Lisa M. Sonatore

A peptide-based fluorescence resonance energy transfer assay for Bacillus anthracis lethal factor protease

A fluorescence resonance energy transfer assay has been developed for monitoring Bacillus anthracis lethal factor (LF) protease activity. A fluorogenic 16-mer peptide based on the known LF protease substrate MEK1 was synthesized and found to be cleaved by the enzyme at the anticipated site. Extension of this work to a fluorogenic 19-mer peptide, derived, in part, from a consensus sequence of known LF protease targets, produced a much better substrate, cleaving approximately 100 times more efficiently. This peptide sequence was modified further on resin to incorporate donor/quencher pairs to generate substrates for use in fluorescence resonance energy transfer-based appearance assays. All peptides cleaved at similar rates with signal/background ranging from 9–16 at 100% turnover. One of these substrates, denoted (Cou)Consensus(K(QSY-35)GG)-NH2, was selected for additional assay optimization. A plate-based assay requiring only low nanomolar levels of enzyme was developed for screening and inhibitor characterization.

1,3,8-Triazaspiro[4.5]decane-2,4-diones as efficacious pan-inhibitors of hypoxia-inducible factor prolyl hydroxylase 1-3 (HIF PHD1-3) for the treatment of anemia.

The discovery of 1,3,8-triazaspiro[4.5]decane-2,4-diones (spirohydantoins) as a structural class of pan-inhibitors of the prolyl hydroxylase (PHD) family of enzymes for the treatment of anemia is described. The initial hit class, spirooxindoles, was identified through affinity selection mass spectrometry (AS-MS) and optimized for PHD2 inhibition and optimal PK/PD profile (short-acting PHDi inhibitors). 1,3,8-Triazaspiro[4.5]decane-2,4-diones (spirohydantoins) were optimized as an advanced lead class derived from the original spiroindole hit. A new set of general conditions for C-N coupling, developed using a high-throughput experimentation (HTE) technique, enabled a full SAR analysis of the spirohydantoins. This rapid and directed SAR exploration has resulted in the first reported examples of hydantoin derivatives with good PK in preclinical species. Potassium channel off-target activity (hERG) was successfully eliminated through the systematic introduction of acidic functionality to the molecular structure. Undesired upregulation of alanine aminotransferese (ALT) liver enzymes was mitigated and a robust on-/off-target margin was achieved. Spirohydantoins represent a class of highly efficacious, short-acting PHD1-3 inhibitors causing a robust erythropoietin (EPO) upregulation in vivo in multiple preclinical species. This profile deems spirohydantoins as attractive short-acting PHDi inhibitors with the potential for treatment of anemia.

Pharmacological inhibition of diacylglycerol acyltransferase 1 reduces body weight and modulates gut peptide release—Potential insight into mechanism of action

Investigation was conducted to understand the mechanism of action of diacylglycerol acyltransferase 1 (DGAT1) using small molecules DGAT1 inhibitors, compounds K and L.

Microscale High-Throughput Experimentation as an Enabling Technology in Drug Discovery: Application in the Discovery of (Piperidinyl)pyridinyl-1H-benzimidazole Diacylglycerol Acyltransferase 1 Inhibitors

Miniaturization and parallel processing play an important role in the evolution of many technologies. We demonstrate the application of miniaturized high-throughput experimentation methods to resolve synthetic chemistry challenges on the frontlines of a lead optimization effort to develop diacylglycerol acyltransferase (DGAT1) inhibitors. Reactions were performed on ∼1 mg scale using glass microvials providing a miniaturized high-throughput experimentation capability that was used to study a challenging SNAr reaction. The availability of robust synthetic chemistry conditions discovered in these miniaturized investigations enabled the development of structure-activity relationships that ultimately led to the discovery of soluble, selective, and potent inhibitors of DGAT1.

The Catalytic Flexibility of tRNAIle-lysidine Synthetase Can Generate Alternative tRNA Substrates for Isoleucyl-tRNA Synthetase

