(R)-β-lysine Modified Elongation Factor P Functions in Translation Elongation

Post-translational modification of bacterial elongation factor P (EF-P) with (R)-β-lysine at a conserved lysine residue activates the protein in vivo and increases puromycin reactivity of the ribosome in vitro. The additional hydroxylation of EF-P at the same lysine residue by the YfcM protein has also recently been described. The roles of modified and unmodified EF-P during different steps in translation, and how this correlates to its physiological role in the cell, have recently been linked to the synthesis of polyproline stretches in proteins. Polysome analysis indicated that EF-P functions in translation elongation, rather than initiation as proposed previously. This was further supported by the inability of EF-P to enhance the rate of formation of fMet-Lys or fMet-Phe, indicating that the role of EF-P is not to specifically stimulate formation of the first peptide bond. Investigation of hydroxyl-(β)-lysyl-EF-P showed 30% increased puromycin reactivity but no differences in dipeptide synthesis rates when compared with the β-lysylated form. Unlike disruption of the other genes required for EF-P modification, deletion of yfcM had no phenotypic consequences in Salmonella. Taken together, our findings indicate that EF-P functions in translation elongation, a role critically dependent on post-translational β-lysylation but not hydroxylation

An Allosteric Inhibitor of Protein Arginine Methyltransferase 3

PRMT3, a protein arginine methyltransferase, has been shown to influence ribosomal biosynthesis by catalyzing the dimethylation of the 40S ribosomal protein S2. Although PRMT3 has been reported to be a cytosolic protein, it has been shown to methylate histone H4 peptide (H4 1-24) in vitro. Here, we report the identification of a PRMT3 inhibitor (1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(2-cyclohexenylethyl)urea; compound 1) with IC50 value of 2.5 μM by screening a library of 16,000 compounds using H4 (1-24) peptide as a substrate. The crystal structure of PRMT3 in complex with compound 1 as well as kinetic analysis reveals an allosteric mechanism of inhibition. Mutating PRMT3 residues within the allosteric site or using compound 1 analogs that disrupt interactions with allosteric site residues both abrogated binding and inhibitory activity. These data demonstrate an allosteric mechanism for inhibition of protein arginine methyltransferases, an emerging class of therapeutic targets

Brønsted Acid-Catalyzed Direct Substitution of 2-Ethoxytetrahydrofuran with Trifluoroborate Salts

Metal-free transformations of organotrifluoroborates are advantageous since they avoid the use of frequently expensive and sensitive transition metals. Lewis acid-catalyzed reactions involving potassium trifluoroborate salts have emerged as an alternative to metal-catalyzed protocols. However, the drawbacks to these methods are that they rely on the generation of unstable boron dihalide species, thereby resulting in low functional group tolerance. Recently, we discovered that in the presence of a Brønsted acid, trifluoroborate salts react rapidly with in situ generated oxocarbenium ions. Here, we report Brønsted acid-catalyzed direct substitution of 2-ethoxytetrahydrofuran using potassium trifluoroborate salts. The reaction occurs when tetrafluoroboric acid is used as a catalyst to afford functionalized furans in moderate to excellent yields. A variety of alkenyl- and alkynyltrifluoroborate salts readily participate in this transformation

Metal-Free Synthesis of Ynones from Acyl Chlorides and Potassium Alkynyltrifluoroborate Salts

A straightforward method for the preparation of ynones from acyl chlorides and potassium alkynyltrifluoroborate salts has been developed. The one-pot reaction proceeds rapidly in the presence of a Lewis acid without exclusion of air and moisture

Brønsted Acid-Catalyzed Reactions of Trifluoroborate Salts with Benzhydryl Alcohols

Brønsted acid-catalyzed carbon–carbon bond forming methodology using potassium alkynyl- and alkenyltrifluoroborate salts has been developed. Organotrifluoroborates react with benzhydryl alcohols to afford a broad range of alkynes and alkenes in good to excellent yields. This protocol features good atom economy because organotrifluoroborate salts and alcohols react in a 1:1 ratio. Furthermore, a variety of unprotected functional groups were tolerated under the developed conditions, including amide, aldehyde, free hydroxyl, and carboxylic acid

(R)-β-lysine-modified elongation factor P functions in translation elongation

Post-translational modification of bacterial elongation factor P (EF-P) with (R)-β-lysine at a conserved lysine residue activates the protein in vivo and increases puromycin reactivity of the ribosome in vitro. The additional hydroxylation of EF-P at the same lysine residue by the YfcM protein has also recently been described. The roles of modified and unmodified EF-P during different steps in translation, and how this correlates to its physiological role in the cell, have recently been linked to the synthesis of polyproline stretches in proteins. Polysome analysis indicated that EF-P functions in translation elongation, rather than initiation as proposed previously. This was further supported by the inability of EF-P to enhance the rate of formation of fMet-Lys or fMet-Phe, indicating that the role of EF-P is not to specifically stimulate formation of the first peptide bond. Investigation of hydroxyl-(β)-lysyl-EF-P showed 30% increased puromycin reactivity but no differences in dipeptide synthesis rates when compared with the β-lysylated form. Unlike disruption of the other genes required for EF-P modification, deletion of yfcM had no phenotypic consequences in Salmonella. Taken together, our findings indicate that EF-P functions in translation elongation, a role critically dependent on post-translational β-lysylation but not hydroxylation

