Ryan G. Kruger

Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27)

Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive posttranslational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the polycomb repressive complex 2 and is overexpressed in many cancers. In B-cell lymphomas, its substrate preference is frequently altered through somatic mutation of the EZH2 Y641 residue. Herein, we identify mutation of EZH2 A677 to a glycine (A677G) among lymphoma cell lines and primary tumor specimens. Similar to Y641 mutant cell lines, an A677G mutant cell line revealed aberrantly elevated H3K27me3 and decreased monomethylated H3K27 (H3K27me1) and dimethylated H3K27 (H3K27me2). A677G EZH2 possessed catalytic activity with a substrate specificity that was distinct from those of both WT EZH2 and Y641 mutants. Whereas WT EZH2 displayed a preference for substrates with less methylation [unmethylated H3K27 (H3K27me0):me1:me2 kcat/Km ratio = 9:6:1] and Y641 mutants preferred substrates with greater methylation (H3K27me0:me1:me2 kcat/Km ratio = 1:2:13), the A677G EZH2 demonstrated nearly equal efficiency for all three substrates (H3K27me0:me1:me2 kcat/Km ratio = 1.1:0.6:1). When transiently expressed in cells, A677G EZH2, but not WT EZH2, increased global H3K27me3 and decreased H3K27me2. Structural modeling of WT and mutant EZH2 suggested that the A677G mutation acquires the ability to methylate H3K27me2 through enlargement of the lysine tunnel while preserving activity with H3K27me0/me1 substrates through retention of the Y641 residue that is crucial for orientation of these smaller substrates. This mutation highlights the interplay between Y641 and A677 residues in the substrate specificity of EZH2 and identifies another lymphoma patient population that harbors an activating mutation of EZH2.

Assembly of the SIR Complex and Its Regulation by O-Acetyl-ADP-Ribose, a Product of NAD-Dependent Histone Deacetylation

Assembly of silent chromatin domains in budding yeast involves the deacetylation of histone tails by Sir2 and the association of the Sir3 and Sir4 proteins with hypoacetylated histone tails. Sir2 couples deacetylation to NAD hydrolysis and the synthesis of a metabolite, O-acetyl-ADP-ribose (AAR), but the functional significance of NAD hydrolysis or AAR, if any, is unknown. Here we examine the association of the Sir2, Sir3, and Sir4 proteins with each other and histone tails. Our analysis reveals that deacetylation of histone H4-lysine 16 (K16), which is critical for silencing in vivo, is also critical for the binding of Sir3 and Sir4 to histone H4 peptides in vitro. Moreover, AAR itself promotes the association of multiple copies of Sir3 with Sir2/Sir4 and induces a dramatic structural rearrangement in the SIR complex. These results suggest that Sir2 activity modulates the assembly of the SIR complex through both histone deacetylation and AAR synthesis.

A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models.

Protein arginine methyltransferase-5 (PRMT5) is reported to have a role in diverse cellular processes, including tumorigenesis, and its overexpression is observed in cell lines and primary patient samples derived from lymphomas, particularly mantle cell lymphoma (MCL). Here we describe the identification and characterization of a potent and selective inhibitor of PRMT5 with antiproliferative effects in both in vitro and in vivo models of MCL. EPZ015666 (GSK3235025) is an orally available inhibitor of PRMT5 enzymatic activity in biochemical assays with a half-maximal inhibitory concentration (IC50) of 22 nM and broad selectivity against a panel of other histone methyltransferases. Treatment of MCL cell lines with EPZ015666 led to inhibition of SmD3 methylation and cell death, with IC50 values in the nanomolar range. Oral dosing with EPZ015666 demonstrated dose-dependent antitumor activity in multiple MCL xenograft models. EPZ015666 represents a validated chemical probe for further study of PRMT5 biology and arginine methylation in cancer and other diseases.

