Mohammad Eram - Academia.edu (original) (raw)

Papers by Mohammad Eram

Research paper thumbnail of Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress response

Nature Communications

Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increas... more Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective, and cell-active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 results in drastically reduced levels of arginine monomethylated HSP70 family stress-associated proteins. Structural and biochemical analyses reveal that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibits methylation of both constitutive and inducible forms of HSP70, and leads to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response.

Research paper thumbnail of A First-in-class, Highly Selective and Cell-active Allosteric Inhibitor of Protein Arginine Methyltransferase 6 (PRMT6)

PRMT6 catalyzes monomethylation and asymmetric dimethylation of arginine residues in various prot... more PRMT6 catalyzes monomethylation and asymmetric dimethylation of arginine residues in various proteins, plays important roles in biological processes and is associated with multiple cancers. While there are several reported PRMT6 inhibitors, a highly selective PRMT6 inhibitor has not been reported to date. Furthermore, allosteric inhibitors of protein methyltransferases are rare. Here we report the discovery and characterization of a first-in-class, highly selective allosteric inhibitor of PRMT6, SGC6870. SGC6870 is a potent PRMT6 inhibitor (IC50 = 77 ± 6 nM) with outstanding selectivity for PRMT6 over a broad panel of other methyltransferases and non-epigenetic targets. Notably, the crystal structure of the PRMT6–SGC6870 complex and kinetic studies revealed SGC6870 binds a unique, induced allosteric pocket. Additionally, SGC6870 engages PRMT6 and potently inhibits its methyltransferase activity in cells. Moreover, SGC6870’s enantiomer, SGC6870N, is inactive against PRMT6 and can be ...

Research paper thumbnail of Abstract LB-108: A potent and selective small molecule degrader of STAT5 for the treatment of hematological malignancies

Bioinformatics, Convergence Science, and Systems Biology

Research paper thumbnail of Author Correction: Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress response

Nature Communications

An amendment to this paper has been published and can be accessed via a link at the top of the pa... more An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Research paper thumbnail of Discovery of a chemical probe for PRDM9

Nature Communications

PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its nor... more PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its normal expression is restricted to germ cells and attenuation of its activity results in altered meiotic gene transcription, impairment of double-stranded breaks and pairing between homologous chromosomes. There is growing evidence for a role of aberrant expression of PRDM9 in oncogenesis and genome instability. Here we report the discovery of MRK-740, a potent (IC50: 80 ± 16 nM), selective and cell-active PRDM9 inhibitor (Chemical Probe). MRK-740 binds in the substrate-binding pocket, with unusually extensive interactions with the cofactor S-adenosylmethionine (SAM), conferring SAM-dependent substrate-competitive inhibition. In cells, MRK-740 specifically and directly inhibits H3K4 methylation at endogenous PRDM9 target loci, whereas the closely related inactive control compound, MRK-740-NC, does not. The discovery of MRK-740 as a chemical probe for the PRDM subfamily of methyltransferases...

Research paper thumbnail of Chemical Probes for Protein Arginine Methyltransferases

Research paper thumbnail of Structural and functional consequences of the STAT5BN642H driver mutation

Nature Communications

Hyper-activated STAT5B variants are high value oncology targets for pharmacologic intervention. S... more Hyper-activated STAT5B variants are high value oncology targets for pharmacologic intervention. STAT5B N642H , a frequently-occurring oncogenic driver mutation, promotes aggressive T-cell leukemia/lymphoma in patient carriers, although the molecular origins remain unclear. Herein, we emphasize the aggressive nature of STAT5B N642H in driving T-cell neoplasia upon hematopoietic expression in transgenic mice, revealing evidence of multiple T-cell subset organ infiltration. Notably, we demonstrate STAT5B N642H-driven transformation of γδ T-cells in in vivo syngeneic transplant models, comparable to STAT5B N642H patient γδ T-cell entities. Importantly, we present human STAT5B and STAT5B N642H crystal structures, which propose alternative mutation-mediated SH2 domain conformations. Our biophysical data suggests STAT5B N642H can adopt a hyper-activated and hyper-inactivated state with resistance to dephosphorylation. MD simulations support sustained interchain cross-domain interactions in STAT5B N642H , conferring kinetic stability to the mutant antiparallel dimer. This study provides a molecular explanation for the STAT5B N642H activating potential, and insights into pre-clinical models for targeted intervention of hyper-activated STAT5B.

Research paper thumbnail of Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress response

Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increas... more Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective and cell active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells, is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 resulted in drastically reduced levels of arginine monomethylation of HSP70 family members and other stress-associated proteins. Structural and biochemical analysis revealed that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibited methylation of both constitutive and inducible forms of HSP70, and led to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response.

