James Janc - Academia.edu (original) (raw)

Papers by James Janc

Research paper thumbnail of Clinical Results with a B Cell Activating Anti-CD73 Antibody for the Immunotherapy of COVID-19

ABSTRACTRobust polyclonal humoral immune responses have the potential to generate a diverse set o... more ABSTRACTRobust polyclonal humoral immune responses have the potential to generate a diverse set of antibodies to neutralize and eliminate viruses such as SARS-CoV-2 and protect against transmission, re-infection and the evolution of variants that evade immunity. CD73 is present on subsets of human B and T cells where it plays a role in lymphocyte activation and migration. CD73 also functions as an ectoenzyme that converts AMP into immunosuppressive adenosine. We have developed a humanized anti-CD73 antibody, mupadolimab (CPI-006), that blocks CD73 enzymatic activity and activates CD73POS B cells, thereby inducing differentiation into plasmablasts, immunoglobulin class switching, and antibody secretion independent of the adenosine modulatory activity. These effects suggest mupadolimab may enhance the magnitude, diversity, and duration of anti-viral responses in patients with COVID-19. This hypothesis was tested in a dose escalation phase 1 trial in 29 hospitalized patients with COVID...

Research paper thumbnail of A Selective Small Molecule Factor VIIa Inhibitor, CRA-027483, Suppresses Acute Inflammation in Mice

Blood

Factor VIIa (FVIIa) is a well known member of the extrinsic coagulation cascade and therefore an ... more Factor VIIa (FVIIa) is a well known member of the extrinsic coagulation cascade and therefore an attractive therapeutic target for thrombosis. CRA-027483 is a potent, selective, reversible small molecule inhibitor of human FVIIa that displays anti-thrombotic activities in vitro and in vivo. In addition to the well-characterized role of the FVIIa/tissue factor (TF) complex in coagulation, FVIIa/TF complex has more recently been implicated in inflammation via intracellular signaling initiated by the cleavage of protease activated receptors (PARs) by the complex. Therefore, we assessed the ability of CRA-027483 to inhibit acute inflammation using a mouse model of endotoxemia. The compound was delivered subcutaneously one hour prior to LPS challenge and serum or plasma was harvested at varying time points post-challenge. Soluble mediators of coagulation and inflammation induced by LPS were measured by ELISA. Additionally, prothrombin time (PT) and compound exposure were determined. CRA-...

Research paper thumbnail of Preliminary Clinical Data from a Phase 1 Trial with CPI-818, a Selective ITK Inhibitor That Preferentially Blocks the Growth of T Lymphoma Cells

Blood

Introduction ITK is a tyrosine kinase critical to T cell receptor (TCR) signaling. Overexpression... more Introduction ITK is a tyrosine kinase critical to T cell receptor (TCR) signaling. Overexpression of this gene has been reported in cutaneous T-cell lymphoma (CTCL) and peripheral T-cell lymphoma (PTCL). Genomic analyses have demonstrated the contribution of aberrant TCR signaling in the pathogenesis of T-cell lymphomas (TCL). RLK, a closely related kinase, is co-expressed with ITK in T and NK cells, and is partially functionally redundant with ITK signaling. In NK cells, ITK has been shown to be involved in FcγRIII signaling and antibody-dependent cellular cytotoxicity (ADCC). However, the relative contribution of ITK vs RLK in ADCC is not well understood. Thus, selective inhibition of ITK, but not other signal transduction components such as RLK, may be an effective strategy to treat TCL while preserving normal T and NK cell functions. CPI-818 is an orally bioavailable, covalent inhibitor of ITK with >100-fold selectivity over RLK and BTK. It was well tolerated and exhibited an...

Research paper thumbnail of Abstract 1313: CPI-818: A selective inhibitor of interleukin-2-inducible T-cell kinase (ITK) that inhibits T-cell receptor signaling, promotes Th1 skewing, and achieves objective tumor responses when administered to dogs with T cell lymphomas

Experimental and Molecular Therapeutics

Research paper thumbnail of A Novel Approach to Serine Protease Inhibition: Kinetic Characterization of Inhibitors Whose Potencies and Selectivities Are Dramatically Enhanced by Zinc(II)

