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Objective: Irisin is known to be an important metabolic regulator of glucose and lipid metabolism. The aims of the present study are to assess the role of mouse Irisin in obesity and energy metabolism and its glucose and lipid-lowering effects in a high-fat diet-induced obesity (DIO) mice model., Methods: DIO mice were treated with recombinant murine Irisin or vehicle, and parameters such as body weight, feed intake, glucose, and lipid levels, obesity, energy consumption, and insulin sensitivity were assessed. mRNA and protein levels of UCP1 and different thermogenesis biomarker were evaluated by quantitative real-time PCR and Western blot, respectively, in tissues and major metabolic organs., Results: Irisin decreased body weight and whole-body fat mass in DIO mice in a dose dependent manner due to marked increases in total energy expenditure. It also lowered blood glucose, insulin, and lipid levels and possibly reversed hepatic steatosis. Irisin improved hepatic and peripheral insulin sensitivity in DIO mice along with body weight reduction and adiposity. Gene expression of UCP1 in different organs (adipose tissue and major organs, i.e., liver, kidney, heart, brain, and spleen) have suggested the role of irisin is global. Gene expression profile of different biomarkers in spleen suggest a profound role of Irisin in inflammation. Liver tissue have also shown significant increase of UCP1 expression in dose dependent manner which suggest a role of irisin in liver., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Conceptual design and modification of urea moiety in chemotype PF-3845/04457845, the bench marking irreversible inhibitor of fatty acid amide hydrolase (FAAH), led to discovery of a novel nicotinamide-based lead 12a having reversible mechanism of action. Focused SAR around the pyridine heterocycle (Ar) in 12a (Tables 1 and 2) resulted into four shortlisted compounds, (-)-12a, (-)-12i, (-)-12l-m. The required (-)-enantiomers were obtained via diastereomeric resolution of a novel chiral dissymmetric intermediate 15. Based on comparative profile of FAAH potency, metabolic stability in liver microsome, liability of inhibiting major hCYP450 isoforms, rat PK, and brain penetration ability, two SAR optimized compounds, (-)-12l and (-)-12m, were selected for efficacy study in rat model of chemotherapy-induced peripheral neuropathy (CIPN). Both the compounds exhibited dose related antihyperalgesic effects, when treated with 3-30 mg/kg po for 7 days. The effects at 30 mg/kg are comparable to that of PF-04457845 (10 mg/kg) and Tramadol (40 mg/kg)., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

1. Budesonide, a potent topical corticosteroid, reported to have low oral bioavailability in mice, rat, dog and human due to rapid first pass metabolism. However, there is insufficient information available in literature regarding the role of intestine and or liver responsible for the first pass metabolism of budesonide. 2. Current study in rats investigates the role of intestine and liver in first pass metabolism of budesonide using two in vivo models. Additionally, budesonide was also evaluated in in vitro assays such as thermodynamic solubility, permeability in Caco-2 cells and stability in simulated gastric (SGF), intestinal fluids (SIF) to understand the underlaying cause for low oral bioavailability. 3. Budesonide showed low oral, intra-duodenal and high intra-portal bioavailability in rat. In a dual vein cannulated rat model, intestinal and hepatic extraction ratios calculated based upon intestinal availability (Fa·Fg) and hepatic availability (Fh), suggests hepatic extraction of budesonide is minimal compared to intestinal. 4. In vitro results suggest, solubility and permeability may not be a barrier for the observed low oral bioavailability in rats. 5. Correlating the in vitro and in vivo data together, it can be concluded that, intestine might be playing major role in first pass metabolism of budesonide.