Research paper thumbnail of TP-064, a potent and selective small molecule inhibitor of PRMT4 for multiple myeloma

Oncotarget, Jan 6, 2018

Protein arginine methyltransferase (PRMT) 4 (also known as coactivator-associated arginine methyl... more Protein arginine methyltransferase (PRMT) 4 (also known as coactivator-associated arginine methyltransferase 1; CARM1) is involved in a variety of biological processes and is considered as a candidate oncogene owing to its overexpression in several types of cancer. Selective PRMT4 inhibitors are useful tools for clarifying the molecular events regulated by PRMT4 and for validating PRMT4 as a therapeutic target. Here, we report the discovery of TP-064, a potent, selective, and cell-active chemical probe of human PRMT4 and its co-crystal structure with PRMT4. TP-064 inhibited the methyltransferase activity of PRMT4 with high potency (half-maximal inhibitory concentration, IC < 10 nM) and selectivity over other PRMT family proteins, and reduced arginine dimethylation of the PRMT4 substrates BRG1-associated factor 155 (BAF155; IC= 340 ± 30 nM) and Mediator complex subunit 12 (MED12; IC = 43 ± 10 nM). TP-064 treatment inhibited the proliferation of a subset of multiple myeloma cell li...

Research paper thumbnail of Pyruvate decarboxylase activity of the acetohydroxyacid synthase of Thermotoga maritima

Biochemistry and Biophysics Reports

Acetohydroxyacid synthase (AHAS) catalyzes the production of acetolactate from pyruvate. The enzy... more Acetohydroxyacid synthase (AHAS) catalyzes the production of acetolactate from pyruvate. The enzyme from the hyperthermophilic bacterium Thermotoga maritima has been purified and characterized (k cat $ 100 s À 1). It was found that the same enzyme also had the ability to catalyze the production of acetaldehyde and CO 2 from pyruvate, an activity of pyruvate decarboxylase (PDC) at a rate approximately 10% of its AHAS activity. Compared to the catalytic subunit, reconstitution of the individually expressed and purified catalytic and regulatory subunits of the AHAS stimulated both activities of PDC and AHAS. Both activities had similar pH and temperature profiles with an optimal pH of 7.0 and temperature of 85°C. The enzyme kinetic parameters were determined, however, it showed a non-Michaelis-Menten kinetics for pyruvate only. This is the first report on the PDC activity of an AHAS and the second bifunctional enzyme that might be involved in the production of ethanol from pyruvate in hyperthermophilic microorganisms.

Research paper thumbnail of Targeting human SET1/MLL family of proteins

Protein Science

The SET1 family of proteins, and in particular MLL1, are essential regulators of transcription an... more The SET1 family of proteins, and in particular MLL1, are essential regulators of transcription and key mediators of normal development and disease. Here, we summarize the detailed characterization of the methyltransferase activity of SET1 complexes and the role of the key subunits, WDR5, RbBP5, ASH2L, and DPY30. We present new data on full kinetic characterization of human MLL1, MLL3, SET1A, and SET1B trimeric, tetrameric, and pentameric complexes to elaborate on substrate specificities and compare our findings with what has been reported before. We also review exciting recent work identifying potent inhibitors of oncogenic MLL1 function through disruption of protein-protein interactions within the MLL1 complex.

Research paper thumbnail of Molecular basis for the methylation specificity of ATXR5 for histone H3

Nucleic acids research, Jan 5, 2017

In plants, the histone H3.1 lysine 27 (H3K27) mono-methyltransferases ARABIDOPSIS TRITHORAX RELAT... more In plants, the histone H3.1 lysine 27 (H3K27) mono-methyltransferases ARABIDOPSIS TRITHORAX RELATED PROTEIN 5 and 6 (ATXR5/6) regulate heterochromatic DNA replication and genome stability. Our initial studies showed that ATXR5/6 discriminate between histone H3 variants and preferentially methylate K27 on H3.1. In this study, we report three regulatory mechanisms contributing to the specificity of ATXR5/6. First, we show that ATXR5 preferentially methylates the R/F-K*-S/C-G/A-P/C motif with striking preference for hydrophobic and aromatic residues in positions flanking this core of five amino acids. Second, we demonstrate that post-transcriptional modifications of residues neighboring K27 that are typically associated with actively transcribed chromatin are detrimental to ATXR5 activity. Third, we show that ATXR5 PHD domain employs a narrow binding pocket to selectively recognize unmethylated K4 of histone H3. Finally, we demonstrate that deletion or mutation of the PHD domain reduce...

