IsoCleft Finder – a web-based tool for the detection and analysis of protein binding-site geometric and chemical similarities

IsoCleft Finder is a web-based tool for the detection of local geometric and chemical similarities between potential small-molecule binding cavities and a non-redundant dataset of ligand-bound known small-molecule binding-sites. The non-redundant dataset developed as part of this study is composed of 7339 entries representing unique Pfam/PDB-ligand (hetero group code) combinations with known levels of cognate ligand similarity. The query cavity can be uploaded by the user or detected automatically by the system using existing PDB entries as well as user-provided structures in PDB format. In all cases, the user can refine the definition of the cavity interactively via a browser-based Jmol 3D molecular visualization interface. Furthermore, users can restrict the search to a subset of the dataset using a cognate-similarity threshold. Local structural similarities are detected using the IsoCleft software and ranked according to two criteria (number of atoms in common and Tanimoto score of local structural similarity) and the associated Z-score and p-value measures of statistical significance. The results, including predicted ligands, target proteins, similarity scores, number of atoms in common, etc., are shown in a powerful interactive graphical interface. This interface permits the visualization of target ligands superimposed on the query cavity and additionally provides a table of pairwise ligand topological similarities. Similarities between top scoring ligands serve as an additional tool to judge the quality of the results obtained. We present several examples where IsoCleft Finder provides useful functional information. IsoCleft Finder results are complementary to existing approaches for the prediction of protein function from structure, rational drug design and x-ray crystallography. IsoCleft Finder can be found at: http://bcb.med.usherbrooke.ca/isocleftfinder

Novel pharmacological maps of protein lysine methyltransferases: key for target deorphanization

Epigenetic therapies are being investigated for the treatment of cancer, cognitive disorders, metabolic alterations and autoinmune diseases. Among the diferent epigenetic target families, protein lysine methyltransferases (PKMTs), are especially interesting because it is believed that their inhibition may be highly specifc at the functional level. Despite its relevance, there are currently known inhibitors against only 10 out of the 50 SET-domain containing members of the PKMT family. Accordingly, the identifcation of chemical probes for the validation of the therapeutic impact of epigenetic modulation is key. Moreover, little is known about the mechanisms that dictate their substrate specifcity and ligand selectivity. Consequently, it is desirable to explore novel methods to characterize the pharmacological similarity of PKMTs, going beyond classical phylogenetic relationships. Such characterization would enable the prediction of ligand of-target efects caused by lack of ligand selectivity and the repurposing of known compounds against alternative targets. This is particularly relevant in the case of orphan targets with unreported inhibitors. Here, we frst perform a systematic study of binding modes of cofactor and substrate bound ligands with all available SET domain-containing PKMTs. Protein ligand interaction fngerprints were applied to identify conserved hot spots and contact-specifc residues across subfamilies at each binding site; a relevant analysis for guiding the design of novel, selective compounds. Then, a recently described methodology (GPCR-CoINPocket) that incorporates ligand contact information into classical alignment-based comparisons was applied to the entire family of 50 SET-containing proteins to devise pharmacological similarities between them. The main advantage of this approach is that it is not restricted to proteins for which crystallographic data with bound ligands is available. The resulting family organization from the separate analysis of both sites (cofactor and substrate) was retrospectively and prospectively validated. Of note, three hits (inhibition>50% at 10 µM) were identifed for the orphan NSD1

Probing the SAM Binding Site of SARS-CoV-2 nsp14 in vitro Using SAM Competitive Inhibitors Guides Developing Selective bi-substrate Inhibitors

The COVID-19 pandemic has clearly brought the healthcare systems world-wide to a breaking point along with devastating socioeconomic consequences. The SARS-CoV-2 virus which causes the disease uses RNA capping to evade the human immune system. Non-structural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small molecule inhibitors of nsp14 methyltransferase (MT) activity, we developed and employed a radiometric MT assay to screen a library of 161 in house synthesized S-adenosylmethionine (SAM) competitive methyltransferase inhibitors and SAM analogs. Among seven identified screening hits, SS148 inhibited nsp14 MT activity with an IC50 value of 70 ± 6 nM and was selective against 20 human protein lysine methyltransferases indicating significant differences in SAM binding sites. Interestingly, DS0464 with IC50 value of 1.1 ± 0.2 μM showed a bi-substrate competitive inhibitor mechanism of action. Modeling the binding of this compound to nsp14 suggests that the terminal phenyl group extends into the RNA binding site. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein methyltransferases. The structure-activity relationship provided by these compounds should guide the optimization of selective bi-substrate nsp14 inhibitors and may provide a path towards a novel class of antivirals against COVID-19, and possibly other coronaviruses

