The protein product of an essential gene of unknown function from Streptococcus pneumoniae was ex... more The protein product of an essential gene of unknown function from Streptococcus pneumoniae was expressed and purified for screening in the ThermoFluor affinity screening assay. This assay can detect ligand binding to proteins of unknown function. The recombinant protein was found to be in a dimeric, native-like folded state and to unfold cooperatively. ThermoFluor was used to screen the protein against a library of 3000 compounds that were specifically selected to provide information about possible biological functions. The results of this screen identified pyridoxal phosphate and pyridoxamine phosphate as equilibrium binding ligands (K(d) approximately 50 pM, K(d) approximately 2.5 microM, respectively), consistent with an enzymatic cofactor function. Several nucleotides and nucleotide sugars were also identified as ligands of this protein. Sequence comparison with two enzymes of known structure but relatively low overall sequence homology established that several key residues directly involved in pyridoxal phosphate binding were strictly conserved. Screening a collection of generic drugs and natural products identified the antifungal compound canescin A as an irreversible covalent modifier of the enzyme. Our investigation of this protein indicates that its probable biological role is that of a nucleoside diphospho-keto-sugar aminotransferase, although the preferred keto-sugar substrate remains unknown. These experiments demonstrate the utility of a generic affinity-based ligand binding technology in decrypting possible biological functions of a protein, an approach that is both independent of and complementary to existing genomic and proteomic technologies.
The discovery of a back-up to the hepatitis C virus NS3 protease inhibitor asunaprevir (2) is des... more The discovery of a back-up to the hepatitis C virus NS3 protease inhibitor asunaprevir (2) is described. The objective of this work was the identification of a drug with antiviral properties and toxicology parameters similar to 2, but with a preclinical pharmacokinetic (PK) profile that was predictive of once-daily dosing. Critical to this discovery process was the employment of an ex vivo cardiovascular (CV) model which served to identify compounds that, like 2, were free of the CV liabilities that resulted in the discontinuation of BMS-605339 (1) from clinical trials. Structure-activity relationships (SARs) at each of the structural subsites in 2 were explored with substantial improvement in PK through modifications at the P1 site, while potency gains were found with small, but rationally designed structural changes to P4. Additional modifications at P3 were required to optimize the CV profile, and these combined SARs led to the discovery of BMS-890068 (29).
2-Aryl-2,2-difluoroacetamido-proline and pipecolate esters are high affinity FKBP12 ligands whose... more 2-Aryl-2,2-difluoroacetamido-proline and pipecolate esters are high affinity FKBP12 ligands whose rotamase inhibitory activity is comparable to that seen for the corresponding ketoamides. X-ray structural studies suggest that the fluorine atoms participate in discrete interactions with the Phe36 phenyl ring and the Tyr26 hydroxyl group, with the latter resembling a moderate-to-weak hydrogen bond.
Kynurenine aminotransferases convert kynurenine to kynurenic acid and play an important role in t... more Kynurenine aminotransferases convert kynurenine to kynurenic acid and play an important role in tryptophan degradation pathway. Kynurenic acid levels in brain have been hypothesized to be linked to a number of central nervous system (CNS) disorders. Kynurenine aminotransferase II (KATII) has proven to be a key modulator of kynurenic acid levels in brain and thus is an attractive target to treat CNS diseases. A sensitive, high-throughput, label-free rapidfire mass spectrometry assay was developed for human KATII. Unlike other assays, this method is directly applicable to KATII enzymes from different animal species, which allows us to select proper animal model(s) to evaluate human KATII inhibitors. We also established a coupled fluorescence assay for human KATII. The short assay time and kinetic capability of the fluorescence assay provide a useful tool for orthogonal inhibitor validation and mechanistic studies.
The Journal of pharmacology and experimental therapeutics, 2001
Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the s... more Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. BMS-229724 (4-[4-[2-[2-[bis(4-chlorophenyl)methoxy]ethyl-sulfonyl]ethoxy]phenyl]-1,1,1-trifluoro-2-butanone) was found to be a selective inhibitor of cPLA2 (IC50 = 2.8 microM) in that it did not inhibit secreted phospholipase A2 in vitro, nor phospholipase C and phospholipase D in cells. The compound was active in inhibiting arachidonate and eicosanoid production in U937 cells, neutrophils, platelets, monocytes, and mast cells. With a synthetic covesicle substrate system, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the lipid/water interface. The apparent equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface (K(I)*(app)) was determined to be 1. 10(-5) mol% versus an apparent dissocia...
