Papers by Jacques Ermolieff
Heparin accelerates the inhibition of cathepsin G by mucus proteinase inhibitor: potent effect of O-butyrylated heparin
The Biochemical journal, Jan 15, 1998
Heparin tightly binds cathepsin G and so protects the enzyme from inhibition by alpha1-antichymot... more Heparin tightly binds cathepsin G and so protects the enzyme from inhibition by alpha1-antichymotrypsin, alpha1-proteinase inhibitor and eglin c, three proteins which do not bind heparin [Ermolieff J., Boudier C., Laine A., Meyer B. and Bieth J.G. (1994) J. Biol. Chem. 269, 29502-29508]. Here we show that heparin no longer protects cathepsin G from inhibition when the enzyme is reacted with mucus proteinase inhibitor (MPI), a heparin-binding protein. Heparin fragments of Mr=4500 and 8100 and O-butyrylated heparin of Mr=8000 form tight complexes with cathepsin G (Kd=0.5-2.2 nM) and MPI (Kd=0. 4-0.8 muM) and accelerate the MPI-promoted inhibition of cathepsin G by a factor of 17-26. They also accelerate the inhibition of neutrophil elastase and pancreatic chymotrypsin. The rate acceleration is due to the binding of heparin to MPI. Butyrylation of heparin slightly decreases its affinity for cathepsin G and MPI but sharply decreases the ionic interactions between the positively charged ...
Analytical Biochemistry, Mar 1, 2011
The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorig... more The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorigenesis. Signal transduction through this pathway requires the assembly and activation of PDK1 and AKT at the plasma membrane. On activation of the pathway, PDK1 and AKT1/2 translocate to the membrane and bind to phosphatidylinositol-(3,4,5)-trisphosphate (PIP 3 ) through interaction with their pleckstrinhomology domains. A biochemical method was developed to measure the kinase activity of PDK1 and AKT1/2, utilizing nickel-chelating coated lipid vesicles as a way to mimic the membrane environment. The presence of these vesicles in the reaction buffer enhanced the specific activity of the His-tagged PDK1 (full-length, and the truncated kinase domain) and the activity of the full-length His-tagged AKT1 and AKT2 when assayed in a cascade-type reaction. This enhanced biochemical assay is also suitable for measuring the inhibition of PDK1 by several selective compounds from the carbonyl-4-aminopyrrolopyrimidine (CAP) series. One of these inhibitors, PF-5168899, was further evaluated using a high content cell-based assay in the presence of CHO cells engineered with GFP-PDK1.
Biochemical and Biophysical Research Communications, Jun 1, 2007
Assay conditions for the 11b-hydroxysteroid dehydrogenase have been optimized by adding phospholi... more Assay conditions for the 11b-hydroxysteroid dehydrogenase have been optimized by adding phospholipids in the media buffer to increase and stabilize the enzymatic activity. The presence of phospholipids greatly facilitates the study of the binding of cortisone and NADPH at the enzyme catalytic site. Kinetic analyses conducted with the human and rabbit enzyme isoforms suggest that both enzymes behave according to an ordered sequential bi-bi mechanism where the NADPH is the first to bind at the active site followed by cortisone. The equilibrium dissociation constant, K i a as well as the apparent Michaelis-Menten constants K m a, K m b, k cat a, and k cat b for NADPH and cortisone, have been determined to be 147.5 lM, 14.4 lM, 43.8 nM, 0.21 min À1 , and 0.27 min À1 , respectively, for the human enzyme and 41.1 lM, 3.1 lM, 161.7 nM, 0.49 min À1 , and 0.52 min À1 , respectively, for the rabbit enzyme.
