Structure-guided engineering of a receptor-agonist pair for inducible activation of the ABA adaptive response to drought.

Strategies to activate abscisic acid (ABA) receptors and boost ABA signaling by small molecules that act as ABA receptor agonists are promising biotechnological tools to enhance plant drought tolerance. Protein structures of crop ABA receptors might require modifications to improve recognition of chemical ligands, which in turn can be optimized by structural information. Through structure-based targeted design, we have combined chemical and genetic approaches to generate an ABA receptor agonist molecule (iSB09) and engineer a CsPYL1 ABA receptor, named CsPYL15m, which efficiently binds iSB09. This optimized receptor-agonist pair leads to activation of ABA signaling and marked drought tolerance. No constitutive activation of ABA signaling and hence growth penalty was observed in transformed Arabidopsis thaliana plants. Therefore, conditional and efficient activation of ABA signaling was achieved through a chemical-genetic orthogonal approach based on iterative cycles of ligand and receptor optimization driven by the structure of ternary receptor-ligand-phosphatase complexes.

Reactivity of a Recombinant Esterase from Thermus thermophilus HB27 in Aqueous and Organic Media.

The thermoalkalophilic membrane-associated esterase E34Tt from Thermus thermophilus HB27 was cloned and expressed in Kluyveromyces lactis (KLEST-3S esterase). The recombinant enzyme was tested as a biocatalyst in aqueous and organic media. It displayed a high thermal stability and was active in the presence of 10% (v/v) organic solvents and 1% (w/v) detergents. KLEST-3S hydrolysed triglycerides of various acyl chains, which is a rare characteristic among carboxylic ester hydrolases from extreme thermophiles, with maximum activity on tributyrin. It also displayed interfacial activation towards triacetin. KLEST-3S was also tested as a biocatalyst in organic media. The esterase provided high yields for the acetylation of alcohols. In addition, KLEST-3S catalyzed the stereoselective hydrolysis of (R,S)-ibuprofen methyl ester (87% ee). Our results indicate that KLEST-3S may be a robust and efficient biocatalyst for application in industrial bioconversions.

Discovery of V-0219: A Small-Molecule Positive Allosteric Modulator of the Glucagon-Like Peptide-1 Receptor toward Oral Treatment for "Diabesity".

Peptidic agonists of the glucagon-like peptide-1 receptor (GLP-1R) have gained a prominent role in the therapy of type-2 diabetes and are being considered for reducing food intake in obesity. Potential advantages of small molecules acting as positive allosteric modulators (PAMs) of GLP-1R, including oral administration and reduced unwanted effects, could improve the utility of this class of drugs. Here, we describe the discovery of compound 9 (4-{[1-({3-[4-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5-yl}methyl)piperidin-3-yl]methyl}morpholine, V-0219) that exhibits enhanced efficacy of GLP-1R stimulation, subnanomolar potency in the potentiation of insulin secretion, and no significant off-target activities. The identified GLP-1R PAM shows a remarkable in vivo activity, reducing food intake and improving glucose handling in normal and diabetic rodents. Enantioselective synthesis revealed oral efficacy for (S)-9 in animal models. Compound 9 behavior bolsters the interest of a small-molecule PAM of GLP-1R as a promising therapeutic approach for the increasingly prevalent obesity-associated diabetes.

Biochemical and Structural Characterization of a novel thermophilic esterase EstD11 provide catalytic insights for the HSL family.

A novel esterase, EstD11, has been discovered in a hot spring metagenomic library. It is a thermophilic and thermostable esterase with an optimum temperature of 60°C. A detailed substrate preference analysis of EstD11 was done using a library of chromogenic ester substrate that revealed the broad substrate specificity of EstD11 with significant measurable activity against 16 substrates with varied chain length, steric hindrance, aromaticity and flexibility of the linker between the carboxyl and the alcohol moiety of the ester. The tridimensional structures of EstD11 and the inactive mutant have been determined at atomic resolutions. Structural and bioinformatic analysis, confirm that EstD11 belongs to the family IV, the hormone-sensitive lipase (HSL) family, from the α/ß-hydrolase superfamily. The canonical α/ß-hydrolase domain is completed by a cap domain, composed by two subdomains that can unmask of the active site to allow the substrate to enter. Eight crystallographic complexes were solved with different substrates and reaction products that allowed identification of the hot-spots in the active site underlying the specificity of the protein. Crystallization and/or incubation of EstD11 at high temperature provided unique information on cap dynamics and a first glimpse of enzymatic activity in vivo. Very interestingly, we have discovered a unique Met zipper lining the active site and the cap domains that could be essential in pivotal aspects as thermo-stability and substrate promiscuity in EstD11.

Adsorption of Pharmaceutical Pollutants from Water Using Covalent Organic Frameworks.

Capture of pharmaceutical pollutants from water was studied using a novel fluorine-bearing covalent organic framework TpBD-(CF3 )2 , which showed ibuprofen adsorption capacity of 119 mg g-1 at neutral pH. This value is further enhanced at pH 2, highlighting the potential of this class of materials to serve as adsorbents even under harsh conditions. The adsorbed pharmaceutical can be recovered from TpBD-(CF3 )2 in high yield, offering the option of recycling both the adsorbent and the pharmaceutical. The high efficiency of ibuprofen capture as compared to other less lipophilic pharmaceuticals suggests that COFs can be pre-designed for selective capture of contaminants.

A Positive Allosteric Modulator of the Serotonin 5-HT2C Receptor for Obesity.

The 5-HT2CR agonist lorcaserin, clinically approved for the treatment of obesity, causes important side effects mainly related to subtype selectivity. In the search for 5-HT2CR allosteric modulators as safer antiobesity drugs, a chemical library from Vivia Biotech was screened using ExviTech platform. Structural modifications of identified hit VA240 in synthesized analogues 6-41 afforded compound 11 (N-[(1-benzyl-1H-indol-3-yl)methyl]pyridin-3-amine, VA012), which exhibited dose-dependent enhancement of serotonin efficacy, no significant off-target activities, and low binding competition with serotonin or other orthosteric ligands. PAM 11 was very active in feeding inhibition in rodents, an effect that was not related to the activation of 5-HT2AR. A combination of 11 with the SSRI sertraline increased the anorectic effect. Subchronic administration of 11 reduced food intake and body weight gain without causing CNS-related malaise. The behavior of compound 11 identified in this work supports the interest of a serotonin 5-HT2CR PAM as a promising therapeutic approach for obesity.

Selective monomethylation of anilines by Cu(OAc)2-promoted cross-coupling with MeB(OH)2.

N-Methylanilines are readily synthesized in high yields through the copper(II)-promoted coupling of anilines and methylboronic acid. This method represents a new approach for the selective monomethylation of anilines, and it is the first reported example of a Chan-Lam coupling involving the use of methylboronic acid. An incubation period of the substrate with the copper reagent is needed before addition of the methylboronic acid.