Inhibition of signal transduction downstream of the IL-23 receptor represents an intriguing appro... more Inhibition of signal transduction downstream of the IL-23 receptor represents an intriguing approach to the treatment of autoimmunity. Using a chemogenomics approach marrying kinome-wide inhibitory profiles of a compound library with the cellular activity against an IL-23-stimulated transcriptional response in T lymphocytes, a class of inhibitors was identified which bind to and stabilize the pseudokinase domain of the Janus kinase tyrosine kinase 2 (Tyk2), resulting in blockade of receptor-mediated activation of the adjacent catalytic domain. These Tyk2 pseudokinase domain stabilizers were also shown to inhibit Tyk2-dependent signaling through the Type I interferon receptor (IFNAR), but not Tyk2-independent signaling and transcriptional cellular assays including stimulation through the receptors for IL-2 (JAK1 and JAK3 dependent) and thrombopoietin (JAK2 dependent) demonstrating the high functional selectivity of this approach. A crystal structure of the pseudokinase domain ligande...
2-Aryl-2,2-difluoroacetamido-proline and pipecolate esters are high affinity FKBP12 ligands whose... more 2-Aryl-2,2-difluoroacetamido-proline and pipecolate esters are high affinity FKBP12 ligands whose rotamase inhibitory activity is comparable to that seen for the corresponding ketoamides. X-ray structural studies suggest that the fluorine atoms participate in discrete interactions with the Phe36 phenyl ring and the Tyr26 hydroxyl group, with the latter resembling a moderate-to-weak hydrogen bond.
The discovery of asunaprevir (BMS-650032, 24) is described. This tripeptidic acylsulfonamide inhi... more The discovery of asunaprevir (BMS-650032, 24) is described. This tripeptidic acylsulfonamide inhibitor of the NS3/4A enzyme is currently in phase III clinical trials for the treatment of hepatitis C virus infection. The discovery of 24 was enabled by employing an isolated rabbit heart model to screen for the cardiovascular (CV) liabilities (changes to HR and SNRT) that were responsible for the discontinuation of an earlier lead from this chemical series, BMS-605339 (1), from clinical trials. The structure−activity relationships (SARs) developed with respect to CV effects established that small structural changes to the P2* subsite of the molecule had a significant impact on the CV profile of a given compound. The antiviral activity, preclincial PK profile, and toxicology studies in rat and dog supported clinical development of BMS-650032 (24). . Effects of BMS-605339 (1) on HR and SNRT in a isolated rabbit heart preparation perfused with 0.3, 1, 3, and 10 μM of drug in proteinfree medium. Hearts were perfused with each concentration of 1 (unbound) for ∼12 min. Mean ± SEM, n = 4 hearts/group. *P < 0.05, **P < 0.01 versus vehicle control.
Described herein are structure-activity relationship studies that resulted in the optimization of... more Described herein are structure-activity relationship studies that resulted in the optimization of the activity of members of a class of cyclopropyl-fused indolobenzazepine HCV NS5B polymerase inhibitors. Subsequent iterations of analogue design and syntheses successfully addressed off-target activities, most notably human pregnane X receptor (hPXR) transactivation, and led to significant improvements in the physicochemical properties of lead compounds. Those analogues exhibiting improved solubility and membrane permeability were shown to have notably enhanced pharmacokinetic profiles. Additionally, a series of alkyl bridged piperazine carboxamides was identified as being of particular interest, and from which the compound BMS-791325 (2) was found to have distinguishing antiviral, safety, and pharmacokinetic properties that resulted in its selection for clinical evaluation.
Inosine monophosphate dehydrogenase (IMPDH) is a key enzyme that is involved in the de novo synth... more Inosine monophosphate dehydrogenase (IMPDH) is a key enzyme that is involved in the de novo synthesis of purine nucleotides. Novel 2-aminooxazoles were synthesized and tested for inhibition of IMPDH catalytic activity. Multiple analogues based on this chemotype were found to inhibit IMPDH with low nanomolar potency. One of the analogues (compound 23) showed excellent in vivo activity in the inhibition of antibody production in mice and in the adjuvant induced arthritis model in rats.
A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase is... more A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase is described. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are presented.
