 validated RECQL5 binding nanobodies, one of which is a potent inhibitor of RECQL5 ATPase activity and is suitable for use as a tool reagent to investigate inhibition of RECQL5 and its complexes in vitro.

Human Lysine Demethylase JMJD1B (KDM3B); A Target Enabling Package

There are 3 members of the Lysine Demethylase JMJD1 (KDM3) family, JMJD1A-C. SGC Oxford has expressed, purified and crystallized the catalytic domains of JMJD1A, JMJD1B and JMJD1C as part of the probe programme. Fragment screening and X-ray crystallography identified a large number of binders, some of which were progressed into a medicinal chemistry programme. Despite significant effort molecules with probe properties were not obtained. Consequently it has been decided to put the information generated into the public domain.

Human Lysine Demethylase JMJD2D (KDM4D); A Target Enabling Package

There are 4 members of the Lysine Demethylase JMJD2 (KDM4) family. SGC Oxford has expressed, purified and crystallized the catalytic domains of JMJD2A, JMJD2B, JMJD2C and JMJD2D as part of the probe programme. Fragment screening and X-ray crystallography identified a large number of binders, some of which were progressed into a medicinal chemistry programme. Despite significant effort molecules with probe properties were not obtained. Consequently it has been decided to put the information generated into the public domain.

opencc-by-4.0Jun 2019View details →

Human Kelch-like ECH Associated Protein 1 (KEAP1); A Target Enabling Package

KEAP1 is a highly redox-sensitive member of the BTB-Kelch family that assembles with the CUL3 protein to form a Cullin-RING E3 ligase complex for the degradation of NRF2. Oxidative stress disables KEAP1 allowing NRF2 protein levels to accumulate for the transactivation of critical stress response genes. Consequently, the KEAP1-NRF2 system is a highly attractive target for the development of protein-protein interaction inhibitors that will stabilise NRF2 for therapeutic effect in conditions of neurodegeneration and inflammation. As part of this TEP we have solved the first crystal structure of a KEAP1-CUL3 complex as well as a structure of the apo-Kelch domain suitable for small molecule soaking. We further established a selectivity assay panel of 17 human Kelch domain-containing proteins and have shown that non-covalent KEAP1 inhibitors from the literature are highly selective for KEAP1. This protein panel offers a resource for future work on KEAP1 as well as 16 other human Kelch proteins.

opencc-by-4.0Jun 2021View details →

Urea Transporter B (UT-B; SLC14A1); A Target Enabling Package

UT-B is a member of urea transporter family, which consists of two members (UT-A and UT-B). UT-B is primarily a urea channel transporting urea molecules across plasma membrane by concentration gradient. While UT-B is widely expressed, it is particularly important in red blood cells (RBCs). UT-B is associated with improved response to hydroxyurea treatment in sickle cell diseases due to its transport of hydroxyurea into RBCs, and it is the primary antigen for Kidd (Jk) blood group as well as an accessory protein for ABO blood group. UT-B is responsible for urea transport in the vasa recta of kidney, with secondary effect on controlling the urine volume. This makes UT-B a potential target for the development of a new class of diuretics. This TEP presents the structure of UT-B in apo and inhibitor-bound states, determined by crystallography and cryo-electron microscopy, respectively. We have also confirmed the binding of an inhibitor molecule to UT-B in vitro using a biophysical assay. These enable the molecular characterisation of UT-B as an important blood antigen and will also aid further improvement of the inhibitors as a novel therapy for diuretics and potentially neurodegenerative disorders.

opencc-by-4.0Jun 2021View details →

Major facilitator superfamily domain-containing protein 10 (MFSD10) A Target Enabling Package (TEP)

MFSD10 (also known as TETRAN in humans) has been proposed to function as an organic anion efflux pump and as a transporter for some NSAIDs. We have produced milligram quantities of purified recombinant protein and solved its structure in an outward-facing state at 2.6 Å resolution by X-ray crystallography. The structure - the first example for a human atypical SLC - provides the initial clues to understanding the broad specificity of its putative substrate-binding site.