11ΒHSD1 Crystal Structures for Structure Based Drug Design
Structure of guinea pig 11� steroid dehydrogenase 1 with glycyrrhetinic acid
Acta Crystallogr a, 2005
Journal of Computer-Aided Molecular Design, 2011
Phosphoinositide-dependent kinase-1 (PDK1) is a critical enzyme in the PI3K/AKT pathway and to th... more Phosphoinositide-dependent kinase-1 (PDK1) is a critical enzyme in the PI3K/AKT pathway and to the activation of AGC family protein kinases, including S6K, SGK, and PKC. Dysregulation of this pathway plays a key role in cancer cell growth, survival and tumor angiogenesis. As such, inhibitors of PDK1 offer the promise of a new therapeutic modality for cancer treatment. Fragment based drug screening has recently become a viable entry point for hit identification. In this work, NMR spectroscopy fragment screening of PDK1 afforded novel chemotypes as orthogonal starting points from HTS screening hits. Compounds identified as hits by NMR spectroscopy were tested in a biochemical assay, and fragments with activity in both assays were clustered. The Pfizer compound file was mined via substructure and 2D similarity search, and the chemotypes were prioritized by ligand efficiency (LE), SAR mining, chemical attractiveness, and chemical enablement of promising vectors. From this effort, an isoquinolone fragment hit, 5 (IC 50 870 lM, LE = 0.39), was identified as a novel, ligand efficient inhibitor of PDK1 and a suitable scaffold for further optimization. Initially in the absence of crystallographic data, a fragment growing approach efficiently explored four vectors of the isoquinolone scaffold via parallel synthesis to afford a compound with crystallographic data, 16 (IC 50 41.4 lM, LE = 0.33). Subsequent lead optimization efforts provided 24 (IC 50 1.8 lM, LE = 0.42), with greater than fivefold selectivity against other key pathway kinases. Keywords Isoquinolone Á PDK1 inhibitors Á Fragment based lead discovery Á Ligand efficiency Abbreviations PDK1 Phosphoinositide-dependent kinase-1 PI3K Phosphoinositide 3-kinase LE Ligand efficiency HTS High throughput screening MW Molecular weight STD Saturation transfer difference NO Nitrogen and oxygen count TPSA Total polar surface area HBD Hydrogen bond donor count SSHE Substructure similarity hit expansion
Biochemistry, 1995
Heparin accelerates the inhibition of neutrophil elastase by mucus proteinase inhibitor (MPI), th... more Heparin accelerates the inhibition of neutrophil elastase by mucus proteinase inhibitor (MPI), the physiological antielastase of airways as a result of its binding with the inhibitor [Faller, B., MCly, Y., GCrard, D., & Bieth, J. G. (1992) Biochemistry 31, 8285-82901. To explore the heparin-binding site of the inhibitor, we have modified the lysine and arginine residues of MPI and its isolated C-terminal domain by using 4-N,N-(dimethylamino)azobenzene-4'-isothiocyano-2'-sulfonic acid (S-DABITC) [Chang, J. Y. (1989) J. Biol. Chem. 264, 31 11-31 151 and (p-hydroxypheny1)glyoxal (HPG) (Yamasaki, R. B., Vega, A., & Feeney, R. E. (1980) Anal. Biochem. 109, 32-40], respectively. The derivatizations were done in the absence and presence of a 4.5 kDa heparin fraction with a low degree of polydispersity. The effect
Biochemical and Biophysical Research Communications, 2007
Assay conditions for the 11b-hydroxysteroid dehydrogenase have been optimized by adding phospholi... more Assay conditions for the 11b-hydroxysteroid dehydrogenase have been optimized by adding phospholipids in the media buffer to increase and stabilize the enzymatic activity. The presence of phospholipids greatly facilitates the study of the binding of cortisone and NADPH at the enzyme catalytic site. Kinetic analyses conducted with the human and rabbit enzyme isoforms suggest that both enzymes behave according to an ordered sequential bi-bi mechanism where the NADPH is the first to bind at the active site followed by cortisone. The equilibrium dissociation constant, K i a as well as the apparent Michaelis-Menten constants K m a, K m b, k cat a, and k cat b for NADPH and cortisone, have been determined to be 147.5 lM, 14.4 lM, 43.8 nM, 0.21 min À1 , and 0.27 min À1 , respectively, for the human enzyme and 41.1 lM, 3.1 lM, 161.7 nM, 0.49 min À1 , and 0.52 min À1 , respectively, for the rabbit enzyme.