Cytosolic phospholipase A2 (cPLA2) hydrolyzes the sn-2 ester of phospholipids and is believed to ... more Cytosolic phospholipase A2 (cPLA2) hydrolyzes the sn-2 ester of phospholipids and is believed to be responsible for the receptor-regulated release of arachidonic acid from phospholipid pools. The enzyme was assayed using vesicles containing arachidonate-containing phospholipid substrate, such as 1-palmitoyl-2-arachidonoylphosphatidylcholine (PAPC) or 1-stearoyl-2-arachidonoylphosphatidylinositol (SAPI), dispersed within vesicles of 1,2-dimyristoylphosphatidylmethanol (DMPM). We report here that the enzyme shows an apparent cooperative effect with respect to the mole fraction of arachidonate-containing phospholipids within these covesicles. The data can be fit to a modified Hill equation yielding Hill coefficients, n, of 2-3. This effect is unusual in that it is dependent on the nature of the sn-2 ester as opposed to the phosphoglycerol head group. This cooperativity is independent of both the concentration of glycerol, which greatly increases enzyme activity and stability, and the concentration of calcium, which facilitates the fusion of the covesicles. Surprisingly, 1-palmitoyl-2-arachidonoylphosphatidylethanolamine (PAPE) does not show the same cooperative effect, although the rate at which it is hydrolyzed is much greater when PAPC is present. Moreover, PAPE has a dissociation constant from the active site (KD* = 0.7 mol %) which is comparable to that of PAPC and SAPI (KD* values of 0.3 and 0.3 mol %, respectively). These results are consistent with the presence of an allosteric site that, when occupied, induces a change in the enzyme which facilitates enzymatic hydrolysis. If so, PAPC and SAPI, but not PAPE, must be able to bind to this allosteric site. Alternatively, this effect may result from changes in the physical nature of the bilayer which result upon increasing the bilayer concentration of arachidonate-containing phospholipids. This previously unobserved effect may represent another mechanism by which cells can regulate the activity of cPLA2.
to this artificial substrate, membranes from U937 cells were isolated and used as substrate. With... more to this artificial substrate, membranes from U937 cells were isolated and used as substrate. With these mem-Cytosolic phospholipase A 2 catalyzes the selective branes, the dephosphorylated enzyme was only 21% release of arachidonic acid from the sn-2 position of less active than the phosphorylated enzyme. In the phospholipids and is believed to play a key cellular presence of glycerol, there was no detectable differrole in the generation of arachidonic acid. The enzyence the two enzyme forms, and the rate of hydrolysis matic activity of cPLA 2 is affected by several mechawas increased by 300-390% over that measured in the nisms, including substrate presentation and the phosabsence of glycerol. These results suggest that the catphorylation state of the enzyme. Using covesicles of alytic efficiency of the phosphorylated enzyme is not 1-palmitoyl-2-arachidonoyl-[arachidonoyl-1-14 C]-snparticularly relevant to its activation in vivo. Moreglycero-3-phosphocholine and 1,2-dimyristoyl-phosover, it may be that glycerol is mimicking the effect of phatidylmethanol as substrate, the effects of phossome unidentified factor which greatly enhances the phorylation on the interfacial binding and catalytic catalytic efficiency of the enzyme. ᭧ 1997 Academic Press constants were investigated. Phosphorylated and de-Key Words: Cytosolic phospholipase A 2 ; serine phosphosphorylated enzyme forms were shown to have phorylation; glycerol; kinetic constants. identical values of 2.6 mM for K app M , an equilibrium dissociation constant which consists of the intrinsic dissociation constant from the lipid/water interface (K S ) and the dissociation constant for phospholipid from Cytosolic phospholipase A 2 (cPLA 2 ) 2 catalyzes the hythe active site (K* M ). Moreover, the values of K* M for drolysis of the sn-2 arachidonoyl ester of phospholipids phosphorylated and dephosphorylated enzyme did not (1, 2). Because of its apparent role in the generation of differ significantly (0.4 { 0.1 and 0.2 { 0.1, respecleukotrienes and prostaglandins, which are metabotively). However, dephosphorylation of the enzyme relized from arachidonate, cPLA 2 has received considerduced the value of k cat by 39%. The phosphorylation able medicinal interest. state of the enzyme had no effect on either the coopera-The cPLA 2 is a calcium-dependent enzyme which is tivity shown by this enzyme or the thermal stability of present in a number of different tissues and cells inthe enzyme. Surprisingly, the presence of glycerol (4 M) masks the effect of phosphorylation on k cat . Instead, cluding monocytes, neutrophils, and platelets (3-5). glycerol increased the value of k cat by 440% for the The enzyme is normally located in the cytosol, but phosphorylated