Bioinformatics in translational drug discovery

Bioinformatics approaches are becoming ever more essential in translational drug discovery both in academia and within the pharmaceutical industry. Computational exploitation of the increasing volumes of data generated during all phases of drug discovery is enabling key challenges of the process to be addressed. Here, we highlight some of the areas in which bioinformatics resources and methods are being developed to support the drug discovery pipeline. These include the creation of large data warehouses, bioinformatics algorithms to analyse ‘big data’ that identify novel drug targets and/or biomarkers, programs to assess the tractability of targets, and prediction of repositioning opportunities that use licensed drugs to treat additional indications

N6-Methyladenosine (m6A): A Promising New Molecular Target in Acute Myeloid Leukemia

Recent studies have uncovered an important role for RNA modifications in gene expression regulation, which led to the birth of the epitranscriptomics field. It is now acknowledged that RNA modifiers play a crucial role in the control of differentiation of stem and progenitor cells and that changes in their levels are a relevant feature of different types of cancer. To date, among more than 160 different RNA chemical modifications, the more relevant in cancer biology is the reversible and dynamic N6-methylation of adenosine, yielding N6-methyladenosine (m6A). m6A is the more abundant internal modification in mRNA, regulating the expression of the latter at different levels, from maturation to translation. Here, we will describe the emerging role of m6A modification in acute myeloid leukemia (AML), which, among first, has demonstrated how mis-regulation of the m6A modifying system can contribute to the development and progression of cancer. Moreover, we will discuss how AML is paving the way to the development of new therapeutic options based on the inhibition of m6A deposition

