Identification of 2,2-Dimethylbutanoic Acid (HST5040), a Clinical Development Candidate for the Treatment of Propionic Acidemia and Methylmalonic Acidemia.

Propionic acidemia (PA) and methylmalonic acidemia (MMA) are rare autosomal recessive disorders of propionyl-CoA (P-CoA) catabolism, caused by a deficiency in the enzymes P-CoA carboxylase and methylmalonyl-CoA (M-CoA) mutase, respectively. PA and MMA are classified as intoxication-type inborn errors of metabolism because the intramitochondrial accumulation of P-CoA, M-CoA, and other metabolites results in secondary inhibition of multiple pathways of intermediary metabolism, leading to organ dysfunction and failure. Herein, we describe the structure-activity relationships of a series of short-chain carboxylic acids which reduce disease-related metabolites in PA and MMA primary hepatocyte disease models. These studies culminated in the identification of 2,2-dimethylbutanoic acid (10, HST5040) as a clinical candidate for the treatment of PA and MMA. Additionally, we describe the in vitro and in vivo absorption, distribution, metabolism, and excretion profile of HST5040, data from preclinical studies, and the synthesis of the sodium salt of HST5040 for clinical trials.

A novel small molecule approach for the treatment of propionic and methylmalonic acidemias.

Propionic Acidemia (PA) and Methylmalonic Acidemia (MMA) are inborn errors of metabolism affecting the catabolism of valine, isoleucine, methionine, threonine and odd-chain fatty acids. These are multi-organ disorders caused by the enzymatic deficiency of propionyl-CoA carboxylase (PCC) or methylmalonyl-CoA mutase (MUT), resulting in the accumulation of propionyl-coenzyme A (P-CoA) and methylmalonyl-CoA (M-CoA in MMA only). Primary metabolites of these CoA esters include 2-methylcitric acid (MCA), propionyl-carnitine (C3), and 3-hydroxypropionic acid, which are detectable in both PA and MMA, and methylmalonic acid, which is detectable in MMA patients only (Chapman et al., 2012). We deployed liver cell-based models that utilized PA and MMA patient-derived primary hepatocytes to validate a small molecule therapy for PA and MMA patients. The small molecule, HST5040, resulted in a dose-dependent reduction in the levels of P-CoA, M-CoA (in MMA) and the disease-relevant biomarkers C3, MCA, and methylmalonic acid (in MMA). A putative working model of how HST5040 reduces the P-CoA and its derived metabolites involves the conversion of HST5040 to HST5040-CoA driving the redistribution of free and conjugated CoA pools, resulting in the differential reduction of the aberrantly high P-CoA and M-CoA. The reduction of P-CoA and M-CoA, either by slowing production (due to increased demands on the free CoA (CoASH) pool) or enhancing clearance (to replenish the CoASH pool), results in a net decrease in the CoA-derived metabolites (C3, MCA and MMA (MMA only)). A Phase 2 study in PA and MMA patients will be initiated in the United States.

A novel fluorogenic substrate for the measurement of endothelial lipase activity.

Endothelial lipase (EL) is a phospholipase A1 (PLA1) enzyme that hydrolyzes phospholipids at the sn-1 position to produce lysophospholipids and free fatty acids. Measurement of the PLA1 activity of EL is usually accomplished by the use of substrates that are also hydrolyzed by lipases in other subfamilies such as PLA2 enzymes. In order to distinguish PLA1 activity of EL from PLA2 enzymatic activity in cell-based assays, cell supernatants, and other nonhomogeneous systems, a novel fluorogenic substrate with selectivity toward PLA1 hydrolysis was conceived and characterized. This substrate was preferred by PLA1 enzymes, such as EL and hepatic lipase, and was cleaved with much lower efficiency by lipases that exhibit primarily triglyceride lipase activity, such as LPL or a lipase with PLA2 activity. The phospholipase activity detected by the PLA1 substrate could be inhibited with the small molecule esterase inhibitor ebelactone B. Furthermore, the PLA1 substrate was able to detect EL activity in human umbilical vein endothelial cells in a cell-based assay. This substrate is a useful reagent for identifying modulators of PLA1 enzymes, such as EL, and aiding in characterizing their mechanisms of action.