enzyme and by 760% for the dephosphorylated form. Moreover, addition of glycerol had 2 Abbreviations are: BSA, bovine serum albumin; CaI-PLA 2 , calonly small effects on K app M . The increase in the k cat upon cium-independent phospholipase A 2 ; [ 14 C]PAPC, 1-palmitoyl-2-araaddition of glycerol results from a substantial dechidonoyl-[arachidonoyl-1-14 C]-sn-glycero-3-phosphocholine; cPLA 2 , crease in the activation energy from 29.4 to 14.8 kcalr cytosolic phospholipase A 2 ; DMPM, 1,2-dimyristoyl-sn-glycero-3mol 01 . To determine whether the effects of phosphoryphosphomethanol; EDTA, ethylenedinitroilotetraacetic acid; EGTA, lation of the enzyme or addition of glycerol are unique ethylene bis(oxyethylenenitrilo)tetraacetic acid; HBSS, Hanks' buffered salt solution;
Cytosolic phospholipase A 2 (cPLA 2 ) is normally located in the cytosol, but in response to cell... more Cytosolic phospholipase A 2 (cPLA 2 ) is normally located in the cytosol, but in response to cellular activation the enzyme binds to the membrane at the lipid/ water interface where it catalyzes the hydrolysis of the sn-2 ester of arachidonate-containing phospholipids. Synthetic phospholipid vesicle systems have been used in kinetic and mechanistic analyses of cPLA 2 , but these systems result in a rapid loss of enzyme activity. In the present research, covesicles of 1,2-dimyristoylsn-glycero-3-phosphomethanol (DMPM) containing <10 mol% 1-palmitoyl-2-arachidonoyl-sn-glycero-3phosphocholine (PAPC) as substrate were used to show that this premature cessation of enzyme-catalyzed hydrolysis is dependent on vesicle size with 25nm-diameter vesicles supporting little activity as compared to 100-, 200-, and 400-nm vesicles. This suggests that the curvature of the vesicle may shift a conformational equilibrium toward an enzyme state which does not support activity. Interestingly, the presence of 30% (v/v) glycerol greatly enhanced the activity of the enzyme, although vesicle size-dependent premature cessation of hydrolysis was still observed. While the premature cessation of hydrolysis in the absence of glycerol is accompanied by enzyme inactivation, little inactivation occured in the presence of glycerol, indicating that premature cessation and inactivation are not absolutely coupled. When using this covesicle substrate system under conditions (6 -10 mM CaCl 2 ) where the vesicles are fusing, no premature cessation of hydrolysis has been observed. This is despite a mean vesicle diameter of 400 -450 nm under vesicle-fusing conditions, which is comparable to the largest vesicles used under nonfusing conditions (0.5 mM CaCl 2 ) where considerable premature cessation of hydrolysis was observed. Since DMPM has an intrinsic active site dissociation constant at least 330 times larger than that of PAPC, the optimum conditions for conducting kinetic and mechanistic analyses of cPLA 2 with this covesicle substrate is one in which cPLA 2 is assayed in the presence of glycerol and with fusion-inducing concentrations of calcium. The use of 1,2-dioleoyl-sn-glycero-3-phosphomethanol (DOPM) instead of DMPM in this system supports much less activity and adds the complication of a strong affinity of DOPM for the active site.
Amyloid precursor protein (APP) cleaving enzyme (BACE) is the enzyme responsible for b-site cleav... more Amyloid precursor protein (APP) cleaving enzyme (BACE) is the enzyme responsible for b-site cleavage of APP, leading to the formation of the amyloid-b peptide that is thought to be pathogenic in AlzheimerÕs disease (AD). Hence, BACE is an attractive pharmacological target, and numerous research groups have begun searching for potent and selective inhibitors of this enzyme as a potential mechanism for therapeutic intervention in AD. The mature enzyme is composed of a globular catalytic domain that is Nlinked glycosylated in mammalian cells, a single transmembrane helix that anchors the enzyme to an intracellular membrane, and a short C-terminal domain that extends outside the phospholipid bilayer of the membrane. Here we have compared the substrate and active site-directed inhibitor binding properties of several recombinant constructs of human BACE. The constructs studied here address the importance of catalytic domain glycosylation state, inclusion of domains other than the catalytic domain, and incorporation into a membrane bilayer on the interactions of the enzyme active site with peptidic ligands. We find no significant differences in ligand binding properties among these various constructs. These data demonstrate that the nonglycosylated, soluble catalytic domain of BACE faithfully reflects the ligand binding properties of the full-length mature enzyme in its natural membrane environment. Thus, the use of the nonglycosylated, soluble catalytic domain of BACE is appropriate for studies aimed at understanding the determinants of ligand recognition by the enzyme active site.