Biochemistry Usa, 2000

Serine proteases play a role in a variety of disease states and thus are attractive targets for t... more Serine proteases play a role in a variety of disease states and thus are attractive targets for therapeutic intervention. We report the kinetic characterization of a class of serine protease inhibitors whose potencies and selectivities are dramatically enhanced in the presence of Zn(II). The structural basis for Zn(II)-mediated inhibition of trypsin-like proteases has recently been reported [Katz, B. A., Clark, J. M., Finer-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Moore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612]. A case study of the kinetic behavior of human tryptase inhibitors is provided to illustrate the general phenomenon of Zn(II)-mediated inhibition. Tryptase, Zn(II), and the inhibitor form a ternary complex which exhibits classic tight-binding inhibition. The half-life for release of inhibitor from the tryptase-Zn(II)-inhibitor complex has been measured for a number of inhibitors. Consistent with tight-binding behavior, potent tryptase inhibitors are characterized by extremely slow rates of dissociation from the ternary complex with half-lives on the order of hours. A model of human serum, designed to reproduce physiological levels of Zn(II), has been employed to evaluate the performance of Zn(II)-potentiated tryptase inhibitors under physiological conditions. We demonstrate that Zn(II)-mediated inhibition can be achieved at physiological Zn(II) levels.

Research paper thumbnail of Expression and characterization of recombinant gamma-tryptase

Protein Expression and Purification, Oct 1, 2006

Research paper thumbnail of The Biosynthetic Gene Cluster for a Monocyclic �-Lactam Antibiotic, Nocardicin A

Research paper thumbnail of Expression and characterization of recombinant ?-tryptase

Protein Express Purif, 2006

Research paper thumbnail of Cathepsin cysteine protease inhibitors

Research paper thumbnail of Small molecule-mediated inhibition of Factor VIIa catalytic activity blocks fVIIa:TF cellular signaling and chemokine secretion in human tumor cell lines

In addition to its role in coagulation, Factor VIIa (fVIIa) binding to TF has been demonstrated t... more In addition to its role in coagulation, Factor VIIa (fVIIa) binding to TF has been demonstrated to activate a variety of intracellular signaling pathways including MAPK, AKT, and Src. This activity has been shown to require TF binding as well as fVIIa protease activity. TF overexpression in tumor epithelial cells and tumor vasculature has been linked to prognosis. However a precise role for fVIIa:TF signaling in tumor cell growth and survival has not yet been established. We have demonstrated that fVIIa is able to activate MAPK, AKT, and early-response gene induction in the BxPC3 pancreatic human tumor cell line at physiologic (0.1-10nM) concentrations. TF expression in the tumor cell line is required for this intracellular signaling. The chemically-inactivated form of fVIIa, fVIIai, is unable to activate these signaling pathways. In addition, preincubation of fVIIa with potent, fVIIa-selective, reversible small molecule inhibitors of fVIIa protease activity inhibited the activation...

Research paper thumbnail of Efficacy of telavancin, a lipoglycopeptide antibiotic, in experimental models of Gram-positive infection

Expert review of anti-infective therapy, 2014

Telavancin is a parenteral lipoglycopeptide antibiotic with a dual mechanism of action contributi... more Telavancin is a parenteral lipoglycopeptide antibiotic with a dual mechanism of action contributing to bactericidal activity against multidrug-resistant Gram-positive pathogens. It has been approved for the treatment of complicated skin and skin structure infections due to susceptible Gram-positive bacteria and hospital-acquired/ventilator-associated bacterial pneumonia due to Staphylococcus aureus when other alternatives are unsuitable. Telavancin has been demonstrated to be efficacious in multiple animal models of soft tissue, cardiac, systemic, lung, bone, brain and device-associated infections involving clinically relevant Gram-positive pathogens, including methicillin-resistant S. aureus, glycopeptide-intermediate S. aureus, heterogeneous vancomycin-intermediate S. aureus and daptomycin non-susceptible methicillin-resistant S. aureus. The AUC0-24h/MIC ratio is the primary pharmacodynamically-linked pharmacokinetic parameter. The preclinical data for telavancin supports further ...

Research paper thumbnail of Expression and characterization of recombinant γ-tryptase

Protein Expression and Purification, 2006

Tryptases are trypsin-like serine proteases whose expression is restricted to cells of hematopoie... more Tryptases are trypsin-like serine proteases whose expression is restricted to cells of hematopoietic origin, notably mast cells. γ-Tryptase, a recently described member of the family also known as transmembrane tryptase (TMT), is a membrane-bound serine protease found in the secretory granules or on the surface of degranulated mast cells. The 321 amino acid protein contains an 18 amino acid propeptide

Research paper thumbnail of Impact of telavancin on prothrombin time and activated partial thromboplastin time as determined using point-of-care coagulometers