Characterizing the effects of inorganic acid and alkaline shock on the Staphylococcus aureus transcriptome and messenger RNA turnover.

Staphylococcus aureus pathogenesis can be attributed partially to its ability to adapt to otherwise deleterious host-associated stresses. Here, Affymetrix GeneChips® were used to examine the S. aureus responses to inorganic acid and alkaline shock and to assess whether stress-dependent changes in mRNA turnover are likely to facilitate the organism's ability to tolerate a pH challenge. The results indicate that S. aureus adapts to pH shock by eliciting responses expected of cells coping with pH alteration, including neutralizing cellular pH, DNA repair, amino acid biosynthesis, and virulence factor expression. Further, the S. aureus response to alkaline conditions is strikingly similar to that of stringent response-induced cells. Indeed, we show that alkaline shock stimulates the accumulation of the stringent response activator (p)ppGpp. The results also revealed that pH shock significantly alters the mRNA properties of the cell. A comparison of the mRNA degradation properties of transcripts whose titers either increased or decreased in response to a sudden pH change revealed that alterations in mRNA degradation may, in part, account for the changes in the mRNA levels of factors predicted to mediate pH tolerance. A set of small stable RNA molecules were induced in response to acid- or alkaline-shock conditions and may mediate adaptation to pH stress.

Functional, biophysical, and structural bases for antibacterial activity of tigecycline.

Tigecycline is a novel glycylcycline antibiotic that possesses broad-spectrum activity against many clinically relevant species of bacterial pathogens. The mechanism of action of tigecycline was delineated using functional, biophysical, and molecular modeling experiments in this study. Functional assays showed that tigecycline specifically inhibits bacterial protein synthesis with potency 3- and 20-fold greater than that of minocycline and tetracycline, respectively. Biophysical analyses demonstrated that isolated ribosomes bind tigecycline, minocycline, and tetracycline with dissociation constant values of 10(-8), 10(-7), and >10(-6) M, respectively. A molecular model of tigecycline bound to the ribosome was generated with the aid of a 3.40-angstrom resolution X-ray diffraction structure of the 30S ribosomal subunit from Thermus thermophilus. This model places tigecycline in the A site of the 30S subunit and involves substantial interactions with residues of H34 of the ribosomal subunit. These interactions were not observed in a model of tetracycline binding. Modeling data were consistent with the biochemical and biophysical data generated in this and other recent studies and suggested that tigecycline binds to bacterial ribosomes in a novel way that allows it to overcome tetracycline resistance due to ribosomal protection.

Design and syntheses of 1,6-naphthalene derivatives as selective HCMV protease inhibitors.

Through high throughput screening of various libraries, substituted styryl naphthalene 6 was identified as an HCMV protease inhibitor. Optimization of various regions of the lead molecule using parallel synthesis resulted in 1,6-substituted naphthalenes 19d-i. These compounds displayed good potency and were selective over elastase, trypsin, and chymotrypsin. The optimization approach on lead compound 6 in three different regions of the molecule using parallel solution-phase synthesis and the corresponding SAR are discussed in detail.

Characterization of the monomer-dimer equilibrium of human cytomegalovirus protease by kinetic methods.

Herpesviruses encode a serine protease that is essential for the maturation of infectious virions. This protease has a unique polypeptide backbone fold and contains a novel Ser-His-His catalytic triad. It exists in a monomer-dimer equilibrium in solution, but only the dimer form of the enzyme is catalytically active. The stability of this dimer is affected by the presence of anti-chaotropic agents. Most of the reported Kd values for this dimer (between 0.6 and 6 microM) are inconsistent with the fact that the protease is routinely assayed at 20-50 nM concentrations, as only monomeric species would be expected with such Kd values. We have characterized the monomer-dimer equilibrium of HCMV protease using a new method, which observes the exchange between dimers of the wild-type enzyme and the active-site Ser132Ala mutant in a titration experiment. The Kd of the dimer was determined to be 8 microM and 31 nM in the absence or presence of anti-chaotropic agents (10% glycerol and 0.5 M Na2SO4), respectively. Detailed kinetic analysis also showed that, in addition to the 260-fold stabilization of the dimer, the anti-chaotropic agents produced a 7-fold enhancement in the catalytic activity of the dimer.