A series of terminal nonyl chain and nucleobase modified analogues of (+)-EHNA (III) were synthesized and evaluated for their ability to inhibit adenosine deaminase (ADA). The constrained carbon analogues of (+)-EHNA, 7a-7h, 10a-c, 12, 13, 14 and 17a-c appeared very potent with Ki values in the low nanomolar range. Thio-analogues of (+)-EHNA 24a-e wherein 5'C of nonyl chain replaced by sulfur atom found to be less potent compared to (+)-EHNA. Docking of the representative compounds into the active site of ADA was performed to understand structure-activity relationships. Compounds 7a (Ki: 1.1nM) 7b (Ki: 5.2nM) and 26a (Ki: 5.9nM) showed suitable balance of potency, microsomal stability and demonstrated better pharmacokinetic properties as compared to (+)-EHNA and therefore may have therapeutic potential for various inflammatory diseases, hypertension and cancer., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Adenosine induces bronchial hyperresponsiveness and inflammation in asthmatics through activation of A 2B adenosine receptor (A 2B AdoR). Selective antagonists have been shown to attenuate airway reactivity and improve inflammatory conditions in pre-clinical studies. Hence, the identification of novel, potent and selective A 2B AdoR antagonist may be beneficial for the potential treatment of asthma and Chronic Obstructive Pulmonary Disease (COPD). Towards this effort, we explored several prop-2-ynylated C8-aryl or heteroaryl substitutions on xanthine chemotype and found that 1-prop-2-ynyl-1H-pyrazol-4-yl moiety was better tolerated at the C8 position. Compound 59, exhibited binding affinity (K i ) of 62 nM but was non-selective for A 2B AdoR over other AdoRs. Incorporation of substituted phenyl on the terminal acetylene increased the binding affinity (K i ) significantly to <10 nM. Various substitutions on terminal phenyl group and different alkyl substitutions on N-1 and N-3 were explored to improve the potency, selectivity for A 2B AdoR and the solubility. In general, compounds with meta-substituted phenyl provided better selectivity for A 2B AdoR compared to that of para-substituted analogs. Substitutions such as basic amines like pyrrolidine, piperidine, piperazine or cycloalkyls with polar group were tried on terminal acetylene, keeping in mind the poor solubility of xanthine analogs in general. However, these substitutions led to a decrease in affinity compared to compound 59. Subsequent SAR optimization resulted in identification of compound 46 with high human A 2B AdoR affinity (K i  = 13 nM), selectivity against other AdoR subtypes and with good pharmacokinetic properties. It was found to be a potent functional A 2B AdoR antagonist with a K i of 8 nM in cAMP assay in hA 2B -HEK293 cells and an IC 50 of 107 nM in IL6 assay in NIH-3T3 cells. Docking study was performed to rationalize the observed affinity data. Structure-activity relationship (SAR) studies also led to identification of compound 36 as a potent A 2B AdoR antagonist with K i of 1.8 nM in cAMP assay and good aqueous solubility of 529 μM at neutral pH. Compound 46 was further tested for in vivo efficacy and found to be efficacious in ovalbumin-induced allergic asthma model in mice., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Adenosine A 2A receptor (A 2A AdoR) antagonism is a nondopaminergic approach to Parkinson's disease treatment that is under development. Earlier we had reported the therapeutic potential of 7-methoxy-4-morpholino-benzothiazole derivatives as A 2A AdoR antagonists. We herein described a novel series of [1,2,4]triazolo[5,1- f ]purin-2-one derivatives that displays functional antagonism of the A 2A receptor with a high degree of selectivity over A 1 , A 2B , and A 3 receptors. Compounds from this new scaffold resulted in the discovery of highly potent, selective, stable, and moderate brain penetrating compound 33 . Compound 33 endowed with satisfactory in vitro and in vivo pharmacokinetics properties. Compound 33 demonstrated robust oral efficacies in two commonly used models of Parkinson's disease (haloperidol-induced catalepsy and 6-OHDA lesioned rat models) and depression (TST and FST mice models).

Glucokinase activators (GKAs) are among the emerging drug candidates for the treatment of type 2 diabetes (T2D). Despite effective blood glucose lowering in clinical trials, many pan-GKAs "acting both in pancreas and liver" have been discontinued from clinical development mainly because of their potential to cause hypoglycemia. Pan-GKAs over sensitize pancreatic GK, resulting in insulin secretion even at sub-normoglycemic level which might be a possible explanation for hypoglycemia. An alternative approach to minimize the risk of hypoglycemia is to use liver-directed GKAs, which are reported to be advancing well in clinical development. Here, we report the discovery and structure-activity relationship (SAR) studies on a novel 2-phenoxy-acetamide series with the aim of identifying a liver-directed GKA. Incorporation of a carboxylic acid moiety as an active hepatocyte uptake recognizing element at appropriate position of 2-phenoxy-acetamide core led to the identification of 26, a potent GKA with predominant liver-directed pharmacokinetics in mice. Compound 26 on oral administration significantly reduced blood glucose levels during an oral glucose tolerance test (oGTT) performed in diet-induced obese (DIO) mice, while showing no sign of hypoglycemia in normal C57 mice over a 10-fold dose range, even when dosed at fasted condition. Together, these data demonstrate a liver-directed GKA has beneficial effect on glucose homeostasis with reduced risk of hypoglycemia., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