Bacteria decode the isoleucine codon AUA using a tRNA species that is posttranscriptionally modified at the wobble position of the anticodon with a lysine-containing cytidine derivative called lysidine. The lysidine modification of tRNAIle2 is an essential identity determinant for proper aminoacylation by isoleucyl tRNA synthetase (IleRS) and codon recognition on the ribosome. The ATP- and lysine-dependent formation of lysidine is catalyzed by tRNAIle-lysidine synthetase. Using the purified recombinant enzyme from Escherichia coli and an in vitro transcribed tRNA substrate, we have confirmed that lysidine modification is both necessary and sufficient to convert tRNAIle2 into a substrate for IleRS. A series of lysine analogs were tested as potential inhibitors during the mechanistic characterization of tRNAIle-lysidine synthetase. Gel electrophoresis revealed that many of these analogs, including some simple alkyl amines, were alternative substrates. Incorporation of these amines into alternative tRNA products was confirmed by mass spectrometry. The availability of tRNAIle2 with differential modifications enabled an exploration of the structural requirements of the anticodon for aminoacylation by methionyl tRNA synthetase and IleRS. All of the modifications were effective at creating negative determinants for methionyl tRNA synthetase and positive determinants for IleRS, although the tolerance of IleRS differed between the enzymes from E. coli and Bacillus subtilis.

Discovery of a Potent and Selective DGAT1 Inhibitor with a Piperidinyl-oxy-cyclohexanecarboxylic Acid Moiety.

We report the discovery of a novel series of DGAT1 inhibitors in the benzimidazole class with a piperdinyl-oxy-cyclohexanecarboxylic acid moiety. This novel series possesses significantly improved selectivity against the A2A receptor, no ACAT1 off-target activity at 10 μM, and higher aqueous solubility and free fraction in plasma as compared to the previously reported pyridyl-oxy-cyclohexanecarboxylic acid series. In particular, 5B was shown to possess an excellent selectivity profile by screening it against a panel of more than 100 biological targets. Compound 5B significantly reduces lipid excursion in LTT in mouse and rat, demonstrates DGAT1 mediated reduction of food intake and body weight in mice, is negative in a 3-strain Ames test, and appears to distribute preferentially in the liver and the intestine in mice. We believe this lead series possesses significant potential to identify optimized compounds for clinical development.

Rapid development of two factor IXa inhibitors from hit to lead.

Two high-throughput screening hits were investigated for SAR against human factor IXa. Both hits feature a benzamide linked to a [6-5]-heteroaryl via an alkyl amine. In the case where this system is a benzimidazolyl-ethyl amine the binding potency for the hit was improved >500-fold, from 9 μM to 0.016 μM. For the other hit, which contains a tetrahydropyrido-indazole amine, potency was improved 20-fold, from 2 μM to 0.09 μM. X-ray crystal structures were obtained for an example of each class which improved understanding of the binding, and will enable further drug discovery efforts.

Development of a novel tricyclic class of potent and selective FIXa inhibitors.

Using structure based drug design, a novel class of potent coagulation factor IXa (FIXa) inhibitors was designed and synthesized. High selectivity over FXa inhibition was achieved. Selected compounds were evaluated in rat IV/PO pharmacokinetic (PK) studies and demonstrated desirable oral PK profiles. Finally, the pharmacodynamics (PD) of this class of molecules were evaluated in thrombin generation assay (TGA) in Corn Trypsin Inhibitor (CTI) citrated human plasma and demonstrated characteristics of a FIXa inhibitor.

Development of a novel class of potent and selective FIXa inhibitors

Using structure based drug design (SBDD), a novel class of potent coagulation Factor IXa (FIXa) inhibitors was designed and synthesized. High selectivity over FXa inhibition was achieved. Selected compounds demonstrated oral bioavailability in rat IV/PO pharmacokinetic (PK) studies. Finally, the pharmacodynamics (PD) of this class of molecules was evaluated in Thrombin Generation Assay (TGA) in Corn Trypsin Inhibitor (CTI) citrated human plasma and demonstrated characteristics of a FIXa inhibitor.

Potent DGAT1 Inhibitors in the Benzimidazole Class with a Pyridyl-oxy-cyclohexanecarboxylic Acid Moiety

We report the design and synthesis of a series of novel DGAT1 inhibitors in the benzimidazole class with a pyridyl-oxy-cyclohexanecarboxylic acid moiety. In particular, compound 11A is a potent DGAT1 inhibitor with excellent selectivity against ACAT1. Compound 11A significantly reduces triglyceride excursion in lipid tolerance tests (LTT) in both mice and dogs at low plasma exposure. An in vivo study in mice with des-fluoro analogue 10A indicates that this series of compounds appears to distribute in intestine preferentially over plasma. The propensity to target intestine over plasma could be advantageous in reducing potential side effects since lower circulating levels of drug are required for efficacy. However, in the preclinical species, compound 11A undergoes cis/trans epimerization in vivo, which could complicate further development due to the presence of an active metabolite.