Research paper thumbnail of The SUV4-20 inhibitor A-196 verifies a role for epigenetics in genomic integrity

Nature chemical biology, Mar 23, 2017

Protein lysine methyltransferases (PKMTs) regulate diverse physiological processes including tran... more Protein lysine methyltransferases (PKMTs) regulate diverse physiological processes including transcription and the maintenance of genomic integrity. Genetic studies suggest that the PKMTs SUV420H1 and SUV420H2 facilitate proficient nonhomologous end-joining (NHEJ)-directed DNA repair by catalyzing the di- and trimethylation (me2 and me3, respectively) of lysine 20 on histone 4 (H4K20). Here we report the identification of A-196, a potent and selective inhibitor of SUV420H1 and SUV420H2. Biochemical and co-crystallization analyses demonstrate that A-196 is a substrate-competitive inhibitor of both SUV4-20 enzymes. In cells, A-196 induced a global decrease in H4K20me2 and H4K20me3 and a concomitant increase in H4K20me1. A-196 inhibited 53BP1 foci formation upon ionizing radiation and reduced NHEJ-mediated DNA-break repair but did not affect homology-directed repair. These results demonstrate the role of SUV4-20 enzymatic activity in H4K20 methylation and DNA repair. A-196 represents a...

Research paper thumbnail of Discovery of a Potent, Selective and Cell-active Dual Inhibitor of Protein Arginine Methyltransferase 4 and Protein Arginine Methyltransferase 6

Journal of medicinal chemistry, Oct 1, 2016

Well-characterized selective inhibitors of protein arginine methyltransferases (PRMTs) are invalu... more Well-characterized selective inhibitors of protein arginine methyltransferases (PRMTs) are invaluable chemical tools for testing biological and therapeutic hypotheses. Based on 4, a fragment-like inhibitor of type I PRMTs, we conducted structure-activity relationship (SAR) studies and explored three regions of this scaffold. The studies led to the discovery of a potent, selective and cell-active dual inhibitor of PRMT4 and PRMT6, 17 (MS049). As compared to 4, 17 displayed much improved potency for PRMT4 and PRMT6 in both biochemical and cellular assays. It was selective for PRMT4 and PRMT6 over other PRMTs and a broad range of other epigenetic modifiers and non-epigenetic targets. We also developed 46 (MS049N), which was inactive in biochemical and cellular assays, as a negative control for chemical biology studies. Considering possible overlapping substrate specificity of PRMTs, 17 and 46 are valuable chemical tools for dissecting specific biological functions and dysregulation of ...

Research paper thumbnail of Structural basis of arginine asymmetrical dimethylation by PRMT6

The Biochemical journal, Oct 1, 2016

PRMT6 is a type I protein arginine methyltransferase, generating the asymmetric dimethylarginine ... more PRMT6 is a type I protein arginine methyltransferase, generating the asymmetric dimethylarginine mark on proteins such as histone H3R2. Asymmetric dimethylation of histone H3R2 by PRMT6 acts as a repressive mark that antagonizes trimethylation of H3 lysine 4 by the MLL histone H3K4 methyltransferase. PRMT6 is overexpressed in several cancer types, including prostate, bladder and lung cancers; therefore, it is of great interest to develop potent and selective inhibitors for PRMT6. Here we report the synthesis of a potent bi-substrate inhibitor GMS (6'-methyleneamine sinefungin, an analogue of sinefungin), and the crystal structures of human PRMT6 in complex respectively with SAH and the bi-substrate inhibitor GMS that shed light on the significantly improved inhibition effect of GMS on methylation activity of PRMT6 compared to SAH and a SAM competitive methyltransferase inhibitor sinefungin (SNF). In addition, we also crystallized PRMT6 in complex with SAH and a short arginine co...

Research paper thumbnail of Discovery of a Potent and Selective Coactivator Associated Arginine Methyltransferase 1 (CARM1) Inhibitor by Virtual Screening

Journal of medicinal chemistry, Jul 8, 2016

Protein arginine methyltransferases (PRMTs) represent an emerging target class in oncology and ot... more Protein arginine methyltransferases (PRMTs) represent an emerging target class in oncology and other disease areas. So far, the most successful strategy to identify PRMT inhibitors has been to screen large to medium-size chemical libraries. Attempts to develop PRMT inhibitors using receptor-based computational methods have met limited success. Here, using virtual screening approaches, we identify 11 CARM1 (PRMT4) inhibitors with ligand efficiencies ranging from 0.28 to 0.84. CARM1 selective hits were further validated by orthogonal methods. Two structure-based rounds of optimization produced 27 (SGC2085), a CARM1 inhibitor with an IC50 of 50 nM and more than hundred-fold selectivity over other PRMTs. These results indicate that virtual screening strategies can be successfully applied to Rossmann-fold protein methyltransferases.