In a pursuit to identify reversible and selective BTK inhibitors, two series based on 7H-pyrrolo[2,3-d]pyrimidine and 1H-pyrrolo[2,3-b]pyridine as the hinge binding core, have been identified. Structure activity relationship (SAR) exploration led to identification of two advanced lead molecules, 11 and 13, which demonstrated desired BTK inhibitory potency in different cellular assays, excellent selectivity in a panel of 50 diverse kinases, favorable in vivo PK properties in mice and anti-arthritic effect in a mouse model of CIA., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Multipronged approach was used to synthesize a library of diverse C-8 cyclopentyl hypoxanthine analogs from a common intermediate III. Several potent and selective compounds were identified and evaluated for pharmacokinetic (PK) properties in Wistar rats. One of the compounds 14 with acceptable PK parameters was selected for testing in in vivo primary acute diuresis model. The compound demonstrated significant diuretic activity in this model., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A 2BA doR is a low affinity adenosine receptor that functions by Gs mediated elevation of cAMP and subsequent downstream signaling. The receptor has been implicated in lung inflammatory disorders like COPD and asthma. Several potent and selective A 2B AdoR antagonists have been reported in literature, however most of the compounds suffer from poor pharmacokinetic profile. Therefore, with the aim to identify novel, potent and selective A 2B AdoR antagonists with improved pharmacokinetic properties, we first explored more constrained form of MRS-1754 (4). To improve the metabolic stability, several linker modifications were attempted as replacement of amide linker along with different phenyl or other heteroaryls between C8 position of xanthine head group and terminal phenyl ring. SAR optimization resulted in identification of two novel A 2B AdoR antagonists, 8-{1-[5-Oxo-1-(4-trifluoromethyl-phenyl)-pyrrolidin-3-ylmethyl]-1H-pyrazol-4-yl}-1,3-dipropyl-xanthine (31) and 8-(1-{2-Oxo-2-[4-(3-trifluoromethyl-phenyl)-piperazin-1-yl]-ethyl}-1H-pyrazol-4-yl)-1,3-dipropyl-xanthine (65), with high binding affinity (K i  = 1 and 1.5 nM, respectively) and selectivity for A 2B AdoR with very good functional potency of 0.9 nM and 4 nM, respectively. Compound 31 and 65 also displayed good pharmacokinetic properties in mice with 27% and 65% oral bioavailability respectively. When evaluated in in vivo mice model of asthma, compound 65 also inhibited airway inflammation and airway reactivity in ovalbumin induced allergic asthma at 3 mpk dose., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Our initial structure-activity relationship studies on 7-methoxy-4-morpholino-benzothiazole derivatives featured by aryloxy-2-methylpropanamide moieties at the 2-position led to identification of compound 25 as a potent and selective A 2A adenosine receptor (A 2A AdoR) antagonist with reasonable ADME and pharmacokinetic properties. However, poor intrinsic solubility and low to moderate oral bioavailability made this series unsuitable for further development. Further optimization using structure-based drug design approach resulted in discovery of potent and selective adenosine A 2A receptor antagonists bearing substituted 1-methylcyclohexyl-carboxamide groups at position 2 of the benzothiazole scaffold and endowed with better solubility and oral bioavailability. Compounds 41 and 49 demonstrated a number of positive attributes with respect to in vitro ADME properties. Both compounds displayed good pharmacokinetic properties with 63% and 61% oral bioavailability, respectively, in rat. Further, compound 49 displayed oral efficacy in 6-OHDA lesioned rat model of Parkinson diseases.

Long chain L-2-hydroxy acid oxidase 2 (Hao2) is a peroxisomal enzyme expressed in the kidney and the liver. Hao2 was identified as a candidate gene for blood pressure (BP) quantitative trait locus (QTL) but the identity of its physiological substrate and its role in vivo remains largely unknown. To define a pharmacological role of this gene product, we report the development of selective inhibitors of Hao2. We identified pyrazole carboxylic acid hits 1 and 2 from screening of a compound library. Lead optimization of these hits led to the discovery of 15-XV and 15-XXXII as potent and selective inhibitors of rat Hao2. This report details the structure activity relationship of the pyrazole carboxylic acids as specific inhibitors of Hao2., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

l-2-Hydroxy acid oxidase (Hao2) is a peroxisomal enzyme with predominant expression in the liver and kidney. Hao2 was recently identified as a candidate gene for blood pressure quantitative trait locus in rats. To investigate a pharmacological role of Hao2 in the management of blood pressure, selective Hao2 inhibitors were developed. Optimization of screening hits 1 and 2 led to the discovery of compounds 3 and 4 as potent and selective rat Hao2 inhibitors with pharmacokinetic properties suitable for in vivo studies in rats. Treatment with compound 3 or 4 resulted in a significant reduction or attenuation of blood pressure in an established or developing model of hypertension, deoxycorticosterone acetate-treated rats. This is the first report demonstrating a pharmacological benefit of selective Hao2 inhibitors in a relevant model of hypertension.

GPR91, a 7TM G-Protein-Coupled Receptor, has been recently deorphanized with succinic acid as its endogenous ligand. Current literature indicates that GPR91 plays role in various pathophysiology including renal hypertension, autoimmune disease and retinal angiogenesis. Starting from a small molecule high-throughput screening hit 1 (hGPR91 IC(50): 0.8 μM)-originally synthesized in Merck for Bradykinin B(1) Receptor (BK(1)R) program, systematic structure-activity relationship study led us to discover potent and selective hGPR91 antagonists e.g. 2c, 4c, and 5 g (IC(50): 7-35 nM; >1000 fold selective against hGPR99, a closest related GPCR; >100 fold selective in Drug Matrix screening). This initial work also led to identification of two structurally distinct and orally bio-available lead compounds: 5g (%F: 26) and 7e (IC(50): 180 nM; >100 fold selective against hGPR99; %F: 87). A rat pharmacodynamic assay was developed to characterize the antagonists in vivo using succinate induced increase in blood pressure. Using two representative antagonists, 2c and 4c, the GPR91 target engagement was subsequently demonstrated using the designed pharmacodynamic assay., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A novel, highly potent, orally active, nonsteroidal tissue selective androgen receptor (AR) modulator (BMS-564929) has been identified, and this compound has been advanced to clinical trials for the treatment of age-related functional decline. BMS-564929 is a subnanomolar AR agonist in vitro, is highly selective for the AR vs. other steroid hormone receptors, and exhibits no significant interactions with SHBG or aromatase. Dose response studies in castrated male rats show that BMS-564929 is substantially more potent than testosterone (T) in stimulating the growth of the levator ani muscle, and unlike T, highly selective for muscle vs. prostate. Key differences in the binding interactions of BMS-564929 with the AR relative to the native hormones were revealed through x-ray crystallography, including several unique contacts located in specific helices of the ligand binding domain important for coregulatory protein recruitment. Results from additional pharmacological studies effectively exclude alternative mechanistic contributions to the observed tissue selectivity of this unique, orally active androgen. Because concerns regarding the potential hyperstimulatory effects on prostate and an inconvenient route of administration are major drawbacks that limit the clinical use of T, the potent oral activity and tissue selectivity exhibited by BMS-564929 are expected to yield a clinical profile that provides the demonstrated beneficial effects of T in muscle and other tissues with a more favorable safety window.

