Several protein engineering approaches were combined to optimize the selectivity and activity of Vibrio fluvialis aminotransferase (Vfat) for the synthesis of (3S,5R)-ethyl 3-amino-5-methyloctanoate; a key intermediate in the synthesis of imagabalin, an advanced candidate for the treatment of generalized anxiety disorder. Starting from wild-type Vfat, which had extremely low activity catalyzing the desired reaction, we engineered an improved enzyme with a 60-fold increase in initial reaction velocity for transamination of (R)-ethyl 5-methyl 3-oxooctanoate to (3S,5R)-ethyl 3-amino-5-methyloctanoate. To achieve this, <450 variants were screened, which allowed accurate assessment of enzyme performance using a low-throughput ultra performance liquid chromatography assay. During the course of this work, crystal structures of Vfat wild type and an improved variant (Vfat variant r414) were solved and they are reported here for the first time. This work also provides insight into the critical residues for substrate specificity for the transamination of (R)-ethyl 5-methyl 3-oxooctanoate and structurally related β-ketoesters.

LY-450139 is a ␥-secretase inhibitor shown to have efficacy in multiple cellular and animal models. Paradoxically, robust elevations of plasma amyloid-␤ (A␤) have been reported in dogs and humans after administration of subefficacious doses. The present study sought to further evaluate A␤ responses to LY-450139 in the guinea pig, a nontransgenic model that has an A␤ sequence identical to that of human. Male guinea pigs were treated with LY-450139 (0.2-60 mg/kg), and brain, cerebrospinal fluid, and plasma A␤ levels were characterized at 1, 3, 6, 9, and 14 h postdose. Low doses significantly elevated plasma A␤ levels at early time points, with return to baseline within hours. Higher doses inhibited A␤ levels in all compartments at early time points, but elevated plasma A␤ levels at later time points. To determine whether this phenomenon occurs under steadystate drug exposure, guinea pigs were implanted with subcutaneous minipumps delivering LY-450139 (0.3-30 mg/kg/day) for 5 days. Plasma A␤ was significantly inhibited at 10 -30 mg/kg/day, but significantly elevated at 1 mg/kg/day. To further understand the mechanism of A␤ elevation by LY-450139, H4 cells overexpressing the Swedish mutant of amyloid-precursor protein and a mouse embryonic stem cell-derived neuronal cell line were studied. In both cellular models, elevated levels of secreted A␤ were observed at subefficacious concentrations, whereas dose-responsive inhibition was observed at higher concentrations. These results suggest that LY-450139 modulates the ␥-secretase complex, eliciting A␤ lowering at high concentrations but A␤ elevation at low concentrations.The pathological accumulation of amyloid-␤ peptide into dense core plaques in the brains of Alzheimer's disease patients is the ultimate target of multiple disease-modifying drug discovery efforts. One strategy that has entered the clinic is the use of a ␥-secretase inhibitor to reduce central A␤ production. Preclinically, multiple ␥-secretase inhibitors have demonstrated central and peripheral A␤-lowering activity in transgenic mouse lines overexpressing human mutant amyloid precursor protein (Dovey et al., 2001;Cirrito et al., 2003;Lanz et al., 2003Lanz et al., , 2004Wong et al., 2004;, as well as nontransgenic species (Anderson et al., 2005;Best et al., 2006;El Mouedden et al., 2006). Whereas acute treatment of old, plaque-bearing mice should have little immediate impact on plaque load (insoluble A␤), these inhibitors have been shown to inhibit A␤ in CSF (Lanz et al., 2003;Barten et al., 2005) and interstitial fluid (Cirrito et al., 2003) similarly in both plaque-free and plaque-bearing mice. In addition, plasma A␤ has been shown to be reduced similarly by ␥-secretase inhibition in both young and old Tg2576 mice (Lanz et al., 2003;Barten et al., 2005). These findings indicate that despite the presence or absence of insoluble A␤ plaques, these compounds had similar potency in reducing soluble, secreted A␤ in young and old transgenic mice.The ability of plasma and CSF A␤ to track pharmacologic...

Two autocrine proteins of 14 and 12 kilodaltons that induce the synthesis of rabbit fibroblast collagenase were identified. The proteins were purified from serum-free culture medium taken from rabbit synovial fibroblasts stimulated with phorbol myristate acetate. The amino-terminal sequences of the 14- and 12-kilodalton species were approximately 60 to 80 percent homologous with serum amyloid A and beta 2 microglobulin, respectively. The polyacrylamide gel-eluted proteins retained the ability to induce collagenase synthesis in rabbit and human fibroblasts. These autocrine proteins may provide a means to modulate collagenase synthesis in normal remodeling as well as in inflammation and disease states.