Research paper thumbnail of PR Domain-Containing Protein 7 (PRDM7) is a Histone 3 Lysine 4 Trimethyltransferase

Journal of Biological Chemistry, 2016

PR domain-containing protein 7 (PRDM7) is a primate-specific histone methyltransferase that is th... more PR domain-containing protein 7 (PRDM7) is a primate-specific histone methyltransferase that is the result of a recent gene duplication of PRDM9. The two proteins are highly homologous, especially in the catalytic PR/SET domain, where they differ by only three amino acid residues. Here we report that PRDM7 is an efficient methyltransferase that selectively catalyzes the trimethylation of H3 lysine 4 (H3K4) both in vitro and in cells. Through selective mutagenesis we have dissected the functional roles of each of the three divergent residues between the PR domains of PRDM7 and PRDM9. These studies indicate that after a single serine to tyrosine mutation at residue 357 (S357Y), PRDM7 regains the substrate specificities and catalytic activities similar to its evolutionary predecessor, including the ability to efficiently methylate H3K36.

Research paper thumbnail of Detection of Burkholderia cepacia complex in patients with cystic fibrosis

Tanaffos, 2004

Background: Damaging of the lung function in patients with cystic fibrosis is the most frequent c... more Background: Damaging of the lung function in patients with cystic fibrosis is the most frequent cause of death in these patients. Recently, Burkholderia cepacia has been emerged as an important opportunistic pathogen in these patients; because of its increased isolation from patients with cystic fibrosis since late1970s, the capacity for spread of infection among the cystic fibrosis patient community, its role in damaging lung functions, and its innate multiantibiotic resistance. These different aspects make isolation of Burkholderia cepacia an important task in cystic fibrosis health care settings. Materials and Methods: We examined the capacity of Burkholderia cepacia selective agar (BCSA) as a medium for primary isolation of Burkholderia cepacia samples. Biochemical tests were used to confirm the identification. Results: Burkholderia cepacia strains were isolated from 6 out of 53 respiratory samples as confirmed with biochemical tests. Conclusion: Results of the present study suggest that BCSA can be used as a selective medium with high specificity for primary isolation and identification of Burkholderia cepacia complex bacteria.

Research paper thumbnail of Discovery of a Potent Class I Protein Arginine Methyltransferase Fragment Inhibitor

Journal of Medicinal Chemistry, 2016

Protein methyltransferases (PMTs) are a promising target class in oncology and other disease area... more Protein methyltransferases (PMTs) are a promising target class in oncology and other disease areas. They are composed of SET domain methyltransferases and structurally unrelated Rossman-fold enzymes that include protein arginine methyltransferases (PRMTs). In the absence of a well-defined medicinal chemistry tool-kit focused on PMTs, most current inhibitors were identified by screening large and diverse libraries of leadlike molecules. So far, no successful fragment-based approach was reported against this target class. Here, by deconstructing potent PRMT inhibitors, we find that chemical moieties occupying the substrate arginine-binding site can act as efficient fragment inhibitors. Screening a fragment library against PRMT6 produced numerous hits, including a 300 nM inhibitor (ligand efficiency of 0.56) that decreased global histone 3 arginine 2 methylation in cells, and can serve as a warhead for the development of PRMT chemical probes.

Research paper thumbnail of A Potent, Selective and Cell-active Inhibitor of Human Type I Protein Arginine Methyltransferases

ACS chemical biology, Jan 24, 2015

Protein arginine methyltransferases (PRMTs) play a crucial role in a variety of biological proces... more Protein arginine methyltransferases (PRMTs) play a crucial role in a variety of biological processes. Overexpression of PRMTs has been implicated in various human diseases including cancer. Consequently, selective small-molecule inhibitors of PRMTs have been pursued by both academia and pharmaceutical industry as chemical tools for testing biological and therapeutic hypotheses. PRMTs are divided into three categories: type I PRMTs which catalyze mono- and asymmetric dimethylation of arginine residues, type II PRMTs which catalyze mono- and symmetric dimethylation of arginine residues, and type III PRMT which catalyzes only monomethylation of arginine residues. Here, we report the discovery of a potent, selective and cell-active inhibitor of human type I PRMTs, MS023, and characterization of this inhibitor in a battery of biochemical, biophysical and cellular assays. MS023 displayed high potency for type I PRMTs including PRMT1, 3, 4, 6 and 8, but was completely inactive against type...