Analytical Biochemistry, 2011
The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorig... more The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorigenesis. Signal transduction through this pathway requires the assembly and activation of PDK1 and AKT at the plasma membrane. On activation of the pathway, PDK1 and AKT1/2 translocate to the membrane and bind to phosphatidylinositol-(3,4,5)-trisphosphate (PIP 3 ) through interaction with their pleckstrinhomology domains. A biochemical method was developed to measure the kinase activity of PDK1 and AKT1/2, utilizing nickel-chelating coated lipid vesicles as a way to mimic the membrane environment. The presence of these vesicles in the reaction buffer enhanced the specific activity of the His-tagged PDK1 (full-length, and the truncated kinase domain) and the activity of the full-length His-tagged AKT1 and AKT2 when assayed in a cascade-type reaction. This enhanced biochemical assay is also suitable for measuring the inhibition of PDK1 by several selective compounds from the carbonyl-4-aminopyrrolopyrimidine (CAP) series. One of these inhibitors, PF-5168899, was further evaluated using a high content cell-based assay in the presence of CHO cells engineered with GFP-PDK1.
Structure of guinea pig 11β steroid dehydrogenase 1 with glycyrrhetinic acid
Acta Crystallographica Section A Foundations of Crystallography, 2005
Design of Potent Inhibitors for Human Brain Memapsin 2 (�-Secretase)
J Am Chem Soc, 2000
The Effect of Substrates on the Kinetics and the in Vivo Threshold Activity of Mutant HIV-1 Proteases
Advances in Experimental Medicine and Biology, Feb 1, 1998
Biochemistry Usa, Oct 1, 2009
Checkpoint kinase 1 (CHK1) is a key element in the DNA damage response pathway and plays a crucia... more Checkpoint kinase 1 (CHK1) is a key element in the DNA damage response pathway and plays a crucial role in the S-G 2 -phase checkpoint. Inhibiting CHK1 is a therapeutic strategy involving abrogation of the G2/M mitotic checkpoint defense of tumor cells toward lethal damage induced by DNA-directed chemotherapeutic agents. To date, most CHK1 inhibition approaches have involved targeting the ATP site of this kinase. In this study, we provide crystallographic and kinetic characterization of two small molecule inhibitors that bind to an allosteric site in the proximity of the CHK1 substrate site. Analysis of kinetic and biophysical data has led to the conclusion that these small molecule allosteric site inhibitors of CHK1 are reversible and are neither ATP-nor peptide substrate-competitive. K i values of 1.89 and 0.15 μM, respectively, have been determined for these compounds using a mixed inhibitor kinetic analysis. Cocrystal structures of the inhibitors bound to CHK1 reveal an allosteric site, unique to CHK1, located in the C-terminal domain and consisting of a shallow groove linked to a small hydrophobic pocket. The pocket displays induced fit characteristics in the presence of the two inhibitors. These findings establish the potential for the development of highly selective CHK1 inhibitors.
Bioorg Medicinal Chem Letter, 2010
The design and development of a series of highly selective pyrrolidine carboxamide 11b-HSD1 inhib... more The design and development of a series of highly selective pyrrolidine carboxamide 11b-HSD1 inhibitors are described. These compounds including PF-877423 demonstrated potent in vitro activity against both human and mouse 11b-HSD1 enzymes. In an in vivo assay, PF-877423 inhibited the conversion of cortisone to cortisol. Structure guided optimization effort yielded potent and stable 11b-HSD1 selective inhibitor 42.
Residence time and kinetic efficiency analysis of extracellular signal-regulated kinase 2 inhibitors
Analytical Biochemistry, 2015
The RAS/RAF/MEK/ERK signal transduction cascade plays an important role in the regulation of crit... more The RAS/RAF/MEK/ERK signal transduction cascade plays an important role in the regulation of critical cellular processes such as cell proliferation, migration, and differentiation. The up-regulation of this pathway can negatively affect cell homeostasis and is responsible for the development of various forms of cancer and inflammation processes. Therefore, there is a strong interest in pursuing drug programs targeting some of the enzymes involved in this pathway. In addition to the determination of Ki, Kd, IC50, and/or EC50, a more thorough kinetic analysis can provide useful information for the selection of the best lead series during the early stage of the drug discovery process. This study describes a medium-throughput fluorescent probe displacement assay for the rapid determination of the koff constant, residence time, and kinetic efficiency for ERK (extracellular signal-regulated kinase) inhibitors. Using this method, we have identified several inhibitors that we have subjected to further kinetic analysis by comparing koff constants determined for these time-dependent inhibitors using either the active or inactive form of ERK2.