The protein product of an essential gene of unknown function from Streptococcus pneumoniae was ex... more The protein product of an essential gene of unknown function from Streptococcus pneumoniae was expressed and purified for screening in the ThermoFluor affinity screening assay. This assay can detect ligand binding to proteins of unknown function. The recombinant protein was found to be in a dimeric, native-like folded state and to unfold cooperatively. ThermoFluor was used to screen the protein against a library of 3000 compounds that were specifically selected to provide information about possible biological functions. The results of this screen identified pyridoxal phosphate and pyridoxamine phosphate as equilibrium binding ligands (K(d) approximately 50 pM, K(d) approximately 2.5 microM, respectively), consistent with an enzymatic cofactor function. Several nucleotides and nucleotide sugars were also identified as ligands of this protein. Sequence comparison with two enzymes of known structure but relatively low overall sequence homology established that several key residues directly involved in pyridoxal phosphate binding were strictly conserved. Screening a collection of generic drugs and natural products identified the antifungal compound canescin A as an irreversible covalent modifier of the enzyme. Our investigation of this protein indicates that its probable biological role is that of a nucleoside diphospho-keto-sugar aminotransferase, although the preferred keto-sugar substrate remains unknown. These experiments demonstrate the utility of a generic affinity-based ligand binding technology in decrypting possible biological functions of a protein, an approach that is both independent of and complementary to existing genomic and proteomic technologies.
The discovery of a back-up to the hepatitis C virus NS3 protease inhibitor asunaprevir (2) is des... more The discovery of a back-up to the hepatitis C virus NS3 protease inhibitor asunaprevir (2) is described. The objective of this work was the identification of a drug with antiviral properties and toxicology parameters similar to 2, but with a preclinical pharmacokinetic (PK) profile that was predictive of once-daily dosing. Critical to this discovery process was the employment of an ex vivo cardiovascular (CV) model which served to identify compounds that, like 2, were free of the CV liabilities that resulted in the discontinuation of BMS-605339 (1) from clinical trials. Structure-activity relationships (SARs) at each of the structural subsites in 2 were explored with substantial improvement in PK through modifications at the P1 site, while potency gains were found with small, but rationally designed structural changes to P4. Additional modifications at P3 were required to optimize the CV profile, and these combined SARs led to the discovery of BMS-890068 (29).
2-Aryl-2,2-difluoroacetamido-proline and pipecolate esters are high affinity FKBP12 ligands whose... more 2-Aryl-2,2-difluoroacetamido-proline and pipecolate esters are high affinity FKBP12 ligands whose rotamase inhibitory activity is comparable to that seen for the corresponding ketoamides. X-ray structural studies suggest that the fluorine atoms participate in discrete interactions with the Phe36 phenyl ring and the Tyr26 hydroxyl group, with the latter resembling a moderate-to-weak hydrogen bond.
Kynurenine aminotransferases convert kynurenine to kynurenic acid and play an important role in t... more Kynurenine aminotransferases convert kynurenine to kynurenic acid and play an important role in tryptophan degradation pathway. Kynurenic acid levels in brain have been hypothesized to be linked to a number of central nervous system (CNS) disorders. Kynurenine aminotransferase II (KATII) has proven to be a key modulator of kynurenic acid levels in brain and thus is an attractive target to treat CNS diseases. A sensitive, high-throughput, label-free rapidfire mass spectrometry assay was developed for human KATII. Unlike other assays, this method is directly applicable to KATII enzymes from different animal species, which allows us to select proper animal model(s) to evaluate human KATII inhibitors. We also established a coupled fluorescence assay for human KATII. The short assay time and kinetic capability of the fluorescence assay provide a useful tool for orthogonal inhibitor validation and mechanistic studies.
The Journal of pharmacology and experimental therapeutics, 2001
Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the s... more Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. BMS-229724 (4-[4-[2-[2-[bis(4-chlorophenyl)methoxy]ethyl-sulfonyl]ethoxy]phenyl]-1,1,1-trifluoro-2-butanone) was found to be a selective inhibitor of cPLA2 (IC50 = 2.8 microM) in that it did not inhibit secreted phospholipase A2 in vitro, nor phospholipase C and phospholipase D in cells. The compound was active in inhibiting arachidonate and eicosanoid production in U937 cells, neutrophils, platelets, monocytes, and mast cells. With a synthetic covesicle substrate system, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the lipid/water interface. The apparent equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface (K(I)*(app)) was determined to be 1. 10(-5) mol% versus an apparent dissocia...