Muraglitazar, a novel dual (alpha/gamma) peroxisome proliferator-activated receptor (PPAR) activator, was investigated for its antidiabetic properties and its effects on metabolic abnormalities in genetically obese diabetic db/db mice. In db/db mice and normal mice, muraglitazar treatment modulates the expression of PPAR target genes in white adipose tissue and liver. In young hyperglycemic db/db mice, muraglitazar treatment (0.03-50 mg . kg(-1) . day(-1) for 2 weeks) results in dose-dependent reductions of glucose, insulin, triglycerides, free fatty acids, and cholesterol. In older hyperglycemic db/db mice, longer-term muraglitazar treatment (30 mg . kg(-1) . day(-1) for 4 weeks) prevents time-dependent deterioration of glycemic control and development of insulin deficiency. In severely hyperglycemic db/db mice, muraglitazar treatment (10 mg . kg(-1) . day(-1) for 2 weeks) improves oral glucose tolerance and reduces plasma glucose and insulin levels. In addition, treatment increases insulin content in the pancreas. Finally, muraglitazar treatment increases abnormally low plasma adiponectin levels, increases high-molecular weight adiponectin complex levels, reduces elevated plasma corticosterone levels, and lowers elevated liver lipid content in db/db mice. The overall conclusions are that in db/db mice, the novel dual (alpha/gamma) PPAR activator muraglitazar 1) exerts potent and efficacious antidiabetic effects, 2) preserves pancreatic insulin content, and 3) improves metabolic abnormalities such as hyperlipidemia, fatty liver, low adiponectin levels, and elevated corticosterone levels.

The importance of glucokinase (GK; EC 2.7.1.12) in glucose homeostasis has been demonstrated by the association of GK mutations with diabetes mellitus in humans and by alterations in glucose metabolism in transgenic and gene knockout mice. Liver GK activity in humans and rodents is allosterically inhibited by GK regulatory protein (GKRP). To further understand the role of GKRP in GK regulation, the mouse GKRP gene was inactivated. With the knockout of the GKRP gene, there was a parallel loss of GK protein and activity in mutant mouse liver. The loss was primarily because of posttranscriptional regulation of GK, indicating a positive regulatory role for GKRP in maintaining GK levels and activity. As in rat hepatocytes, both GK and GKRP were localized in the nuclei of mouse hepatocytes cultured in low-glucose-containing medium. In the presence of fructose or high concentrations of glucose, conditions known to relieve GK inhibition by GKRP in vitro, only GK was translocated into the cytoplasm. In the GKRP-mutant hepatocytes, GK was not found in the nucleus under any tested conditions. We propose that GKRP functions as an anchor to sequester and inhibit GK in the hepatocyte nucleus, where it is protected from degradation. This ensures that glucose phosphorylation is minimal when the liver is in the fasting, glucose-producing phase. This also enables the hepatocytes to rapidly mobilize GK into the cytoplasm to phosphorylate and store or metabolize glucose after the ingestion of dietary glucose. In GKRP-mutant mice, the disruption of this regulation and the subsequent decrease in GK activity leads to altered glucose metabolism and impaired glycemic control.

Squalene synthase catalyzes the reductive condensation of two identical substrate molecules, farnesyl diphosphate, to the hydrocarbon squalene via an obligatory intermediate, presqualene pyrophosphate. Since the kinetic mechanism of the transformation is sequential, two substrate binding pockets that recognize the same molecule must exist in the enzyme active site. This raises the possibility of a choice of binding pockets for inhibitors that are designed as substrate or reaction intermediate analogs and thus may provide some information on the mechanism of differentiation of the two identical molecules. In this report, we have investigated the mechanism of inhibition of a series of farnesyl diphosphate analog inhibitors. The inhibitors fall into two categories. One class of compounds binds to free enzyme as well as the enzyme substrate complex, and the binding is refractory to the concentration of the substrate. The second class binds only to the free enzyme, and its binding is significantly modulated by the substrate concentration. Very modest structural changes in the compounds appear to dictate which class of inhibitor any compound may fall into. The significance of these observations with respect to the mechanism of the enzyme are discussed., (Copyright 1999 Academic Press.)