Nature Reviews Drug Discovery, 2002
The increased incidence of type 2 diabetes mellitus (T2DM) and OBESITY in the population has fuel... more The increased incidence of type 2 diabetes mellitus (T2DM) and OBESITY in the population has fuelled an intensified search for new therapeutic treatment options 1 . This increase in obesity and T2DM was previously observed primarily in adult populations, associated with a sedentary lifestyle, but has now also become a medical problem in children 2,3 . The relationship between obesity and T2DM has a polygenetic component and is associated with insulin resistance 4 . Over time, the stress of obesity on a genetically susceptible background produces insulin resistance and impaired glucose tolerance (IGT) 5 . Increased stress on the pancreas, in combination with gluco-and lipotoxicity, causes the insulin-producing PANCREATIC ISLET to fail to compensate for the insulin resistance 6 . As the islet β-cells fail to compensate, IGT evolves into T2DM and, in effect, produces a disease-state continuum 7 . Failure of the islet β-cells to be able to compensate for the insulin resistance might also have a genetic component when combined with the stress of gluco-and lipotoxicity 8 .
Design of Potent Inhibitors for Human Brain Memapsin 2 (β-Secretase)
Journal of the American Chemical Society, 2000
... 3) (a) Vassar, R.; Bennett, BD; Babu-Khan, S ... Zhaoning Zhu, Zhong-Yue Sun, Yuanzan Ye, Joh... more ... 3) (a) Vassar, R.; Bennett, BD; Babu-Khan, S ... Zhaoning Zhu, Zhong-Yue Sun, Yuanzan Ye, Johannes Voigt, Corey Strickland, Elizabeth M. Smith, Jared Cumming, Lingyan Wang, Jesse Wong, Yu-Sen Wang, Daniel F. Wyss, Xia Chen, Reshma Kuvelkar, Matthew E. Kennedy ...
Polyoxometalate HIV-1 Protease Inhibitors. A New Mode of Protease Inhibition
Journal of the American Chemical Society, 2001
Nb-containing polyoxometalates (POMs) of the Wells-Dawson class inhibit HIV-1 protease (HIV-1P) b... more Nb-containing polyoxometalates (POMs) of the Wells-Dawson class inhibit HIV-1 protease (HIV-1P) by a new mode based on kinetics, binding, and molecular modeling studies. Reaction of alpha(1)-K(9)Li[P(2)W(17)O(61)] or alpha(2)-K(10)[P(2)W(17)O(61)] with aqueous H(2)O(2) solutions of K(7)H[Nb(6)O(19)] followed by treatment with HCl and KCl and then crystallization affords the complexes alpha(1)-K(7)[P(2)W(17)(NbO(2))O(61)] (alpha(1)()1) and alpha(2)-K(7)[P(2)W(17)(NbO(2))O(61)] (alpha(2)()1) in 63 and 86% isolated yields, respectively. Thermolysis of the crude peroxoniobium compounds (72-96 h in refluxing H(2)O) prior to treatment with KCl converts the peroxoniobium compounds to the corresponding polyoxometalates (POMs), alpha(1)-K(7)[P(2)W(17)NbO(62)] (alpha(1)()2) and alpha(2)-K(7)[P(2)W(17)NbO(62)] (alpha(2)()2), in moderate yields (66 and 52%, respectively). The identity and high purity of all four compounds were confirmed by (31)P NMR and (183)W NMR. The acid-induced dimerization of the oxo complexes differentiates sterically between the cap (alpha(2)) site and the belt (alpha(1)) site in the Wells-Dawson structure (alpha(2)()2 dimerizes in high yield; alpha(1)()2 does not). All four POMs exhibit high activity in cell culture against HIV-1 (EC(50) values of 0.17-0.83 microM), are minimally toxic (IC(50) values of 50 to >100 microM), and selectively inhibit purified HIV-1 protease (HIV-1P) (IC(50) values for alpha(1)()1, alpha(2)()1, alpha(1)()2, and alpha(2)()2 of 2.0, 1.2, 1.5, and 1.8 microM, respectively). Thus, theoretical, binding, and kinetics studies of the POM/HIV-1P interaction(s) were conducted. Parameters for [P(2)W(17)NbO(62)](7)(-) were determined for the Kollman all-atom (KAA) force field in Sybyl 6.2. Charges for the POM were obtained from natural population analysis (NPA) at the HF/LANL2DZ level of theory. AutoDock 2.2 was used to explore possible binding locations for the POM with HIV-1P. These computational studies strongly suggest that the POMs function not by binding to the active site of HIV-1P, the mode of inhibition of all other HIV-1P protease inhibitors, but by binding to a cationic pocket on the "hinge" region of the flaps covering the active site (2 POMs and cationic pockets per active homodimer of HIV-1P). The kinetics and binding studies, conducted after the molecular modeling, are both in remarkable agreement with the modeling results: 2 POMs bind per HIV-1P homodimer with high affinities (K(i) = 1.1 +/- 0.5 and 4.1 +/- 1.8 nM in 0.1 and 1.0 M NaCl, respectively) and inhibition is noncompetitive (k(cat) but not K(m) is affected by the POM concentration).