Inhibition of signal transduction downstream of the IL-23 receptor represents an intriguing appro... more Inhibition of signal transduction downstream of the IL-23 receptor represents an intriguing approach to the treatment of autoimmunity. Using a chemogenomics approach marrying kinome-wide inhibitory profiles of a compound library with the cellular activity against an IL-23-stimulated transcriptional response in T lymphocytes, a class of inhibitors was identified which bind to and stabilize the pseudokinase domain of the Janus kinase tyrosine kinase 2 (Tyk2), resulting in blockade of receptor-mediated activation of the adjacent catalytic domain. These Tyk2 pseudokinase domain stabilizers were also shown to inhibit Tyk2-dependent signaling through the Type I interferon receptor (IFNAR), but not Tyk2-independent signaling and transcriptional cellular assays including stimulation through the receptors for IL-2 (JAK1 and JAK3 dependent) and thrombopoietin (JAK2 dependent) demonstrating the high functional selectivity of this approach. A crystal structure of the pseudokinase domain ligande...
2-Aryl-2,2-difluoroacetamido-proline and pipecolate esters are high affinity FKBP12 ligands whose... more 2-Aryl-2,2-difluoroacetamido-proline and pipecolate esters are high affinity FKBP12 ligands whose rotamase inhibitory activity is comparable to that seen for the corresponding ketoamides. X-ray structural studies suggest that the fluorine atoms participate in discrete interactions with the Phe36 phenyl ring and the Tyr26 hydroxyl group, with the latter resembling a moderate-to-weak hydrogen bond.
The discovery of asunaprevir (BMS-650032, 24) is described. This tripeptidic acylsulfonamide inhi... more The discovery of asunaprevir (BMS-650032, 24) is described. This tripeptidic acylsulfonamide inhibitor of the NS3/4A enzyme is currently in phase III clinical trials for the treatment of hepatitis C virus infection. The discovery of 24 was enabled by employing an isolated rabbit heart model to screen for the cardiovascular (CV) liabilities (changes to HR and SNRT) that were responsible for the discontinuation of an earlier lead from this chemical series, BMS-605339 (1), from clinical trials. The structure−activity relationships (SARs) developed with respect to CV effects established that small structural changes to the P2* subsite of the molecule had a significant impact on the CV profile of a given compound. The antiviral activity, preclincial PK profile, and toxicology studies in rat and dog supported clinical development of BMS-650032 (24). . Effects of BMS-605339 (1) on HR and SNRT in a isolated rabbit heart preparation perfused with 0.3, 1, 3, and 10 μM of drug in proteinfree medium. Hearts were perfused with each concentration of 1 (unbound) for ∼12 min. Mean ± SEM, n = 4 hearts/group. *P < 0.05, **P < 0.01 versus vehicle control.
Described herein are structure-activity relationship studies that resulted in the optimization of... more Described herein are structure-activity relationship studies that resulted in the optimization of the activity of members of a class of cyclopropyl-fused indolobenzazepine HCV NS5B polymerase inhibitors. Subsequent iterations of analogue design and syntheses successfully addressed off-target activities, most notably human pregnane X receptor (hPXR) transactivation, and led to significant improvements in the physicochemical properties of lead compounds. Those analogues exhibiting improved solubility and membrane permeability were shown to have notably enhanced pharmacokinetic profiles. Additionally, a series of alkyl bridged piperazine carboxamides was identified as being of particular interest, and from which the compound BMS-791325 (2) was found to have distinguishing antiviral, safety, and pharmacokinetic properties that resulted in its selection for clinical evaluation.
Inosine monophosphate dehydrogenase (IMPDH) is a key enzyme that is involved in the de novo synth... more Inosine monophosphate dehydrogenase (IMPDH) is a key enzyme that is involved in the de novo synthesis of purine nucleotides. Novel 2-aminooxazoles were synthesized and tested for inhibition of IMPDH catalytic activity. Multiple analogues based on this chemotype were found to inhibit IMPDH with low nanomolar potency. One of the analogues (compound 23) showed excellent in vivo activity in the inhibition of antibody production in mice and in the adjuvant induced arthritis model in rats.
A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase is... more A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase is described. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are presented.