Development of SAM-based Chemical Probes for Methyltransferases

Fig.1. SAM analog for the profiling of MTase activity. A. Chemical structure of probe 1; B. General scheme of the labeling and capture strategy. A B Methylation is a fundamental mechanism used in the biological system to modify the structure and function of biomolecules such as proteins, DNA, RNA, and metabolites.1 Methyl groups are installed by a large and diverse class of S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTases), which transfer the sulfonium methyl group of SAM to either carbon, nitrogen, oxygen, or other heteroatoms on biomolecules.2 Dysregulated MTase activity contributes to numerous diseases, including cancer, metabolic disorders, neurodegenerative diseases.3 Presently, there is intense interest in pursuing MTases as therapeutic targets for the treatment of the aforementioned diseases. The human MTase family has more than 200 members.4 However, a large fraction of this family remains poorly characterized in terms of their endogenous substrates and biological functions. The need for innovative chemical probes to evaluate MTase function in the native biological matrix becomes apparent.5–8 In this current project, we are developing an activity-based protein profiling (ABPP) probe to directly investigate MTase activity in a complex biological sample. This probe (probe 1, Fig. 1A) is a structural analog of SAM with several major components: 1) a methylated nitrogen “warhead” mimics the positively charged sulfur in SAM;9 2) a photocrosslinkable azido group for the covalent modification of the target enzyme;8 3) a biotin tag for the subsequent affinity capture by streptavidin beads. The general scheme of the labeling and enrichment strategy is shown in Fig.1B. This strategy will selectively enrich for the active MTase content in a complex cellular context. Side-by-side comparison of functional MTase profiles under different physiological and pathological conditions, combined with proteomics analysis, should unwind the intricate interaction loops between human MTases and various cellular pathways, as well as empower the better manipulation of these enzymes for therapeutic purposes. Our original synthetic plan started with commercially available 8-bromoinsoine and acetyl protection of the 2\u27, 3\u27, and 5\u27-hydroxyl groups of the ribose ring (Fig. 2A).6 It was expected that after the installation of the azido group and the coupling with the biotin tag, the 5’-O-acetyl group could be selectively deprotected to enable the subsequent “warhead” installation. However, the selective deprotection using a published protocol was proven to be unsuccessful.6 Instead of the mono-deprotected probe 2, the mono-protected probe 3 was obtained. In the revised synthetic plan, the 2\u27 and 3\u27-OHs were protected as an acetonide.10The 5\u27-OH can then be selectively protected by the acetyl group. The azido group was readily installed at 8- Fig.2. Synthetic plans for probe 1. A. Original plan involves the tri-O-acetylation of the hydroxyl groups on the ribose ring. However, the selective deprotection of 5\u27-OH was challenging; B. Revised plan features the selective protection/deprotection of 5’-OH. A B position with NaN3. The biotin tag was tethered to the C6-position of the adenine ring through a 1,6-hexadiamine linker.9,11 The 5\u27-OH can then be selectively deprotected to afford the photocrosslink-able adenosine derivative probe 4 (Fig. 2B). The later stage of the synthesis focuses on the installation of the “warhead”. The 5\u27-OH was activated as a mesylate, which can then be replaced by a methylamino group.6 The subsequent reductive amination with tert-butyl (S)-2-[N-(tert-butoxycarbonyl) amino]-4-oxobutanoate allowed the “warhead” to be fully incorporated into the structure. Finally, the global deprotection should provide the desired SAM analog, probe 1. We are still in the process of finalizing the last step of the synthesis. Fig.3. Photoaffinity labeling and affinity capture of recombinant proteins using synthetic probe. A. Schematic representation of the labeling and enrichment protocols; B. Western blots showing the labeling of EHMT1 (left) and ADA (right) with probe 3. The biotinylated proteins were detected with anti-biotin antibody. A B In parallel to the synthetic effort, we also established and optimized the photoaffinity labeling and affinity enrichment assays. Recombinantly expressed and purified human euchromatin histone lysine methyltransferase 1 (EHMT1) was used for the initial assay development. Several parameters such as irradiation time, probe dosage, and elution conditions were fine-tuned to ensure accurate profiling of active enzymes. The protein was incubated with probe 3, followed by UV irradiation to trigger the covalent conjugation (Fig. 3A).8,12 The unbound free probes were then filtered off. Subsequently, streptavidin beads were introduced to the sample to capture the biotinylated protein. Ultimately, EHMT1 was eluted off the beads and analyzed by western blot using anti-biotin antibody. EHMT1 was only strongly labeled by probe 3 at high micromolar concentrations (Fig. 3B, left). We reasoned that probe 3 is an adenosine analog rather than a SAM analog. It may demonstrate selectivity towards adenosine-binding proteins. Indeed, probe 3 labeled adenosine deaminase (ADA) in a concentration-dependent manner (Fig. 3B, right). Even at the lowest probe concentration, ADA can still be labeled and enriched. The labeling can be competed off using a known ADA inhibitor, suggesting the on-target effect of the probe. The labeling strategy was also applied to cell lysates. It has the advantage of enriching the active enzymes independently of protein abundance, allowing the capture of dynamic enzyme activity changes in response to environmental or cellular stimuli. In the current study, the facile synthesis of SAM analogs was developed. Photoaffinity labeling and affinity enrichment protocols were developed. The probes were able to label recombinant proteins, and demonstrated target selectivity. The synthesis of probe 1 will be completed. These innovative chemical probes will be used to profile MTase activity in their native matrix to better understand their roles in different cellular events

The Function of the ASH1L Histone Methyltransferase in Cancer: A Chemical Biology Approach