During the screening of the natural products for their ability to increase the activity of glucokinase by relieving inhibition by long chain fatty acyl CoA esters (FAC), two novel compounds, glucolipsin A (1) and B (2) were isolated from the butanol extracts of Streptomyces purpurogeniscleroticus WC71634 and Nocardia vaccinii WC65712, respectively. The structures of these two compounds were established by spectroscopic methods and chemical degradation. Glucolipsin A (1) and B (2) relieved the inhibition of glucokinase by FAC with RC50 values of 5.4 and 4.6 microM.

Bacteriophage T7 RNA polymerase is a single-subunit enzyme which has a C-terminal amino acid sequence of Phe-Ala-Phe-Ala883 (FAFA883). Closely related hydrophobic sequences are present at the C termini of seven other single-subunit RNA polymerases, including the mitochondrial RNA polymerase. Mutations at any of the four C-terminal residues depress initiation rates of T7 RNA polymerase from 50 to 95%, accompanied by large increases in the K(m) values for the initiating nucleotide, GTP, as well as the K(m)'s for promoter DNA. The dramatic drops in initiation rates shown by the mutant enzymes remain after correcting for any alteration in saturation of the enzyme by the initiating nucleotide or the promoter DNA resulting from the changes in K(m). In contrast, the high processivity of the enzyme is not altered by mutations in the last four residues. However, the propensity for the enzyme to add an untemplated nucleotide at the 3'-ends of transcripts is abolished by the A880AFA883 mutation. The C-terminal FAFA sequence or foot appears to interact both with the initiating NTP and with the most downstream nucleotides of the promoter, possibly through hydrophobic interactions with the minor groove, in the region where free radical footprinting of the polymerase-promoter DNA complex suggests that the enzyme binds across the minor groove.

Glucokinase (GK) plays a central role in the sensing of glucose in pancreatic beta-cells and parenchymal cells of the liver. Glucokinase regulatory protein is a physiological inhibitor of GK in the liver. To understand the role of the interaction of these two proteins in glucose sensing, we carried out a series of experiments to localize the protein in the liver cell. The regulatory protein was found to be present mainly in the nucleus of the cell under a variety of conditions that mimicked the glucose status of the fed and fasted state. GK was localized in the nucleus when the cells were exposed to low glucose concentrations. At higher glucose concentrations or in the presence of low concentrations of fructose, GK translocated to the cytoplasm. The effect of fructose was more robust and rapid than the effect of high glucose concentrations. Furthermore, the effect of fructose and high glucose on the translocation of GK from the nucleus could be partially reversed by glucagon. This unusual localization and behavior suggests a role for GK and its regulatory protein in hepatic energy metabolism that may be broader than glucose phosphorylation.

Glucokinase is a critical component of the physiological glucose sensor found in cell types that are responsive to changes in plasma glucose levels. The acute regulation of glucokinase activity has been shown to occur via a regulatory protein found in liver parenchymal cells (Van Schaftingen E, Detheux M, Da Cunha MV. Faseb J 8:414-419, 1994). The action of this protein is modulated by phosphate esters of fructose. In the presence of fructose-6-phosphate, the protein inhibits glucokinase in an allosteric competitive manner, while fructose-1-phosphate reverses this inhibition. A cDNA potentially encoding the rat liver regulatory protein has been cloned, but its identity is uncertain because of the small amounts of soluble protein obtained by expression in bacteria. We report the heterologous expression of the regulatory protein in Escherichia coli and its purification to homogeneity and high specific activity in a single chromatographic step. The properties of this recombinant protein are very similar to those of the liver protein. Direct demonstration of the binding of the recombinant protein to glucokinase has been obtained in vitro using coprecipitation experiments and in vivo, using the yeast two-hybrid system. These studies establish that the protein encoded by the cDNA is identical to the glucokinase regulatory protein and also validate tools with which to carry out structure-function studies on the interaction of the regulatory protein with glucokinase.

Squalene synthase catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to give presqualene diphosphate (PSPP) and the subsequent reductive rearrangement of PSPP to squalene. Previous studies of the mechanism of addition of FPP to the enzyme have led to conflicting interpretations of initial velocity measurements (Beytia, E., Qureshi, A. A., and Porter, J.W. (1973) J. Biol. Chem. 248, 1856-1867; Agnew, W.S., and Popjak, G. (1978) J. Biol. Chem. 253, 4566-4573). Initial velocities for synthesis of PSPP and squalene were measured over a wider range of FPP and NADPH concentrations than previously reported, using a soluble form of recombinant enzyme. In the absence of NADPH, PSPP formation was activated by FPP at concentrations above approximately 0.5 microM. At fixed levels of NADPH, the dependence of initial rates of PSPP and squalene synthesis on FPP concentrations indicated that the C15 substrate added by a sequential mechanism. In addition, NADPH stimulated synthesis of PSPP by 40-fold at saturating levels of the cofactor. This stimulation is, at least in part, by reduction of PSPP to squalene.