Cytosolic phospholipase A2 (cPLA2) hydrolyzes the sn-2 ester of phospholipids and is believed to ... more Cytosolic phospholipase A2 (cPLA2) hydrolyzes the sn-2 ester of phospholipids and is believed to be responsible for the receptor-regulated release of arachidonic acid from phospholipid pools. The enzyme was assayed using vesicles containing arachidonate-containing phospholipid substrate, such as 1-palmitoyl-2-arachidonoylphosphatidylcholine (PAPC) or 1-stearoyl-2-arachidonoylphosphatidylinositol (SAPI), dispersed within vesicles of 1,2-dimyristoylphosphatidylmethanol (DMPM). We report here that the enzyme shows an apparent cooperative effect with respect to the mole fraction of arachidonate-containing phospholipids within these covesicles. The data can be fit to a modified Hill equation yielding Hill coefficients, n, of 2-3. This effect is unusual in that it is dependent on the nature of the sn-2 ester as opposed to the phosphoglycerol head group. This cooperativity is independent of both the concentration of glycerol, which greatly increases enzyme activity and stability, and the concentration of calcium, which facilitates the fusion of the covesicles. Surprisingly, 1-palmitoyl-2-arachidonoylphosphatidylethanolamine (PAPE) does not show the same cooperative effect, although the rate at which it is hydrolyzed is much greater when PAPC is present. Moreover, PAPE has a dissociation constant from the active site (KD* = 0.7 mol %) which is comparable to that of PAPC and SAPI (KD* values of 0.3 and 0.3 mol %, respectively). These results are consistent with the presence of an allosteric site that, when occupied, induces a change in the enzyme which facilitates enzymatic hydrolysis. If so, PAPC and SAPI, but not PAPE, must be able to bind to this allosteric site. Alternatively, this effect may result from changes in the physical nature of the bilayer which result upon increasing the bilayer concentration of arachidonate-containing phospholipids. This previously unobserved effect may represent another mechanism by which cells can regulate the activity of cPLA2.
to this artificial substrate, membranes from U937 cells were isolated and used as substrate. With... more to this artificial substrate, membranes from U937 cells were isolated and used as substrate. With these mem-Cytosolic phospholipase A 2 catalyzes the selective branes, the dephosphorylated enzyme was only 21% release of arachidonic acid from the sn-2 position of less active than the phosphorylated enzyme. In the phospholipids and is believed to play a key cellular presence of glycerol, there was no detectable differrole in the generation of arachidonic acid. The enzyence the two enzyme forms, and the rate of hydrolysis matic activity of cPLA 2 is affected by several mechawas increased by 300-390% over that measured in the nisms, including substrate presentation and the phosabsence of glycerol. These results suggest that the catphorylation state of the enzyme. Using covesicles of alytic efficiency of the phosphorylated enzyme is not 1-palmitoyl-2-arachidonoyl-[arachidonoyl-1-14 C]-snparticularly relevant to its activation in vivo. Moreglycero-3-phosphocholine and 1,2-dimyristoyl-phosover, it may be that glycerol is mimicking the effect of phatidylmethanol as substrate, the effects of phossome unidentified factor which greatly enhances the phorylation on the interfacial binding and catalytic catalytic efficiency of the enzyme. ᭧ 1997 Academic Press constants were investigated. Phosphorylated and de-Key Words: Cytosolic phospholipase A 2 ; serine phosphosphorylated enzyme forms were shown to have phorylation; glycerol; kinetic constants. identical values of 2.6 mM for K app M , an equilibrium dissociation constant which consists of the intrinsic dissociation constant from the lipid/water interface (K S ) and the dissociation constant for phospholipid from Cytosolic phospholipase A 2 (cPLA 2 ) 2 catalyzes the hythe active site (K* M ). Moreover, the values of K* M for drolysis of the sn-2 arachidonoyl ester of phospholipids phosphorylated and dephosphorylated enzyme did not (1, 2). Because of its apparent role in the generation of differ significantly (0.4 { 0.1 and 0.2 { 0.1, respecleukotrienes and prostaglandins, which are metabotively). However, dephosphorylation of the enzyme relized from arachidonate, cPLA 2 has received considerduced the value of k cat by 39%. The phosphorylation able medicinal interest. state of the enzyme had no effect on either the coopera-The cPLA 2 is a calcium-dependent enzyme which is tivity shown by this enzyme or the thermal stability of present in a number of different tissues and cells inthe enzyme. Surprisingly, the presence of glycerol (4 M) masks the effect of phosphorylation on k cat . Instead, cluding monocytes, neutrophils, and platelets (3-5). glycerol increased the value of k cat by 440% for the The enzyme is normally located in the cytosol, but phosphorylated enzyme and by 760% for the