A Potent, Selective, Small-Molecule Inhibitor of DHX9 Abrogates Proliferation of Microsatellite Instable Cancers with Deficient Mismatch Repair.

DHX9 is a multifunctional DExH-box RNA helicase with important roles in the regulation of transcription, translation, and maintenance of genome stability. Elevated expression of DHX9 is evident in multiple cancer types, including colorectal cancer (CRC). Microsatellite instable-high (MSI-H) tumors with deficient mismatch repair (dMMR) display a strong dependence on DHX9, making this helicase an attractive target for oncology drug discovery.

Development of assays to support identification and characterization of modulators of DExH-box helicase DHX9.

DHX9 is a DExH-box RNA helicase that utilizes hydrolysis of all four nucleotide triphosphates (NTPs) to power cycles of 3' to 5' directional movement to resolve and/or unwind double stranded RNA, DNA, and RNA/DNA hybrids, R-loops, triplex-DNA and G-quadraplexes. DHX9 activity is important for both viral amplification and maintaining genomic stability in cancer cells; therefore, it is a therapeutic target of interest for drug discovery efforts. Biochemical assays measuring ATP hydrolysis and oligonucleotide unwinding for DHX9 have been developed and characterized, and these assays can support high-throughput compound screening efforts under balanced conditions.

A Mass Spectrometric Assay of METTL3/METTL14 Methyltransferase Activity.

A variety of covalent modifications of RNA have been identified and demonstrated to affect RNA processing, stability, and translation. Methylation of adenosine at the N6 position (mA) in messenger RNA (mRNA) is currently the most well-studied RNA modification and is catalyzed by the RNA methyltransferase complex METTL3/METTL14. Once generated, mA can modulate mRNA splicing, export, localization, degradation, and translation.

Crystallographic structure of a small molecule SIRT1 activator-enzyme complex.

SIRT1, the founding member of the mammalian family of seven NAD(+)-dependent sirtuins, is composed of 747 amino acids forming a catalytic domain and extended N- and C-terminal regions. We report the design and characterization of an engineered human SIRT1 construct (mini-hSIRT1) containing the minimal structural elements required for lysine deacetylation and catalytic activation by small molecule sirtuin-activating compounds (STACs). Using this construct, we solved the crystal structure of a mini-hSIRT1-STAC complex, which revealed the STAC-binding site within the N-terminal domain of hSIRT1.

Evidence for a common mechanism of SIRT1 regulation by allosteric activators.

A molecule that treats multiple age-related diseases would have a major impact on global health and economics. The SIRT1 deacetylase has drawn attention in this regard as a target for drug design. Yet controversy exists around the mechanism of sirtuin-activating compounds (STACs).

SIRT1 activation by small molecules: kinetic and biophysical evidence for direct interaction of enzyme and activator.

SIRT1 is a protein deacetylase that has emerged as a therapeutic target for the development of activators to treat diseases of aging. SIRT1-activating compounds (STACs) have been developed that produce biological effects consistent with direct SIRT1 activation. At the molecular level, the mechanism by which STACs activate SIRT1 remains elusive.

Effect of Peptide-Chelate Architecture on Metabolic Stability of Peptide-based MRI Contrast Agents.

A strategy for preparing high relaxivity, metabolically stable peptide-based MR contrast agents is described.

Structure-activity relationship study of EphB3 receptor tyrosine kinase inhibitors.

A structure-activity relationship study for a 2-chloroanilide derivative of pyrazolo[1,5-a]pyridine revealed that increased EphB3 kinase inhibitory activity could be accomplished by retaining the 2-chloroanilide and introducing a phenyl or small electron donating substituents to the 5-position of the pyrazolo[1,5-a]pyridine. In addition, replacement of the pyrazolo[1,5-a]pyridine with imidazo[1,2-a]pyridine was well tolerated and resulted in enhanced mouse liver microsome stability. The structure-activity relationship for EphB3 inhibition of both heterocyclic series was similar.

Structure-activity relationship, kinetic mechanism, and selectivity for a new class of ubiquitin C-terminal hydrolase-L1 (UCH-L1) inhibitors.

3-Amino-2-keto-7H-thieno[2,3-b]pyridin-6-one derivatives were discovered as moderately potent inhibitors of ubiquitin C-terminal hydrolase-L1 (UCH-L1) utilizing an assay that measures hydrolysis of the fluorogenic substrate Ub-AMC. SAR studies revealed that both the carboxylate at the 5-position and the 6-pyridone ring were critical for inhibitory activity. Furthermore, activity was dependent on the nature of the ketone substituent at the 2-position, with 4-Me-Ph and 2-naphthyl being best.