Synergy and duality in peptide antibiotic mechanisms

The molecular mechanisms by which peptide antibiotics disrupt bacterial DNA synthesis, protein biosynthesis, cell wall biosynthesis, and membrane integrity are diverse, yet historically have been understood to follow a theme of one antibiotic, one inhibitory mechanism. In the past year, mechanistic and structural studies have shown a rich diversity in peptide antibiotic mechanism. Novel secondary targeting mechanisms for peptide antibiotics have recently been discovered, and the mechanisms of peptide antibiotics involved in synergistic relationships with antibiotics and proteins have been more clearly defined. In apparent response to selective pressures, antibiotic-producing organisms have elegantly integrated multiple functions and cooperative interactions into peptide antibiotic design for the purpose of improving antimicrobial success.

Complexation of peptidoglycan intermediates by the lipoglycodepsipeptide antibiotic ramoplanin: Minimal structural requirements for intermolecular complexation and fibril formation

The peptide antibiotic ramoplanin inhibits bacterial peptidoglycan (PG) biosynthesis by interrupting late-stage membrane-associated glycosyltransferase reactions catalyzed by the transglycosylase and MurG enzymes. The mechanism of ramoplanin involves sequestration of lipid-anchored PG biosynthesis intermediates, physically occluding these substrates from proper utilization by these enzymes. In this report, we describe the first molecular-level details of the interaction of ramoplanin with PG biosynthesis intermediates. NMR analysis in conjunction with chemical dissection of the PG monomer revealed that the ramoplanin octapeptide d-Hpg-d-Orn-d-alloThr-Hpg-d-Hpg-alloThr-Phe-d-Orn recognizes MurNAc-Ala-γ-d-Glu pyrophosphate, the minimum component of PG capable of high-affinity complexation and fibril formation. Ramoplanin therefore recognizes a PG binding locus different from the N-acyl-d-Ala-d-Ala moiety targeted by vancomycin. Because ramoplanin is structurally less complex than glycopeptide antibiotics such as vancomycin, peptidomimetic chemotherapeutics derived from this recognition sequence may find future use as antibiotics against vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, and related pathogens.

Functional Analysis of the Lipoglycodepsipeptide Antibiotic Ramoplanin

The peptide antibiotic ramoplanin is highly effective against several drug-resistant gram-positive bacteria, including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), two important opportunistic human pathogens. Ramoplanin inhibits bacterial peptidoglycan (PG) biosynthesis by binding to Lipid intermediates I and II at a location different than the N-acyl-D-Ala-D-Ala dipeptide site targeted by vancomycin. Lipid I/II capture physically occludes these substrates from proper utilization by the late-stage PG biosynthesis enzymes MurG and the transglycosylases. Key structural features of ramoplanin responsible for antibiotic activity and PG molecular recognition have been discovered by antibiotic semisynthetic modification in conjunction with NMR analyses. These results help define a minimalist ramoplanin pharmacophore and introduce the possibility of generating ramoplanin-derived peptide or peptidomimetic antibiotics for use against VRE, MRSA, and related pathogens.

Long residence time inhibition of EZH2 in activated polycomb repressive complex 2.

EZH2/PRC2 catalyzes transcriptionally repressive methylation at lysine 27 of histone H3 and has been associated with numerous cancer types. Point mutations in EZH2 at Tyr641 and Ala677 identified in non-Hodgkin lymphomas alter substrate specificity and result in increased trimethylation at histone H3K27. Interestingly, EZH2/PRC2 is activated by binding H3K27me3 marks on histones, and this activation is proposed as a mechanism for self-propagation of gene silencing. Recent work has identified GSK126 as a potent, selective, SAM-competitive inhibitor of EZH2 capable of globally decreasing H3K27 trimethylation in cells. Here we show that activation of PRC2 by an H3 peptide trimethylated at K27 is primarily an effect on the rate-limiting step (kcat) with no effect on substrate binding (Km). Additionally, GSK126 is shown to have a significantly longer residence time of inhibition on the activated form of EZH2/PRC2 as compared to unactivated EZH2/PRC2. Overall inhibition constant (Ki*) values for GSK126 were determined to be as low as 93 pM and appear to be driven by slow dissociation of inhibitor from the activated enzyme. The data suggest that activation of EZH2 allows the enzyme to adopt a conformation that possesses greater affinity for GSK126. The long residence time of GSK126 may be beneficial in vivo and may result in durable target inhibition after drug systemic clearance.