Research paper thumbnail of Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress response

Nature Communications

Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increas... more Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective, and cell-active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 results in drastically reduced levels of arginine monomethylated HSP70 family stress-associated proteins. Structural and biochemical analyses reveal that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibits methylation of both constitutive and inducible forms of HSP70, and leads to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response.

Research paper thumbnail of A First-in-class, Highly Selective and Cell-active Allosteric Inhibitor of Protein Arginine Methyltransferase 6 (PRMT6)

PRMT6 catalyzes monomethylation and asymmetric dimethylation of arginine residues in various prot... more PRMT6 catalyzes monomethylation and asymmetric dimethylation of arginine residues in various proteins, plays important roles in biological processes and is associated with multiple cancers. While there are several reported PRMT6 inhibitors, a highly selective PRMT6 inhibitor has not been reported to date. Furthermore, allosteric inhibitors of protein methyltransferases are rare. Here we report the discovery and characterization of a first-in-class, highly selective allosteric inhibitor of PRMT6, SGC6870. SGC6870 is a potent PRMT6 inhibitor (IC50 = 77 ± 6 nM) with outstanding selectivity for PRMT6 over a broad panel of other methyltransferases and non-epigenetic targets. Notably, the crystal structure of the PRMT6–SGC6870 complex and kinetic studies revealed SGC6870 binds a unique, induced allosteric pocket. Additionally, SGC6870 engages PRMT6 and potently inhibits its methyltransferase activity in cells. Moreover, SGC6870’s enantiomer, SGC6870N, is inactive against PRMT6 and can be ...

Research paper thumbnail of Abstract LB-108: A potent and selective small molecule degrader of STAT5 for the treatment of hematological malignancies

Bioinformatics, Convergence Science, and Systems Biology

Research paper thumbnail of Author Correction: Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress response

Nature Communications

An amendment to this paper has been published and can be accessed via a link at the top of the pa... more An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Research paper thumbnail of Discovery of a chemical probe for PRDM9

Nature Communications

PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its nor... more PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its normal expression is restricted to germ cells and attenuation of its activity results in altered meiotic gene transcription, impairment of double-stranded breaks and pairing between homologous chromosomes. There is growing evidence for a role of aberrant expression of PRDM9 in oncogenesis and genome instability. Here we report the discovery of MRK-740, a potent (IC50: 80 ± 16 nM), selective and cell-active PRDM9 inhibitor (Chemical Probe). MRK-740 binds in the substrate-binding pocket, with unusually extensive interactions with the cofactor S-adenosylmethionine (SAM), conferring SAM-dependent substrate-competitive inhibition. In cells, MRK-740 specifically and directly inhibits H3K4 methylation at endogenous PRDM9 target loci, whereas the closely related inactive control compound, MRK-740-NC, does not. The discovery of MRK-740 as a chemical probe for the PRDM subfamily of methyltransferases...

Research paper thumbnail of Chemical Probes for Protein Arginine Methyltransferases

Research paper thumbnail of Structural and functional consequences of the STAT5BN642H driver mutation

Nature Communications

Hyper-activated STAT5B variants are high value oncology targets for pharmacologic intervention. S... more Hyper-activated STAT5B variants are high value oncology targets for pharmacologic intervention. STAT5B N642H , a frequently-occurring oncogenic driver mutation, promotes aggressive T-cell leukemia/lymphoma in patient carriers, although the molecular origins remain unclear. Herein, we emphasize the aggressive nature of STAT5B N642H in driving T-cell neoplasia upon hematopoietic expression in transgenic mice, revealing evidence of multiple T-cell subset organ infiltration. Notably, we demonstrate STAT5B N642H-driven transformation of γδ T-cells in in vivo syngeneic transplant models, comparable to STAT5B N642H patient γδ T-cell entities. Importantly, we present human STAT5B and STAT5B N642H crystal structures, which propose alternative mutation-mediated SH2 domain conformations. Our biophysical data suggests STAT5B N642H can adopt a hyper-activated and hyper-inactivated state with resistance to dephosphorylation. MD simulations support sustained interchain cross-domain interactions in STAT5B N642H , conferring kinetic stability to the mutant antiparallel dimer. This study provides a molecular explanation for the STAT5B N642H activating potential, and insights into pre-clinical models for targeted intervention of hyper-activated STAT5B.

Research paper thumbnail of Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress response

Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increas... more Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective and cell active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells, is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 resulted in drastically reduced levels of arginine monomethylation of HSP70 family members and other stress-associated proteins. Structural and biochemical analysis revealed that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibited methylation of both constitutive and inducible forms of HSP70, and led to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response.