The kinetics of hydrolysis of rat tendon type I, bovine nasal septum type II, and human placental type III collagens by class I and class II Clostridium histolyticum collagenases (CHC) have been investigated. To facilitate this study, radioassays developed previously for the hydrolysis of these [3H]acetylated collagens by tissue collagenases have been adapted for use with the CHC. While the CHC are known to make multiple scissions in these collagens, the assays are shown to monitor the initial proteolytic events. The individual kinetic parameters kcat and KM have been determined for the hydrolysis of all three collagens by both class I and class II CHC. The specific activities of these CHC toward fibrillar type I and III collagens have also been measured. In contrast to human tissue collagenases, neither class of CHC exhibits a marked specificity toward any collagen type either in solution or in fibrillar form. The values of the kinetic parameters kcat and KM for the CHC are similar in magnitude to those of the human enzymes acting on their preferred substrates. Thus, the widely held view that the CHC are more potent collagenases is not strictly correct. As with the tissue collagenases, the local collagen structure at the cleavage sites is believed to play an important role in determining the rates of the reactions studied.

Seven collagenases denoted by the letters alpha, beta, gamma, delta, epsilon, zeta and eta have been purified to homogeneity from the culture filtrate of Clostridium histolyticum. All seven enzymes are zinc proteinases that require calcium ions for activity and have essential carboxyl, tyrosyl and lysyl residues. These enzymes can be divided into two classes on the basis of the sequence homologies in their polypeptide chains, as revealed from a comparison of their tryptic digests. This division into classes is also supported by a comparison of their specificities toward peptide substrates, their interaction with substrate-analog inhibitors, and their mode of attack of triple helical collagens. The sequence specificities of these enzymes have been studied in detail. The specificities of the two classes are similar, but complementary. Both classes exhibit both endopeptidase and tripeptidylcarboxypeptidase activities, where the latter is thought to facilitate removal of Gly-X-Y triplets from the C-terminus of collagen fragments. The mode of attack of these collagenases on triple helical type I, II and III collagens is very similar for the enzymes within each class, but different for the two classes. The class I enzymes first hydrolyze loci near the ends of the triple helical domains of these collagen molecules, while the class II enzymes make their initial cleavages in the interior. The sites of these initial cleavages are being sequenced and preliminary results indicate that they do not resemble the tissue collagenase cleavage site with respect to either their imino acid content or distribution. The kinetic parameters for the hydrolysis of type I, II and III collagens have been measured and are similar in magnitude to those for the tissue collagenases. Synthetic peptide substrate-analog inhibitors have been prepared for both classes of collagenases and shown to be transition-state-analog inhibitors.

The role of the C-terminal Phe882-Ala883 residues of bacteriophage T7 RNA polymerase in specific transcription has been investigated by means of site-directed mutagenesis. A mutant enzyme that lacks the C-terminal Phe882-Ala883 residues, denoted the "foot" mutant, has been cloned and overproduced, and the effects of the deletion on promoter recognition, initiation, and elongation have been determined. Gel retardation assays and DNase I footprinting show that the foot mutant specifically recognizes and binds to T7 promoters, although this binding appears to be approximately 30-fold weaker than that of the wild-type enzyme. Transcription assays using oligonucleotide templates that contain the consensus T7 promoter show a dramatic decrease in transcriptional activity for the foot mutant. With templates whose coding region begins CCC..., the mutant synthesizes poly(G) products even in the presence of all four nucleotides. The synthesis of poly(G) products from such templates has previously been observed for the wild-type enzyme when GTP is the sole nucleotide present in the reaction and is thought to occur by a novel mechanism involving slippage of the RNA chain 3' to 5' relative to the template [Martin, C.T., Muller, D.K., & Coleman, J.E. (1988) Biochemistry 27, 3966-3974]. These data suggest that the loss in transcriptional activity by the foot mutant results from a severe decrease in processivity as well as catalytic efficiency of the enzyme. Removal of the C-terminal Phe and Ala residues from the wild-type enzyme with carboxypeptidase A generates the phenotype of the mutant precisely, proving that all of the properties of the foot mutant derive from the loss of the Phe-Ala-COOH moiety.(ABSTRACT TRUNCATED AT 250 WORDS)

A series of sulfhydryl and novel sulfur-based substrate-analog inhibitors has been synthesized and tested against human fibroblast and neutrophil collagenases. Absolute stereospecific synthesis of several sulfhydryl inhibitors establishes that it is the diastereomers with the R-configuration of the P'1 residues, which correspond to the unnatural D-amino acid analogs, that are the most potent inhibitors. The corresponding disulfide, sulfonate, sulfinate, sulfide, sulfoxide and sulfone analogs exhibit widely variable levels of potency, but all less than the sulfhydryl compounds. No correlation between inhibitor potency and any single structural feature of these new compounds is apparent. However, differences in potency can be ascribed to the different affinities of these functional groups for zinc coordination and hydrogen bonding to nearby active site residues.