Synthesis of P1-Citronellyl-P2-α-d-pyranosyl pyrophosphates as potential substrates for the E. coli undecaprenyl-pyrophosphoryl-N-acetylglucoseaminyl transferase MurG

P(1)-Citronellyl-P(2)-alpha-D-pyranosyl pyrophosphates containing alpha-D-N-acetylglucoseaminyl, alpha-D-glucosyl, and alpha-D-N-acetylmuramyl carbohydrates were synthesized and used in substrate specificity studies of the Escherichia coli MurG enzyme. Oxalyl chloride activation of citronellyl phosphate for coupling to alpha-D-pyranose-1-phosphates resulted in markedly improved yields over traditional Khorana-Moffatt and diphenyl chlorophosphate activation strategies.

An economical and preparative orthogonal solid phase synthesis of fluorescein and rhodamine derivatized peptides: FRET substrates for the Staphylococcus aureus sortase SrtA transpeptidase reaction

An economical and preparative-scale orthogonal solid-phase method of incorporating carboxyrhodamine and carboxyfluorescein fluorescence resonance energy transfer (FRET) probes site-specifically into synthetic peptide substrates for the S. aureus Sortase transpeptidase SrtA has been developed.

Steroid-binding site residues dictate optimal substrate positioning in rat 3α-hydroxysteroid dehydrogenase (3α-HSD or AKR1C9)

Abstract Rat liver 3α-hydroxysteroid dehydrogenase (3α-HSD or AKR1C9), a member of the aldo–keto reductase (AKR) superfamily, plays a pivotal role in the inactivation of circulating steroid hormones. It is the most thoroughly characterized HSD of the AKR superfamily and can be used as a template for structure-function studies in other AKR members such as rodent and human 3α-, 17β- and 20α-HSDs. Based on the crystal structure of the E·NADP+ testosterone ternary complex, there are ten residues that line the testosterone binding cavity: T24, L54, Y55, H117, F118, F129, T226, W227, N306 and Y310. Each residue in the cavity, except for the catalytic residues Y55 and H117, was systematically mutated to alanine to determine the role of the individual residues in steroid recognition. Binding data and kinetic parameters (Kd, kcat, Km and kcat/Km) of the homogeneous mutants were compared with that of the wild type (WT) enzyme. Titration of the intrinsic tryptophan fluorescence with NADPH demonstrated that cofactor binding was unaltered. However, binding of the steroid hormones testosterone and progesterone to the E·NADPH binary complex was affected to varying degrees. The largest effects on Kd were an 8-fold decrease in affinity for testosterone and a 50-fold decrease in affinity for progesterone. The mutants bound both hormones in the same rank-order except for W227A, where the binding of progesterone was more adversely affected. A series of 3α-hydroxysteroid substrates (A/B trans- and cis-ring fused C19 and C21 steroids) were used to determine the ability of each mutant to catalyze steroid turnover. The alanine mutants that retained kcat/Km values similar to WT were those in which alanine substituted short polar residues such as T24A and T226A. The mutants with the lowest catalytic efficiencies were those in which alanine substituted aromatic residues such as W227A and F129A. The loss in catalytic efficiency was due to large changes in kcat (up to 1000-fold), but not Km. Molecular modeling of the alanine mutants showed that changes in the reaction trajectory defined by the angles and distances by groups that participate in catalysis correlate with changes in kcat. These results highlight the importance of steroid binding site residues in dictating the proper orientation of substrates to achieve high catalytic turnover while having minimal effects on hormone affinity.