Journal of Thrombosis and Thrombolysis, 2013

Telavancin is approved in the United States, Canada, and Europe (At the time of submission, the t... more Telavancin is approved in the United States, Canada, and Europe (At the time of submission, the telavancin European marketing authorization for nosocomial pneumonia was suspended until Theravance provides evidence of a new European Medicines Agency approved supplier) as an antibiotic to treat certain Gram-positive bacterial skin infections. Telavancin has been shown to prolong plasmatic prothrombin (PT) and activated partial thromboplastin (aPTT) clotting times in clinical diagnostic lab-based assays. In this study, we evaluated the potential for telavancin to prolong whole blood PT/International Normalized Ratio (INR) and aPTT tests on point-of-care (POC) instruments. Whole blood collected from 8 healthy subjects was supplemented with telavancin to final concentrations of 0, 10, 20, and 100 μg/ml. Final concentrations were selected to match trough, twice trough, and peak plasma levels following the approved 10 mg/kg dose. Four widely employed POC coagulation instruments were chosen to be representative of the POC platforms currently in use.. These systems were the Roche Coaguchek XS, the Abbott iSTAT, the ITC Hemochron SIG+, and the Alere INRatio2 POC devices. The PT/INR measured by the Coaguchek XS showed the greatest sensitivity to the presence of telavancin. The PT/INR measured by the Hemochron SIG+ and iSTAT were sensitive to telavancin but to a lesser extent. The INRatio2 was the least sensitive to the presence of telavancin when testing the whole blood PT/INR. Only the Hemochron SIG+ device was capable of measuring aPTT and showed a concentration-dependent increase in aPTT. This study supports the current recommendation that PT and aPTT monitoring be conducted immediately to the next dose of telavancin when coagulation parameters are tested using POC instrumentation.

Research paper thumbnail of Practical Robust Fit of Enzyme Inhibition Data

Methods in Enzymology, 2004

Research paper thumbnail of Contribution of Multicentered Short Hydrogen Bond Arrays to Potency of Active Site-Directed Serine Protease Inhibitors

Journal of the American Chemical Society, 2002

We describe and compare the pH dependencies of the potencies and of the bound structures of two i... more We describe and compare the pH dependencies of the potencies and of the bound structures of two inhibitor isosteres that form multicentered short hydrogen bond arrays at the active sites of trypsin, thrombin, and urokinase type plasminogen activator (urokinase or uPA) over certain ranges of pH. Depending on the pH, short hydrogen bond arrays at the active site are mediated by two waters, one in the oxyanion hole (H(2)O(oxy)) and one on the other (S2) side of the inhibitor (H(2)O(S2)), by one water (H(2)O(oxy)), or by no water. The dramatic variation in the length of the active site hydrogen bonds as a function of pH, of inhibitor, and of enzyme, along with the involvement or absence of ordered water, produces a large structural manifold of active site hydrogen bond motifs. Diverse examples of multicentered and two-centered short hydrogen bond arrays, both at and away from the active site, recently discovered in several protein crystal systems, suggest that short hydrogen bonds in proteins may be more common than has been recognized. The short hydrogen bond arrays resemble one another with respect to ionic nature, highly polar environment, multitude of associated ordinary hydrogen bonds, and disparate pK(a) values of participating groups. Comparison of structures and K(i) values of trypsin complexes at pH values where the multicentered short hydrogen bond arrays mediating inhibitor binding are present or absent indicate that these arrays have a minor effect on inhibitor potency. These features suggest little covalent nature within the short hydrogen bonds, despite their extraordinary shortness (as short as 2.0 A).

Research paper thumbnail of Elaborate Manifold of Short Hydrogen Bond Arrays Mediating Binding of Active Site-directed Serine Protease Inhibitors