No abstract

Enzyme design is an important area of ongoing research with a broad range of applications in protein therapeutics, biocatalysis, bioengineering, and other biomedical areas; however, significant challenges exist in the design of enzymes to catalyze specific reactions of interest. Here, we develop a computational protocol using an approach that combines molecular dynamics, docking, and MM-GBSA scoring to predict the catalytic activity of enzyme variants. Our primary focuses are to understand the molecular basis of substrate recognition and binding in an S-stereoselective ω-aminotransferase (ω-AT), which naturally catalyzes the transamination of pyruvate into alanine, and to predict mutations that enhance the catalytic efficiency of the enzyme. The conversion of (R)-ethyl 5-methyl-3-oxooctanoate to (3S,5R)-ethyl 3-amino-5-methyloctanoate in the context of several ω-AT mutants was evaluated using the computational protocol developed in this work. We correctly identify the mutations that yield the greatest improvements in enzyme activity (20-60-fold improvement over wild type) and confirm that the computationally predicted structure of a highly active mutant reproduces key structural aspects of the variant, including side chain conformational changes, as determined by X-ray crystallography. Overall, the protocol developed here yields encouraging results and suggests that computational approaches can aid in the redesign of enzymes with improved catalytic efficiency.

Rabbit proactivator is a neutral metalloproteinase that activates another metalloproteinase, procollagenase, and degrades noncollagenous matrix. We describe the construction of an activator complementary DNA (cDNA) clone, which is 1.9 kb, that selects a 2.1-kb messenger RNA (mRNA) in Northern blot hybridizations. Nucleic acid sequence studies of the activator cDNA indicate 1) that it encodes protein M , 53,881, 2) that this protein exhibits -80% homology with rat transin, an oncogene-induced protein with a previously unknown function, and 3) that, in the first 172 residues, it is virtually identical to the rabbit metalloproteinase, stromelysin. Homology between rabbit activator and human skin collagenase is approximately 50%. Activator and collagenase mRNA are coordinately regulated; untreated cultures of rabbit synovial fibroblasts produce low levels of each protein, but addition of phorbol myristate acetate ( W M ) results in an increase in mRNA for both proteins by 2.5-5 hours. Adding alltrans-retinoic acid (lo-%) or dexamethasone (lO-'M) to phorbol-stimulated cells coordinately suppresses both activator and collagenase mRNA. Our data suggest the existence of coordinately regulated metalloproteinases that are important in the modulation of connective tissue metabolism.The metalloproteinase, collagenase (EC 3.2.24.7), is the only neutral proteinase able to initiate degradation of the interstitial collagens, types I, 11, and I11 (for review, see refs. 1 and 2). As such, collagenase plays a crucial role in the degradation of collagen that occurs as a result of both normal physiologic and disease processes (3). To study mechanisms that control the synthesis of collagenase, we have developed a model system of cultured rabbit synovial fibroblasts. These cells are readily available from the synovium of the knees of young rabbits and grow well in culture. Of experimental value is the fact that they make only minimal amounts of collagenase unless stimulated with such agents as interleukin-I , crystals of monosodium urate monohydrate, or the tumor promoter, phorbol myristate acetate (PMA) (r18). Addition of an inducer results in an increase in collagenase messenger RNA (mRNA), which is followed by rapid synthesis and secretion of collagenase protein (6,9). Equally valuable is the fact that collagenase synthesis can be antagonized by glucocorticoids and by retinoids (10,ll). It is believed that both compounds act at the level of transcription to decrease collagenase mRNA and protein, but do not affect the half-life of collagenase mRNA (12). This system has thus allowed us to study both the induction and suppression of collagenase synthesis.Rabbit synovial fibroblast collagenase is synthesized and secreted as an inactive polypeptide, procollagenase ( M , 57,000), with a minor species of glycosylated enzyme (M, 61,000) (9,13). Activation of

The development of a commercial route toward the JAK1 inhibitor abrocitinib is described. The application of a late-stage Lossen rearrangement provided the desired cis-diaminocyclobutane, which was subsequently sulfonylated using a novel water-tolerable triazole sulfonylating reagent to provide the active pharmaceutical ingredient.

Imine reductases (IREDs) catalyze the asymmetric reduction of cyclic imines, but also in some cases the coupling of ketones and amines to form secondary amine products in an enzymecatalyzed reductive amination (RedAm) reaction. Enzymatic RedAm reactions have typically used small hydrophobic amines, but many interesting pharmaceutical targets require that larger amines be used in these coupling reactions. Following the identification of IR77 from Ensifer adhaerens as a promising biocatalyst for the reductive amination of cyclohexanone with pyrrolidine, we have characterized the ability of this enzyme to catalyze couplings with larger bicyclic amines such as isoindoline and octahydrocyclopenta(c)pyrrole. By comparing the activity of IR77 with reductions using sodium cyanoborohydride in water, it was shown that, while the coupling of cyclohexanone and pyrrolidine involved at least some element of reductive amination, the amination with the larger amines likely occurred ex situ, with the imine recruited from solution for enzyme reduction. The structure of IR77 was determined, and using this as a basis, structureguided mutagenesis, coupled with point mutations selecting improving amino acid sites suggested by other groups, permitted the identification of a mutant A208N with improved activity for amine product formation. Improvements in conversion were attributed to greater enzyme stability as revealed by X-ray crystallography and nano differential scanning fluorimetry. The mutant IR77-A208N was applied to the preparative scale amination of cyclohexanone at 50 mM concentration, with 1.2 equiv of three larger amines, in isolated yields of up to 93%.