ASH1L (absent, small, or homeotic-like 1) is a histone lysine methyltransferase (KMTase) that is overexpressed in cancer and activates oncogenic HOX genes. Small molecule inhibitors of ASH1L would be invaluable tools to investigate the role of ASH1L in cancer; however, no ASH1L inhibitors have been reported to date. Previous studies of ASH1L’s catalytic SET domain identified an autoinhibitory loop that blocks access of histone substrate to the enzyme active site. We used nuclear magnetic resonance (NMR) and X-ray crystallography to identify conformational heterogeneity in the ASH1L autoinhibitory loop and nearby SET-I loop, two structural features that regulate the enzymatic activity of the SET domain. These studies suggested that conformational heterogeneity in the autoinhibitory loop region of the ASH1L SET domain may create transient pockets into which small molecule ligands could bind. We took a fragment-based drug discovery (FBDD) approach to probe the ASH1L SET domain for potential small molecule binding sites and then construct new ligands specific to ASH1L. FBDD identified a ligand that binds to the ASH1L autoinhibitory loop region. We used information from NMR and crystallographic studies to optimize the fragment-like ligand into a first-in-class potent and specific ASH1L inhibitor. Structural studies indicate that ASH1L inhibitors block enzymatic activity by stabilizing the autoinhibited conformation of the SET domain. Our current most potent compounds inhibit ASH1L activity with IC50 of ~4 μM and bind to ASH1L with ~1 μM affinity, representing greater than 1000-fold improvement over the fragment screening hit. Our work identified the first ASH1L inhibitor and represents the first example of successfully applying FBDD to KMTases. Using our ASH1L inhibitors, we took a combined genetic and pharmacologic approach to investigate the role of ASH1L in leukemia and breast cancer. We found that ASH1L is required for proliferation of breast cancer and leukemia cells, and that ASH1L activates HOXA genes and MEIS1 in leukemia. Moreover, inhibition of the ASH1L SET domain downregulated HOX gene expression and induced differentiation of leukemia cells transformed by MLL-AF9. Our results demonstrate cellular efficacy of ASH1L inhibitors and uncover a new role for the ASH1L SET domain in acute leukemia.PHDMolecular & Cellular Path PhDUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/143998/1/rogawski_1.pd

Design and Synthesis of Inhibitors Targeting DCN1/3 and Inhibitors Targeting MLL1 Methyltransferase

The goal of my work presented in this dissertation was to fill the unmet need for compounds that can be used to interrogate the biology of the Cullin-RING ubiquitin ligases (CRLs) and the MLL1 methyltransferase. We conducted structure-based design, organic synthesis, and biochemical/biophysical assays to discover potent inhibitors targeting DCN1, DCN3 and, MLL1 methyltransferase. In DCN1 project, we describe the design, synthesis, and evaluation of peptidomimetics targeting the DCN1-UBC12 protein-protein interaction. Starting from a 12-residue UBC12 peptide, we successfully obtained a series of peptidomimetic compounds that bind to DCN1 protein with KD values of < 10 nM. Determination of a co-crystal structure of a potent peptidomimetic inhibitor complexed with DCN1 provided the structural basis for their high-affinity interaction. Cellular investigation of one potent DCN1 inhibitor, compound DI-404, reveals that it effectively and selectively inhibits the neddylation of cullin 3 over other cullin members. Further optimization of DI-404 may yield a new class of therapeutics for the treatment of human diseases in which cullin 3 CRL plays a key role. In DCN3 Project, we report the discovery of first-in-class, small-molecule inhibitors targeting the DCN3-UBE2F interaction, hereafter called DCN3 inhibitors. Our efforts have yielded potent and highly selective small-molecule DCN3 inhibitors, exemplified by TC-6304 which binds to DCN3 with a Ki value of 35 nM and fails to bind to DCN1 at concentrations as high as 30 µM. Cellular thermal shift assays showed that TC-6304 engages DCN3 in cells in a dose-dependent manner but does not affect the neddylation of any of the cullins that were examined, indicating the different roles of DCN3 when compared to DCN1. This study provides first-in-class, potent and selective small-molecule inhibitors of DCN3. In MLL project, we describe the design, synthesis and evaluation of a chemical library focused on S-adenosylmethionine-based compounds which led to the discovery of first-in-class, potent small-molecule inhibitors directly targeting the MLL1 SET domain. These are exemplified by compound TC-5115 with an IC50 value of 15 nM against MLL1 methyltransferase, which is 48 times more potent than the pan-HMT inhibitor, SAH. Determination of co-crystal structures for a number of these MLL1 inhibitors reveals that they adopt a unique binding mode that interacts with SET-I domain of MLL1 methyltransferase.PHDMedicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/153483/1/trchern_1.pd