Research paper thumbnail of TP-064, a potent and selective small molecule inhibitor of PRMT4 for multiple myeloma

Oncotarget, Jan 6, 2018

Protein arginine methyltransferase (PRMT) 4 (also known as coactivator-associated arginine methyl... more Protein arginine methyltransferase (PRMT) 4 (also known as coactivator-associated arginine methyltransferase 1; CARM1) is involved in a variety of biological processes and is considered as a candidate oncogene owing to its overexpression in several types of cancer. Selective PRMT4 inhibitors are useful tools for clarifying the molecular events regulated by PRMT4 and for validating PRMT4 as a therapeutic target. Here, we report the discovery of TP-064, a potent, selective, and cell-active chemical probe of human PRMT4 and its co-crystal structure with PRMT4. TP-064 inhibited the methyltransferase activity of PRMT4 with high potency (half-maximal inhibitory concentration, IC < 10 nM) and selectivity over other PRMT family proteins, and reduced arginine dimethylation of the PRMT4 substrates BRG1-associated factor 155 (BAF155; IC= 340 ± 30 nM) and Mediator complex subunit 12 (MED12; IC = 43 ± 10 nM). TP-064 treatment inhibited the proliferation of a subset of multiple myeloma cell li...

Research paper thumbnail of Pyruvate decarboxylase activity of the acetohydroxyacid synthase of Thermotoga maritima

Biochemistry and Biophysics Reports

Acetohydroxyacid synthase (AHAS) catalyzes the production of acetolactate from pyruvate. The enzy... more Acetohydroxyacid synthase (AHAS) catalyzes the production of acetolactate from pyruvate. The enzyme from the hyperthermophilic bacterium Thermotoga maritima has been purified and characterized (k cat $ 100 s À 1). It was found that the same enzyme also had the ability to catalyze the production of acetaldehyde and CO 2 from pyruvate, an activity of pyruvate decarboxylase (PDC) at a rate approximately 10% of its AHAS activity. Compared to the catalytic subunit, reconstitution of the individually expressed and purified catalytic and regulatory subunits of the AHAS stimulated both activities of PDC and AHAS. Both activities had similar pH and temperature profiles with an optimal pH of 7.0 and temperature of 85°C. The enzyme kinetic parameters were determined, however, it showed a non-Michaelis-Menten kinetics for pyruvate only. This is the first report on the PDC activity of an AHAS and the second bifunctional enzyme that might be involved in the production of ethanol from pyruvate in hyperthermophilic microorganisms.

Research paper thumbnail of Targeting human SET1/MLL family of proteins

Protein Science

The SET1 family of proteins, and in particular MLL1, are essential regulators of transcription an... more The SET1 family of proteins, and in particular MLL1, are essential regulators of transcription and key mediators of normal development and disease. Here, we summarize the detailed characterization of the methyltransferase activity of SET1 complexes and the role of the key subunits, WDR5, RbBP5, ASH2L, and DPY30. We present new data on full kinetic characterization of human MLL1, MLL3, SET1A, and SET1B trimeric, tetrameric, and pentameric complexes to elaborate on substrate specificities and compare our findings with what has been reported before. We also review exciting recent work identifying potent inhibitors of oncogenic MLL1 function through disruption of protein-protein interactions within the MLL1 complex.

Research paper thumbnail of Molecular basis for the methylation specificity of ATXR5 for histone H3

Nucleic acids research, Jan 5, 2017

In plants, the histone H3.1 lysine 27 (H3K27) mono-methyltransferases ARABIDOPSIS TRITHORAX RELAT... more In plants, the histone H3.1 lysine 27 (H3K27) mono-methyltransferases ARABIDOPSIS TRITHORAX RELATED PROTEIN 5 and 6 (ATXR5/6) regulate heterochromatic DNA replication and genome stability. Our initial studies showed that ATXR5/6 discriminate between histone H3 variants and preferentially methylate K27 on H3.1. In this study, we report three regulatory mechanisms contributing to the specificity of ATXR5/6. First, we show that ATXR5 preferentially methylates the R/F-K*-S/C-G/A-P/C motif with striking preference for hydrophobic and aromatic residues in positions flanking this core of five amino acids. Second, we demonstrate that post-transcriptional modifications of residues neighboring K27 that are typically associated with actively transcribed chromatin are detrimental to ATXR5 activity. Third, we show that ATXR5 PHD domain employs a narrow binding pocket to selectively recognize unmethylated K4 of histone H3. Finally, we demonstrate that deletion or mutation of the PHD domain reduce...