Discovery of Novel, Potent, and Selective Inhibitors of 3-Phosphoinositide-Dependent Kinase (PDK1)
Journal of Medicinal Chemistry, 2011
Analogues substituted with various amines at the 6-position of the pyrazine ring on (4-amino-7-is... more Analogues substituted with various amines at the 6-position of the pyrazine ring on (4-amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)pyrazin-2-ylmethanone were discovered as potent and selective inhibitors of PDK1 with potential as anticancer agents. An early lead with 2-pyridine-3-ylethylamine as the pyrazine substituent showed moderate potency and selectivity. Structure-based drug design led to improved potency and selectivity against PI3Kα through a combination of cyclizing the ethylene spacer into a saturated, five-membered ring and substituting on the 4-position of the aryl ring with a fluorine. ADME properties were improved by lowering the lipophilicity with heteroatom replacements in the saturated, five-membered ring. The optimized analogues have a PDK1 Ki of 1 nM and >100-fold selectivity against PI3K/AKT-pathway kinases. The cellular potency of these analogues was assessed by the inhibition of AKT phosphorylation (T308) and by their antiproliferation activity against a number of tumor cell lines.
Journal of Endocrinology, 2008
Glucocorticoid excess increases fat mass, preferentially within omental depots; yet circulating c... more Glucocorticoid excess increases fat mass, preferentially within omental depots; yet circulating cortisol concentrations are normal in most patients with metabolic syndrome (MS). At a pre-receptor level, 11b-hydroxysteroid dehydrogenase type 1 (11b-HSD1) activates cortisol from cortisone locally within adipose tissue, and inhibition of 11b-HSD1 in liver and adipose tissue has been proposed as a novel therapy to treat MS by reducing hepatic glucose output and adiposity. Using a transformed human subcutaneous preadipocyte cell line (Chub-S7) and human primary preadipocytes, we have defined the role of glucocorticoids and 11b-HSD1 in regulating adipose tissue differentiation. Human cells were differentiated with 1 . 0 mM cortisol (F), or cortisone (E) with or without 100 nM of a highly selective 11b-HSD1 inhibitor PF-877423. 11b-HSD1 mRNA expression increased across adipocyte differentiation (P!0 . 001, nZ4), which was paralleled by an increase in 11b-HSD1 oxo-reductase activity (from nil on day 0 to 5 . 9G1.9 pmol/mg per h on day 16, P!0 . 01, nZ7). Cortisone enhanced adipocyte differentiation; fatty acid-binding protein 4 expression increased 312fold (P!0 . 001) and glycerol-3-phosphate dehydrogenase 47-fold (P!0 . 001) versus controls. This was abolished by co-incubation with PF-877423. In addition, cellular lipid content decreased significantly. These findings were confirmed in the primary cultures of human subcutaneous preadipocytes. The increase in 11b-HSD1 mRNA expression and activity is essential for the induction of human adipogenesis. Blocking adipogenesis with a novel and specific 11b-HSD1 inhibitor may represent a novel approach to treat obesity in patients with MS.
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Papers by Jacques Ermolieff