Design, synthesis and evaluation of novel small molecule inhibitors of the histone methyltransferase DOT1L and ubiquitination facilitator Keap1

This thesis details the design, synthesis and evaluation of novel small molecule inhibitors of the histone methyltransferase DOT1L and the ubiquitination facilitator Keap1. The thesis is in two parts as outlined below. Part 1: The first part of this thesis details efforts towards identification of novel small molecule inhibitors of DOT1L, a histone methyltransferase which has been implicated in the development and proliferation of mixed lineage leukaemias (MLL). This work aims to optimise the drug-like properties of published DOT1L inhibitors while retaining potency, through further exploration of the nucleobase template. Structure-activity relationships (SARs) identified polar substituents and small heterocycles as favourable replacements for the halogen in 5-ITC, a small molecule inhibitor of DOT1L. Alternative nucleobase templates also demonstrated comparable DOT1L inhibition. To demonstrate proof of concept, a polar nitrile substituent was translated into the inhibitor Br-SAH as a direct replacement of the bromide. Activity was retained and a crystal structure obtained which demonstrated the nitrile occupied the same hydrophobic pocket. This work also demonstrated the use of a nitrile as a non-traditional replacement for heavy halogen atoms. Part 2: The second part of this thesis details identification of novel inhibitors of the Keap1-Nrf2 protein-protein interactions (PPI) using an approach based on kinetic target-guided synthesis (kTGS). Keap1 is a dimeric cytoplasmic protein that mediates the ubiquitination of Nrf2, a transcription factor that acts as a regulator of cellular antioxidant responses. Disruption of the PPI between Keap1 and Nrf2 has been shown to have a therapeutic benefit in diseases associated with oxidative stress and inflammation as well as providing a potential route to chemopreventative agents for cancer. Biased kTGS was applied as proof of concept. A biased ligand was designed and screened against a focused library of azides. The 1,3-dipolar cycloaddition products formed in the presence of the Keap1 Kelch domain were evaluated and validated through chemical synthesis and screening. A novel triazole structure was identified with improved activity over the initial biased fragment thus demonstrating kTGS as a valid approach for identifying novel inhibitors of the Keap1-Nrf2 PPI interaction

Characterisation and structural biology of protein arginine methyltransferases

PhD ThesisPost-translational and epigenetic modifications of proteins and nucleic acids are known to play major roles in influencing cell fate. Enzymes that catalyse modifications such as phosphorylation, acetylation and methylation have been identified as promising drug targets. Protein methyltransferase 2 (PRMT2) and Coactivator-associated arginine methyltransferase 1 (CARM1) belong to the class of Type 1 PRMTs which catalyse the asymmetric dimethylation of substrate arginine residues. CARM1 has been shown to be overexpressed in different cancer types including breast and prostate cancer. PRMT2 has been identified as a potential target for oncology with reported links to androgen receptor signalling, NF-κB signalling and induction of apoptosis. However, selective chemical probes that could be used as tools for target validation and which could potentially be a starting point for drug discovery are still missing. The work presented here aims to identify selective CARM1 and PRMT2 inhibitors that target the cofactor- and substrate-binding sites. Crystal structures of mouse PRMT2 in the apo-state and in complex with Sinefungin are presented. Crystal structures of the catalytic domain of CARM1 in complex with the cofactor S-adenosyl Lhomocysteine (SAH) and different small molecule inhibitors were also determined. Surface plasmon resonance was used to characterise inhibitor binding to CARM1 and identify structure-activity relationships. To further map the CARM1 active site, ligand soaks of CARM1 with a library of small fragments called FragLites were performed. These small fragments can more readily find potential binding pockets than larger more drug-like inhibitors. A direct and label-free mass spectrometry-based assay was developed to measure CARM1 activity and its inhibition. Together these findings can be used to further develop inhibitors that target the PRMT family. These inhibitors will be useful tools to investigate the biology of PRMT2 and CARM1 and to understand their biological role in cancer