Kinetic analysis of the interaction of tissue transglutaminase with a nonpeptidic slow-binding inhibitor.

Tissue transglutaminase (TGase) is a Ca2+-dependent enzyme that catalyzes cross-linking of intracellular proteins through a mechanism that involves isopeptide bond formation between Gln and Lys residues and is allosterically regulated by GTP. TGase is thought to play a pathogenic role in neurodegenerative diseases by promoting aggregation of disease-specific proteins that accumulate as part of these disorders. Given the role that TGase plays in neurodegenerative disorders, we initiated a research program to discover inhibitors of this enzyme that might ultimately be developed into therapeutic agents.

Mechanistic studies of ubiquitin C-terminal hydrolase L1.

Ubiquitin C-terminal hydrolases (UCHs) cleave Ub-X bonds (Ub is ubiquitin and X an alcohol, an amine, or a protein) through a thioester intermediate that is produced by nucleophilic attack of the Cys residue of a Cys-SH/His-Im catalytic diad. We are studying the mechanism of UCH-L1, a UCH that is implicated in Parkinson's disease, and now wish to report our initial findings. (i) Pre-steady-state kinetic studies for UCH-L1-catalyzed hydrolysis of Ub-AMC (AMC, 7-amido-4-methylcoumarin) indicate that k(cat) is rate-limited by acyl-enzyme formation.

Structure-activity relationship study of novel tissue transglutaminase inhibitors.

Thieno[2,3-d]pyrimidin-4-one acylhydrazide derivatives were discovered as moderately potent inhibitors of TGase 2 (tissue transglutaminase) utilizing a fluorescence-based assay that measured TGase 2 catalyzed incorporation of the dansylated Lys derivative alpha-N-Boc-Lys-CH(2)-CH(2)-dansyl into the protein substrate N,N-dimethylated-casein. A SAR study revealed that the acylhydrazide thioether side-chain and the thiophene ring were critical to inhibitory activity.

Development of a mechanism-based assay for tissue transglutaminase--results of a high-throughput screen and discovery of inhibitors.

Tissue transglutaminase (TGase) is a Ca(2+)-dependent enzyme that catalyzes cross-linking of intracellular proteins through a mechanism that involves isopeptide bond formation between Gln and Lys residues. In addition to its transamidation activity, TGase can bind guanosine 5'-triphosphate (GTP) and does so in a manner that is antagonized by calcium. Once bound, GTP undergoes hydrolysis to form guanosine 5'-diphosphate and inorganic phosphate.

Discovery of inhibitors that elucidate the role of UCH-L1 activity in the H1299 lung cancer cell line.

Neuronal ubiquitin C-terminal hydrolase (UCH-L1) has been linked to Parkinson's disease (PD), the progression of certain nonneuronal tumors, and neuropathic pain. Certain lung tumor-derived cell lines express UCH-L1 but it is not expressed in normal lung tissue, suggesting that this enzyme plays a role in tumor progression, either as a trigger or as a response. Small-molecule inhibitors of UCH-L1 would be helpful in distinguishing between these scenarios.

Kinetic analysis of the action of tissue transglutaminase on peptide and protein substrates.

Tissue transglutaminase (TGase) catalyzes transfer of gamma-acyl moieties of Gln residues in peptides or protein substrates to either water or amine nucleophiles through an acyl-enzyme intermediate formed from initial acyl-transfer to an active site Cys residue. Natural substrates for this enzyme include proteins (e.g.

Enzymatic reaction of silent substrates: kinetic theory and application to the serine protease chymotrypsin.

Investigating the selectivity that an enzyme expresses toward its substrates can be technically challenging if reaction of these substrates is not accompanied by a conveniently monitored change in some physicochemical property. In this paper, we describe a simple method for determining steady-state kinetic parameters for enzymatic turnover of such "silent" substrates. According to this method, silent substrate S is allowed to compete for enzymic reaction with signal-generating substrate S*, whose conversion to product can be conveniently monitored.

Mechanistic origins of the substrate selectivity of serine proteases.

Serine proteases catalyze the hydrolysis of amide bonds of their protein and peptide substrates through a mechanism involving the intermediacy of an acyl-enzyme. While the rate constant for formation of this intermediate, k(2), shows a dramatic dependence on peptide chain length, the rate constant for the intermediate's hydrolysis is relatively insensitive to chain length. To probe the mechanistic origins of this phenomenon, we determined temperature dependencies and solvent isotope effects for the alpha-chymotrypsin-catalyzed hydrolysis of Suc-Phe-pNA (K(s) = 1 mM, k(2) = 0.