opencc-by-4.0Jun 2021View details →

Cyclin-Dependent Kinase-Like 5 (CDKL5); A Target Enabling Package

The protein kinase CDKL5 is best known for harbouring loss of function mutations that cause a variant of Rett syndrome, CDKL5 deficiency disorder, that predisposes to seizures and mental retardation. Recent kinome-wide screening has identified CDKL5 as a therapeutic target to ameliorate acute kidney injury, which is a common complication of hospitalisation that can lead to chronic kidney disease. There are currently no proven treatments. We prepared recombinant proteins for the kinase domains of CDKL1, CDKL2, CDKL3 and CDKL5 and solved the structures of these kinases in complex with identified inhibitors at resolutions from 1.5 to 2.4 Å. Overall, the structures capture the kinases with both active and inactive conformations and provide a model to explain the effects of CDKL5 mutation. An in vitro kinase assay showed the importance of a C-terminal αJ helix for the activity of CDKL2 and CDKL3, but not CDKL1 and CDKL5. Functional analyses of the single orthologue in C. elegans CDKL-1 also suggested that CDKL proteins can limit cilia length, which could potentially contribute to the neurological defects in CDKL5 deficiency syndrome. AST-487 and ASC67 present inhibitors of CDKL5 that could be developed for treating acute kidney injury. However, future work is needed to improve selectivity in this drug development.

opencc-by-4.0Aug 2021View details →

TREM2: A Target Enabling Package

Single loss of function mutations in TREM2, a receptor expressed by microglia in the brain, are associated with an increased risk of neurodegeneration, including Alzheimer’s disease and Nasu-Hakola disease.  Therefore, drugs enhancing TREM2 activity represent a potential treatment strategy. We identified 4 TREM2-specific scFv antibody fragments by phage display and solved the crystal structures of two of these in complex with the TREM2 ectodomain. The scFv antibodies altered TREM2 ectodomain shedding and TREM2 downstream signalling, representing promising research tools and therapeutic antibodies.

opencc-by-4.0Mar 2022View details →

PLEC: A Target Enabling Package

Little is known about how PLEC biology is linked to AD pathophysiology, despite the multifactorial indicators pointing to PLEC as a prominent risk factor for AD.  This project is intended to develop resources to help the scientific community delve deeper into PLEC biology within the central nervous system, where PLEC is prominently expressed by both excitatory and inhibitory neurons, as well as astrocytes.  The resource development for PLEC is a basic target enabling package (TEP), which includes the identification and characterization of viable antibodies against PLEC for biochemistry and immunohistology (the antibody report is linked below).  We anticipate that the development of additional resources to investigate PLEC’s role in AD pathogenesis will result in the discovery of new therapeutic approaches to treat AD.

opencc-by-4.0Mar 2022View details →

NDUFS2: A Target Enabling Package

The molecular role of NDUFS2 in mitochondrial metabolism is through its participation in complex I of the respiratory chain within the inner membrane of mitochondria.  Complex I function is critical for mitochondrial ATP production, but is also involved in reactive oxygen species production.  As a core component of complex I, NDUFS2 resides within two hypotheses of opposing mechanisms with the potential to foster AD pathology: AD Hypometabolism Hypothesis and the AD Oxidative Stress Hypothesis.  Each hypothesis is subordinate to a separately identified biological domain (mitochondrial metabolism and oxidative stress).  Consequently, decreasing complex I integrity via down-regulation of NDUFS2 could either promote or decrease the propagation of AD pathology.  The primary aim of this project is to generate resources to more effectively study the role of NDUFS2 in AD pathogenesis, through the generation of a basic TEP suite of resources, including a full antibody characterization report linked below.  Future studies leveraging these resources may shed light onto the molecular mechanism associated with NDUFS2 linkage with AD risk.

opencc-by-4.0Mar 2022View details →

Cathepsin H: A Target Enabling Package

CTSH participates in multiple AD linked biological domains, as noted above, and plays a role in both proteostasis and apoptosis, as well as immune response–three biological domains linked with AD pathology.  Little is known about the precise role that CTSH may play in AD pathology, but it participates in hypotheses linked with all three biological domains.  Given the genetic and omic association with AD risk, elucidating the mechanisms of biological contribution to AD risk may inform future therapeutic development.  The goal of this project is to develop a basic target enabling package to facilitate the future exploration of the role of CTSH in AD biology.