A series of experiments has been carried out to characterize 58-kDa human neutrophil collagenase (HNC) and compare it with human fibroblast collagenase (HFC). N-Terminal sequencing of latent and spontaneously activated HNC shows that it is a distinct collagenase that is homologous to HFC and other members of the matrix metalloproteinase gene family. Activation occurs autolytically by hydrolysis of an M-L bond at a locus homologous to the Q80-F81-V82-L83 autolytic activation site of HFC. This releases a 16-residue propeptide believed to contain the "cysteine switch" residue required for latency. Polyclonal antibody raised against HNC cross-reacts with HFC but with none of the other major human matrix metalloproteinases examined. Treatment of HNC with endoglycosidase F or N-glycosidase F indicates that it is glycosylated at multiple sites. The deglycosylated latent and spontaneously activated enzymes have molecular weights of approximately 44K and 42K, respectively. Differences in the carbohydrate processing of HFC and HNC may determine why HFC is a secreted protein while HNC is stored in intracellular granules. The kinetic parameters kcat and KM for the hydrolysis of the interstitial collagen types I, II, and III in solution by both collagenases have been determined. The strong preferences of HNC for type I collagen and of HFC for type III collagen found in earlier studies have been confirmed. The preference of HNC for type I over type III collagen is almost abolished when fibrillar collagens are used as substrates, but the preference for HFC for type III over type I collagen is only partially decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Latent and active 58-kDa forms of human neutrophil collagenase (HNC) have been purified to homogeneity. Buffy coats were extracted in the presence and absence of phenylmethanesulfonyl fluoride to generate crude starting preparations that contained latent and active HNC, respectively. The buffers used in preparing these extracts and for all subsequent chromatographic steps contained NaCl at a concentration of 0.5 M or greater, 0.05% Brij-35, concentrations of CaCl2 of 5 mM or greater, and (when feasible) 50 microM ZnSO4 to stabilize the HNC. The collagenase activity in the buffy coat extracts was adsorbed to a Reactive Red 120-agarose column at pH 7.5 in 0.5 M NaCl and was eluted when the NaCl concentration was increased to 1 M. The active and p-(chloromercuri)benzoate-activated latent enzymes were next adsorbed to a Sepharose-CH-Pro-Leu-Gly-NHOH affinity resin in 1 M NaCl at pH 7.5 and desorbed at pH 9 to give a fraction containing only HNC and a small amount of neutrophil gelatinase. The latter enzyme was removed by passage over a gelatin-Sepharose column in 1 M NaCl at pH 7.5. The purified samples of active and latent HNC were obtained with typical cumulative yields of 32 and 82% and specific activities toward soluble rat type I collagen at 30 degrees C of 7200 and 12,000 micrograms min-1 mg-1, respectively. These specific activities are markedly higher than previously reported for HNC. Both active and latent HNC exhibit a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis both in the presence and in the absence of 2-mercaptoethanol. The mobility of latent HNC is consistent with a molecular weight of approximately 58K, with the active form exhibiting a slightly lower (less than 1-2K) molecular weight.

A series of chemical modification reactions has been carried out to identify functional constituents of the active site of human neutrophil collagenase. The enzyme is reversibly inhibited by the transition metal chelating agent 1,10-phenanthroline, and inhibition is fully reversed by zinc. Removal of weakly bound metal ions by gel filtration inactivates collagenase, and activity is fully restored on immediate readdition of calcium. The enzyme is unaffected by reagents that modify serine, cysteine, and arginine residues. However, reaction with the carboxyl reagents cyclohexylmorpholinocarbodiimide and Woodward's Reagent K lowers the activity of the enzyme substantially. Acetylimidazole inactivates the enzyme, but activity is completely restored on addition of hydroxylamine. The enzyme is also inactivated by tetranitromethane, indicating that it contains an essential tyrosine residue. Acylation of collagenase with diethyl pyrocarbonate, diketene, acetic anhydride, or trinitrobenzenesulfonate inactivates the enzyme, and activity is not restored on addition of hydroxylamine, indicating the presence of an essential lysine residue.

The action of three class I (beta, gamma, and eta) and three class II (delta, epsilon, and zeta) collagenases from Clostridium histolyticum on two series of peptides with collagen-like sequences has been examined. The peptides in the first series all contain 4-nitrophenylalanyl-Gly-Pro-Ala in subsites P1 through P3', but each is successively lengthened in the N-terminal direction by addition of an appropriate residue until subsite P5 is occupied. The second group of peptides all have cinnamoyl-Leu in subsites P2 and P1, respectively, but each is successively lengthened in the C-terminal direction by partial additions of the Gly-Pro-Leu triplet until subsite P6' is occupied. N-Terminal elongation causes the kcat/KM values to rise markedly and to level off after occupancy of subsite P6 for the class I enzymes and subsite P3 for the class II enzymes. C-Terminal elongation produces the best substrates for both classes of enzymes when subsites P3' or P4' are occupied by amino acids with free carboxyl groups. The kcat/KM values for the hydrolysis of both Leu-Gly bonds of cinnamoyl-Leu-Gly-Pro-Leu-Gly-Pro-Leu have been measured for both classes of enzymes. Both rates are large, but both classes preferentially hydrolyze the Leu-Gly bond of the C-terminal triplet. Thus, both classes of enzymes exhibit both endopeptidase and tripeptidylcarboxypeptidase activities.