Research paper thumbnail of The SUV4-20 inhibitor A-196 verifies a role for epigenetics in genomic integrity

Nature chemical biology, Mar 23, 2017

Protein lysine methyltransferases (PKMTs) regulate diverse physiological processes including tran... more Protein lysine methyltransferases (PKMTs) regulate diverse physiological processes including transcription and the maintenance of genomic integrity. Genetic studies suggest that the PKMTs SUV420H1 and SUV420H2 facilitate proficient nonhomologous end-joining (NHEJ)-directed DNA repair by catalyzing the di- and trimethylation (me2 and me3, respectively) of lysine 20 on histone 4 (H4K20). Here we report the identification of A-196, a potent and selective inhibitor of SUV420H1 and SUV420H2. Biochemical and co-crystallization analyses demonstrate that A-196 is a substrate-competitive inhibitor of both SUV4-20 enzymes. In cells, A-196 induced a global decrease in H4K20me2 and H4K20me3 and a concomitant increase in H4K20me1. A-196 inhibited 53BP1 foci formation upon ionizing radiation and reduced NHEJ-mediated DNA-break repair but did not affect homology-directed repair. These results demonstrate the role of SUV4-20 enzymatic activity in H4K20 methylation and DNA repair. A-196 represents a...

Research paper thumbnail of Discovery of a Potent, Selective and Cell-active Dual Inhibitor of Protein Arginine Methyltransferase 4 and Protein Arginine Methyltransferase 6

Journal of medicinal chemistry, Oct 1, 2016

Well-characterized selective inhibitors of protein arginine methyltransferases (PRMTs) are invalu... more Well-characterized selective inhibitors of protein arginine methyltransferases (PRMTs) are invaluable chemical tools for testing biological and therapeutic hypotheses. Based on 4, a fragment-like inhibitor of type I PRMTs, we conducted structure-activity relationship (SAR) studies and explored three regions of this scaffold. The studies led to the discovery of a potent, selective and cell-active dual inhibitor of PRMT4 and PRMT6, 17 (MS049). As compared to 4, 17 displayed much improved potency for PRMT4 and PRMT6 in both biochemical and cellular assays. It was selective for PRMT4 and PRMT6 over other PRMTs and a broad range of other epigenetic modifiers and non-epigenetic targets. We also developed 46 (MS049N), which was inactive in biochemical and cellular assays, as a negative control for chemical biology studies. Considering possible overlapping substrate specificity of PRMTs, 17 and 46 are valuable chemical tools for dissecting specific biological functions and dysregulation of ...

Research paper thumbnail of Structural basis of arginine asymmetrical dimethylation by PRMT6

The Biochemical journal, Oct 1, 2016

PRMT6 is a type I protein arginine methyltransferase, generating the asymmetric dimethylarginine ... more PRMT6 is a type I protein arginine methyltransferase, generating the asymmetric dimethylarginine mark on proteins such as histone H3R2. Asymmetric dimethylation of histone H3R2 by PRMT6 acts as a repressive mark that antagonizes trimethylation of H3 lysine 4 by the MLL histone H3K4 methyltransferase. PRMT6 is overexpressed in several cancer types, including prostate, bladder and lung cancers; therefore, it is of great interest to develop potent and selective inhibitors for PRMT6. Here we report the synthesis of a potent bi-substrate inhibitor GMS (6'-methyleneamine sinefungin, an analogue of sinefungin), and the crystal structures of human PRMT6 in complex respectively with SAH and the bi-substrate inhibitor GMS that shed light on the significantly improved inhibition effect of GMS on methylation activity of PRMT6 compared to SAH and a SAM competitive methyltransferase inhibitor sinefungin (SNF). In addition, we also crystallized PRMT6 in complex with SAH and a short arginine co...

Research paper thumbnail of Discovery of a Potent and Selective Coactivator Associated Arginine Methyltransferase 1 (CARM1) Inhibitor by Virtual Screening

Journal of medicinal chemistry, Jul 8, 2016

Protein arginine methyltransferases (PRMTs) represent an emerging target class in oncology and ot... more Protein arginine methyltransferases (PRMTs) represent an emerging target class in oncology and other disease areas. So far, the most successful strategy to identify PRMT inhibitors has been to screen large to medium-size chemical libraries. Attempts to develop PRMT inhibitors using receptor-based computational methods have met limited success. Here, using virtual screening approaches, we identify 11 CARM1 (PRMT4) inhibitors with ligand efficiencies ranging from 0.28 to 0.84. CARM1 selective hits were further validated by orthogonal methods. Two structure-based rounds of optimization produced 27 (SGC2085), a CARM1 inhibitor with an IC50 of 50 nM and more than hundred-fold selectivity over other PRMTs. These results indicate that virtual screening strategies can be successfully applied to Rossmann-fold protein methyltransferases.