(R)-β-Lysine-modified Elongation Factor P Functions in Translation Elongation

Post-translational modification of bacterial elongation factor P (EF-P) with (R)-β-lysine at a conserved lysine residue activates the protein in vivo and increases puromycin reactivity of the ribosome in vitro. The additional hydroxylation of EF-P at the same lysine residue by the YfcM protein has also recently been described. The roles of modified and unmodified EF-P during different steps in translation, and how this correlates to its physiological role in the cell, have recently been linked to the synthesis of polyproline stretches in proteins. Polysome analysis indicated that EF-P functions in translation elongation, rather than initiation as proposed previously. This was further supported by the inability of EF-P to enhance the rate of formation of fMet-Lys or fMet-Phe, indicating that the role of EF-P is not to specifically stimulate formation of the first peptide bond. Investigation of hydroxyl-(β)-lysyl-EF-P showed 30% increased puromycin reactivity but no differences in dipeptide synthesis rates when compared with the β-lysylated form. Unlike disruption of the other genes required for EF-P modification, deletion of yfcM had no phenotypic consequences in Salmonella. Taken together, our findings indicate that EF-P functions in translation elongation, a role critically dependent on post-translational β-lysylation but not hydroxylation

Structure-Based Optimization of a Small Molecule Antagonist of the Interaction Between WD Repeat-Containing Protein 5 (WDR5) and Mixed-Lineage Leukemia 1 (MLL1)

WD repeat-containing protein 5 (WDR5) is an important component of the multiprotein complex essential for activating mixed-lineage leukemia 1 (MLL1). Rearrangement of the MLL1 gene is associated with onset and progression of acute myeloid and lymphoblastic leukemias, and targeting the WDR5-MLL1 interaction may result in new cancer therapeutics. Our previous work showed that binding of small molecule ligands to WDR5 can modulate its interaction with MLL1, suppressing MLL1 methyltransferase activity. Initial structure–activity relationship studies identified <i>N</i>-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides as potent and selective antagonists of this protein–protein interaction. Guided by crystal structure data and supported by in silico library design, we optimized the scaffold by varying the C-1 benzamide and C-5 substituents. This allowed us to develop the first highly potent (<i>K</i>_disp < 100 nM) small molecule antagonists of the WDR5-MLL1 interaction and demonstrate that <i>N</i>-(4-(4-methylpiperazin-1-yl)-3′-(morpholinomethyl)-[1,1′-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide <b>16d</b> (OICR-9429) is a potent and selective chemical probe suitable to help dissect the biological role of WDR5

Structure-Based Optimization of a Small Molecule Antagonist of the Interaction Between WD Repeat-Containing Protein 5 (WDR5) and Mixed-Lineage Leukemia 1 (MLL1)

WD repeat-containing protein 5 (WDR5) is an important component of the multiprotein complex essential for activating mixed-lineage leukemia 1 (MLL1). Rearrangement of the MLL1 gene is associated with onset and progression of acute myeloid and lymphoblastic leukemias, and targeting the WDR5-MLL1 interaction may result in new cancer therapeutics. Our previous work showed that binding of small molecule ligands to WDR5 can modulate its interaction with MLL1, suppressing MLL1 methyltransferase activity. Initial structure–activity relationship studies identified <i>N</i>-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides as potent and selective antagonists of this protein–protein interaction. Guided by crystal structure data and supported by in silico library design, we optimized the scaffold by varying the C-1 benzamide and C-5 substituents. This allowed us to develop the first highly potent (<i>K</i>_disp < 100 nM) small molecule antagonists of the WDR5-MLL1 interaction and demonstrate that <i>N</i>-(4-(4-methylpiperazin-1-yl)-3′-(morpholinomethyl)-[1,1′-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide <b>16d</b> (OICR-9429) is a potent and selective chemical probe suitable to help dissect the biological role of WDR5

Synthesis, Optimization, and Evaluation of Novel Small Molecules as Antagonists of WDR5‑MLL Interaction

The WD40-repeat protein WDR5 plays a critical role in maintaining the integrity of MLL complexes and fully activating their methyltransferase function. MLL complexes, the trithorax-like family of SET1 methyltransferases, catalyze trimethylation of lysine 4 on histone 3, and they have been widely implicated in various cancers. Antagonism of WDR5 and MLL subunit interaction by small molecules has recently been presented as a practical way to inhibit activity of the MLL1 complex, and <i>N</i>-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides were reported as potent and selective antagonists of such an interaction. Here, we describe the protein crystal structure guided optimization of prototypic compound <b>2</b> (<i>K</i>_dis = 7 μM), leading to identification of more potent antagonist <b>47</b> (<i>K</i>_dis = 0.3 μM)