dephosphorylated form. Moreover, addition of glycerol had 2 Abbreviations are: BSA, bovine serum albumin; CaI-PLA 2 , calonly small effects on K app M . The increase in the k cat upon cium-independent phospholipase A 2 ; [ 14 C]PAPC, 1-palmitoyl-2-araaddition of glycerol results from a substantial dechidonoyl-[arachidonoyl-1-14 C]-sn-glycero-3-phosphocholine; cPLA 2 , crease in the activation energy from 29.4 to 14.8 kcalr cytosolic phospholipase A 2 ; DMPM, 1,2-dimyristoyl-sn-glycero-3mol 01 . To determine whether the effects of phosphoryphosphomethanol; EDTA, ethylenedinitroilotetraacetic acid; EGTA, lation of the enzyme or addition of glycerol are unique ethylene bis(oxyethylenenitrilo)tetraacetic acid; HBSS, Hanks' buffered salt solution;
Cytosolic phospholipase A 2 (cPLA 2 ) is normally located in the cytosol, but in response to cell... more Cytosolic phospholipase A 2 (cPLA 2 ) is normally located in the cytosol, but in response to cellular activation the enzyme binds to the membrane at the lipid/ water interface where it catalyzes the hydrolysis of the sn-2 ester of arachidonate-containing phospholipids. Synthetic phospholipid vesicle systems have been used in kinetic and mechanistic analyses of cPLA 2 , but these systems result in a rapid loss of enzyme activity. In the present research, covesicles of 1,2-dimyristoylsn-glycero-3-phosphomethanol (DMPM) containing <10 mol% 1-palmitoyl-2-arachidonoyl-sn-glycero-3phosphocholine (PAPC) as substrate were used to show that this premature cessation of enzyme-catalyzed hydrolysis is dependent on vesicle size with 25nm-diameter vesicles supporting little activity as compared to 100-, 200-, and 400-nm vesicles. This suggests that the curvature of the vesicle may shift a conformational equilibrium toward an enzyme state which does not support activity. Interestingly, the presence of 30% (v/v) glycerol greatly enhanced the activity of the enzyme, although vesicle size-dependent premature cessation of hydrolysis was still observed. While the premature cessation of hydrolysis in the absence of glycerol is accompanied by enzyme inactivation, little inactivation occured in the presence of glycerol, indicating that premature cessation and inactivation are not absolutely coupled. When using this covesicle substrate system under conditions (6 -10 mM CaCl 2 ) where the vesicles are fusing, no premature cessation of hydrolysis has been observed. This is despite a mean vesicle diameter of 400 -450 nm under vesicle-fusing conditions, which is comparable to the largest vesicles used under nonfusing conditions (0.5 mM CaCl 2 ) where considerable premature cessation of hydrolysis was observed. Since DMPM has an intrinsic active site dissociation constant at least 330 times larger than that of PAPC, the optimum conditions for conducting kinetic and mechanistic analyses of cPLA 2 with this covesicle substrate is one in which cPLA 2 is assayed in the presence of glycerol and with fusion-inducing concentrations of calcium. The use of 1,2-dioleoyl-sn-glycero-3-phosphomethanol (DOPM) instead of DMPM in this system supports much less activity and adds the complication of a strong affinity of DOPM for the active site.
Amyloid precursor protein (APP) cleaving enzyme (BACE) is the enzyme responsible for b-site cleav... more Amyloid precursor protein (APP) cleaving enzyme (BACE) is the enzyme responsible for b-site cleavage of APP, leading to the formation of the amyloid-b peptide that is thought to be pathogenic in AlzheimerÕs disease (AD). Hence, BACE is an attractive pharmacological target, and numerous research groups have begun searching for potent and selective inhibitors of this enzyme as a potential mechanism for therapeutic intervention in AD. The mature enzyme is composed of a globular catalytic domain that is Nlinked glycosylated in mammalian cells, a single transmembrane helix that anchors the enzyme to an intracellular membrane, and a short C-terminal domain that extends outside the phospholipid bilayer of the membrane. Here we have compared the substrate and active site-directed inhibitor binding properties of several recombinant constructs of human BACE. The constructs studied here address the importance of catalytic domain glycosylation state, inclusion of domains other than the catalytic domain, and incorporation into a membrane bilayer on the interactions of the enzyme active site with peptidic ligands. We find no significant differences in ligand binding properties among these various constructs. These data demonstrate that the nonglycosylated, soluble catalytic domain of BACE faithfully reflects the ligand binding properties of the full-length mature enzyme in its natural membrane environment. Thus, the use of the nonglycosylated, soluble catalytic domain of BACE is appropriate for studies aimed at understanding the determinants of ligand recognition by the enzyme active site.
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Papers by Jeffrey Tredup