Research paper thumbnail of PR Domain-Containing Protein 7 (PRDM7) is a Histone 3 Lysine 4 Trimethyltransferase

Journal of Biological Chemistry, 2016

PR domain-containing protein 7 (PRDM7) is a primate-specific histone methyltransferase that is th... more PR domain-containing protein 7 (PRDM7) is a primate-specific histone methyltransferase that is the result of a recent gene duplication of PRDM9. The two proteins are highly homologous, especially in the catalytic PR/SET domain, where they differ by only three amino acid residues. Here we report that PRDM7 is an efficient methyltransferase that selectively catalyzes the trimethylation of H3 lysine 4 (H3K4) both in vitro and in cells. Through selective mutagenesis we have dissected the functional roles of each of the three divergent residues between the PR domains of PRDM7 and PRDM9. These studies indicate that after a single serine to tyrosine mutation at residue 357 (S357Y), PRDM7 regains the substrate specificities and catalytic activities similar to its evolutionary predecessor, including the ability to efficiently methylate H3K36.

Research paper thumbnail of Detection of Burkholderia cepacia complex in patients with cystic fibrosis

Tanaffos, 2004

Background: Damaging of the lung function in patients with cystic fibrosis is the most frequent c... more Background: Damaging of the lung function in patients with cystic fibrosis is the most frequent cause of death in these patients. Recently, Burkholderia cepacia has been emerged as an important opportunistic pathogen in these patients; because of its increased isolation from patients with cystic fibrosis since late1970s, the capacity for spread of infection among the cystic fibrosis patient community, its role in damaging lung functions, and its innate multiantibiotic resistance. These different aspects make isolation of Burkholderia cepacia an important task in cystic fibrosis health care settings. Materials and Methods: We examined the capacity of Burkholderia cepacia selective agar (BCSA) as a medium for primary isolation of Burkholderia cepacia samples. Biochemical tests were used to confirm the identification. Results: Burkholderia cepacia strains were isolated from 6 out of 53 respiratory samples as confirmed with biochemical tests. Conclusion: Results of the present study suggest that BCSA can be used as a selective medium with high specificity for primary isolation and identification of Burkholderia cepacia complex bacteria.

Research paper thumbnail of Discovery of a Potent Class I Protein Arginine Methyltransferase Fragment Inhibitor

Journal of Medicinal Chemistry, 2016

Protein methyltransferases (PMTs) are a promising target class in oncology and other disease area... more Protein methyltransferases (PMTs) are a promising target class in oncology and other disease areas. They are composed of SET domain methyltransferases and structurally unrelated Rossman-fold enzymes that include protein arginine methyltransferases (PRMTs). In the absence of a well-defined medicinal chemistry tool-kit focused on PMTs, most current inhibitors were identified by screening large and diverse libraries of leadlike molecules. So far, no successful fragment-based approach was reported against this target class. Here, by deconstructing potent PRMT inhibitors, we find that chemical moieties occupying the substrate arginine-binding site can act as efficient fragment inhibitors. Screening a fragment library against PRMT6 produced numerous hits, including a 300 nM inhibitor (ligand efficiency of 0.56) that decreased global histone 3 arginine 2 methylation in cells, and can serve as a warhead for the development of PRMT chemical probes.

Research paper thumbnail of A Potent, Selective and Cell-active Inhibitor of Human Type I Protein Arginine Methyltransferases

ACS chemical biology, Jan 24, 2015

Protein arginine methyltransferases (PRMTs) play a crucial role in a variety of biological proces... more Protein arginine methyltransferases (PRMTs) play a crucial role in a variety of biological processes. Overexpression of PRMTs has been implicated in various human diseases including cancer. Consequently, selective small-molecule inhibitors of PRMTs have been pursued by both academia and pharmaceutical industry as chemical tools for testing biological and therapeutic hypotheses. PRMTs are divided into three categories: type I PRMTs which catalyze mono- and asymmetric dimethylation of arginine residues, type II PRMTs which catalyze mono- and symmetric dimethylation of arginine residues, and type III PRMT which catalyzes only monomethylation of arginine residues. Here, we report the discovery of a potent, selective and cell-active inhibitor of human type I PRMTs, MS023, and characterization of this inhibitor in a battery of biochemical, biophysical and cellular assays. MS023 displayed high potency for type I PRMTs including PRMT1, 3, 4, 6 and 8, but was completely inactive against type...