opencc-by-4.0Jan 2022View details →

CD44: A Target Enabling Package

CD44 is a transmembrane receptor that signals through binding hyaluronic acid (HA) with its ectodomain. The intracellular C-terminal tail associates with a variety of factors, including Moesin (MSN), an F-actin binding protein. Through MSN and HA binding, CD44 can link the extracellular matrix to the cytoskeleton. Investigation of the Alzheimer’s Disease (AD) brain proteome, along with weighted co-expression network analysis, revealed a module enriched with proteins involved in inflammation. CD44 (along with MSN) is a key driver of this module, increasing in abundance in asymptomatic and symptomatic AD patients. The aim of the TEP is to produce reagents to test the hypothesis that inhibiting CD44 ectodomain binding to HA may reduce CD44 signalling and inflammation in the brain, limiting neuronal damage in AD patients.

Pleckstrin Homology domain Interacting Protein (PHIP); A Target Enabling Package

SGC Oxford has expressed, purified and crystallized the second bromodomain of PHIP as part of the probe programme. Fragment screening and X-ray crystallography identified binders, some of which optimised to uM affinity. However, molecules with probe properties were not obtained. Consequently it has been decided to put the information generated into the public domain.

Human Histone Deacetylase 6 (HDAC6); A Target Enabling Package

HDAC6 is a cytosolic deacetylase with diverse roles in cellular trafficking, autophagy and cell signalling. Specifically targeting the unique zinc-finger ubiquitin-binding domain (ZnF-UBD) of HDAC6 may be an attractive strategy in myeloma and lymphoma but no inhibitor has been reported to date. Presented here are 4 co-crystal structures of HDAC6 ZnF-UBD in complex with different compounds, and associated SPR, ITC and FP assays.

Plasmodium bromodomain PfBDP4; A Target Enabling Package

There are 8 bromodomain-containing proteins in each annotated Plasmodium genome, with only one containing dual bromodomains (BRDs) – i.e. a total of 9 BRDs. In collaboration with SGC Oxford, we have expressed, purified and crystallized the BRDs in PfBDP1, PfBDP3, PfBDP4 (namely PfBDP4B) and PfGCN5. By screening all four against a focused BRD inhibitor library (SGC Oxford) using the DSF assay, we identified a small number of hits that were subsequently validated and quantitatively assessed using ITC. Two similar compounds with the dihydropteridinone scaffold, namely BI2536 and BI6727, stood out as potent inhibitors of PfBDP4B (both with Kd below 200 nM). We further collaborated with U of Melbourne to test them against Pf3D7 and found them capable of inhibiting blood stage development at sub-micromolar concentrations. Analysis of the co-structure of PfBDP4B+BI2536 suggests the possibility of increasing the potency and selectivity of inhibition.

Human SET domain bifurcated 1 (SETDB1), Tudor domain; A Target Enabling Package

SETDB1 is a H3K9 methyltransferase involved in transcriptional silencing with a catalytic SET domain and a triple Tudor domain containing a methyl-lysine binding site. SGC Toronto previously solved the apo structure of the Tudor domain (PDB code 3DLM). Amplification of SETDB1 in over 15% lung adenocarcinoma correlates with high mRNA and protein levels and its depletion in SETDB1-amplified cells reduces cancer growth in cell culture and nude mice models, whereas its overexpression increases tumour invasiveness (Rodriguez-Paredes et al. Oncogene 2014, Shah et al. Epigenetic Chromatin 2014). Several histone methyltransferases are known to have non-catalytic functions that might be alternative targeting strategies. For instance, recognition of H3K9 methylation by the ankyrin repeat of the methyltransferase GLP is required for efficient establishment of H3K9 methylation (Liu et al. Genes Dev. 2015). No catalytic domain inhibitor of SETDB1 has been reported to date. The goal of this TEP is to enable the discovery of potent, selective compounds targeting the Tudor domain of SETDB1.