A series of ketone-substrate analogues has been synthesized for the two classes of collagenases from Clostridium histolyticum and shown to be competitive inhibitors. These compounds have sequences that match those of specific peptide substrates for these enzymes. The best inhibitor is the ketone analogue of cinnamoyl-Leu-Gly-Pro-Pro, which has a KI value of 18 nM for epsilon-collagenase, a class II enzyme. This is the tightest binding inhibitor reported for any collagenase to date. Plots of log KI for the inhibitors vs log KM/kcat for the matched substrates for both collagenases are linear with slopes near unity, indicating that the ketones are transition-state analogues. This strongly implies that the ketone carbon atoms of these inhibitors are tetrahedral when bound to the enzymes.

Calf skin and rat tendon type I, bovine cartilage type II, and human amnion type III collagens have been radiolabeled by reaction with [3H]acetic anhydride, [3H]formaldehyde, and succinimidyl 2,3-[3H]propionate. All three reactions produce collagens with high specific activities that are suitable for use as substrates in collagenase assays. The identity of the radiolabel and the labeling indices do not alter the molecular weights or thermal stabilities of the collagens or the solubilities of the collagens or gelatins in dioxane-water mixtures at 4 degrees C. However, in contrast to native or sparsely labeled collagens, those with 40 or more lysine + hydroxylysine residues labeled per molecule do not undergo fibrillogenesis in the presence of 0.2-0.4 M NaCl in the 4-35 degree C temperature range. Thus, the modification reactions not only serve to introduce the radiolabel, but also to keep the collagens soluble over a wide range of temperatures and concentrations. The TCA, TCB fragments produced on partial reaction of each collagen type with tissue collagenases can be selectively denatured by a 10-minute incubation under specific conditions and the intact collagens selectively precipitated by addition of 50% v/v dioxane. This serves as the basis for soluble collagenase assays. The effect of labeling index on the properties of the collagens has been investigated and the results establish the range of conditions over which these collagens can be used as substrates for soluble versus fibrillar collagenase assays.

Accurate and quantitative assays for the hydrolysis of soluble 3H-acetylated rat tendon type I, bovine cartilage type II, and human amnion type III collagens by both bacterial and tissue collagenases have been developed. The assays are carried out at any temperature in the 1-30 degrees C range in a single reaction tube and the progress of the reaction is monitored by withdrawing aliquots as a function of time, quenching with 1,10-phenanthroline, and quantitation of the concentration of hydrolysis fragments. The latter is achieved by selective denaturation of these fragments by incubation under conditions described in the previous paper of this issue. The assays give percentages of hydrolysis of all three collagen types by neutrophil collagenase that agree well with the results of gel electrophoresis experiments. The initial rates of hydrolysis of all three collagens are proportional to the concentration of both neutrophil or Clostridial collagenases over a 10-fold range of enzyme concentrations. All three assays can be carried out at collagen concentrations. that range from 0.06 to 2 mg/ml and give linear double reciprocal plots for both tissue and bacterial collagenases that can be used to evaluate the kinetic parameters Km and kcat or Vmax. The assay developed for the hydrolysis of rat type I collagen by neutrophil collagenase is shown to be more sensitive by at least one order of magnitude than comparable assays that use rat type I collagen fibrils or gels as substrate.

The initial proteolytic events in the hydrolysis of rat tendon type I collagen by the class I and II collagenases from Clostridium histolyticum have been investigated at 15 degrees C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been used to detect the initial cleavage fragments of both the alpha 1(I) and alpha 2 chains, which migrate at different rates in the buffer system employed. Experiments with the class I collagenases indicate that the first cleavage occurs across all three chains of the triple helix close to the C-terminus to produce fragments whose alpha chains have molecular weights of approximately 88,000. The second cleavage occurs near the N-terminus to reduce the molecular weight of the alpha chains to 80,000. Initial proteolysis by the class II collagenases occurs across all three chains at a site in the interior of the collagen triple helix to give N- and C-terminal fragments with alpha-chain molecular weights of 35,000 and 62,000, respectively. The C-terminal fragment is subsequently cleaved to give fragments with alpha-chain molecular weights of 59,000. These results indicate that type I collagen is degraded at several hyperreactive sites by these enzymes. Thus, initial proteolysis by these bacterial collagenases occurs at specific sites, much like the mammalian collagenases. These results with the individual clostridial collagenases provide an explanation for earlier data which indicated that collagen is degraded sequentially from the ends by a crude clostridial collagenase preparation.

A series of phosphonamidates has been synthesized and shown to inhibit human neutrophil collagenase. The compounds all have sequences patterned after the cleavage site in the alpha 1(I) chain of type I collagen, except that the carbonyl group of the Gly residue in subsite P1 has been replaced by a P(= O)(OH) group (abbreviated GlyP). As the central GlyP-Leu unit is lengthened in the N- and C-terminal directions, in accordance with the cleavage sequence found in collagen, inhibition is systematically improved. The best inhibitor is Cbz-GlyP-Leu-Ala-Gly, which inhibits competitively with a KI value of 14 microM. These phosphonamidates are thought to be acting as transition-state analogues.