Journal of Molecular Biology, 2003

An extensive structural manifold of short hydrogen bond-mediated, active site-directed, serine pr... more An extensive structural manifold of short hydrogen bond-mediated, active site-directed, serine protease inhibition motifs is revealed in a set of over 300 crystal structures involving a large suite of small molecule inhibitors (2-(2-phenol)-indoles and 2-(2-phenol)-benzimidazoles) determined over a wide range of pH (3.5-11.4). The active site hydrogen-bonding mode was found to vary markedly with pH, with the steric and electronic properties of the inhibitor, and with the type of protease (trypsin, thrombin or urokinase type plasminogen activator (uPA)). The pH dependence of the active site hydrogen-bonding motif is often intricate, constituting a distinct fingerprint of each complex. Isosteric replacements or minor substitutions within the inhibitor that modulate the pK(a) of the phenol hydroxyl involved in short hydrogen bonding, or that affect steric interactions distal to the active site, can significantly shift the pH-dependent structural profile characteristic of the parent scaffold, or produce active site-binding motifs unique to the bound analog. Ionization equilibria at the active site associated with inhibitor binding are probed in a series of the protease-inhibitor complexes through analysis of the pH dependence of the structure and environment of the active site-binding groups involved in short hydrogen bond arrays. Structures determined at high pH (>11), suggest that the pK(a) of His57 is dramatically elevated, to a value as high as approximately 11 in certain complexes. K(i) values involving uPA and trypsin determined as a function of pH for a set of inhibitors show pronounced parabolic pH dependence, the pH for optimal inhibition governed by the pK(a) of the inhibitor phenol involved in short hydrogen bonds. Comparison of structures of trypsin, thrombin and uPA, each bound by the same inhibitor, highlights important structural variations in the S1 and active sites accessible for engineering notable selectivity into remarkably small molecules with low nanomolar K(i) values.

Research paper thumbnail of Dissecting and Designing Inhibitor Selectivity Determinants at the S1 Site Using an Artificial Ala190 Protease (Ala190 uPA)

Journal of Molecular Biology, 2004

A site-directed mutant of the serine protease urokinase-type plasminogen activator (uPA), was pro... more A site-directed mutant of the serine protease urokinase-type plasminogen activator (uPA), was produced to assess the contribution of the Ser190 side-chain to the affinity and selectivity of lead uPA inhibitors in the absence of other differences present in comparisons of natural proteases. Crystallography and enzymology involving WT and Ala190 uPA were used to calculate free energy binding contributions of hydrogen bonds involving the Ser190 hydroxyl group (O(gamma)(Ser190)) responsible for the remarkable selectivity of 6-halo-5-amidinoindole and 6-halo-5-amidinobenzimidazole inhibitors toward uPA and against natural Ala190 protease anti-targets. Crystal structures of uPA complexes of novel, active site-directed arylguanidine and 2-aminobenzimidazole inhibitors of WT uPA, together with associated K(i) values for WT and Ala190 uPA, also indicate a significant role of Ser190 in the binding of these classes of uPA inhibitors. Structures and associated K(i) values for a lead inhibitor (CA-11) bound to uPA and to five other proteases, as well as for other leads bound to multiple proteases, help reveal the features responsible for the potency (K(i)=11nM) and selectivity of the remarkably small inhibitor, CA-11. The 6-fluoro-5-amidinobenzimidzole, CA-11, is more than 1000-fold selective against natural Ala190 protease anti-targets, and more than 100-fold selective against other Ser190 anti-targets.

Research paper thumbnail of A novel serine protease inhibition motif involving a multi-centered short hydrogen bonding network at the active site

Journal of Molecular Biology, 2001

Research paper thumbnail of Optimization of Subsite Binding to the β5 Subunit of the Human 20S Proteasome Using Vinyl Sulfones and 2-Keto-1,3,4-oxadiazoles:  Syntheses and Cellular Properties of Potent, Selective Proteasome Inhibitors

Journal of Medicinal Chemistry, 2006

Beginning with the peptide sequence Cbz-Ile-Glu(OtBu)-Ala-Leu found in PSI (3), a series of vinyl... more Beginning with the peptide sequence Cbz-Ile-Glu(OtBu)-Ala-Leu found in PSI (3), a series of vinyl sulfones (VS) were synthesized for evaluation as inhibitors of the chymotrypsin-like activity of the 20S proteasome. Variations at the key P3 position confirmed the importance of a long side chain capped with a hydrophobic group for optimal potency, consistent with a model of binding to the S3 subsite. The tert-butyl glutamic ester initially used at P3 gave plasma unstable, insoluble compounds and was replaced with the better isostere, N-beta-neopentyl asparagine. The inhibitors were shortened by replacing the N-terminal Cbz-isoleucine with a p-tosyl group without loss of potency. Small l-amino acids were used at P2, where d-substitution was not tolerated. The resulting optimized P4-P3-P2 sequence was grafted onto a novel proteasome inhibitor warhead, 2-keto-1,3,4-oxadiazoles (KOD), to produce reversible, subnanomolar proteasome inhibitors that were 1000-fold selective versus cathepsin B (CatB), cathepsin S (CatS), and trypsin-like as well as PGPH-like proteasome activity. A number of compounds in both the VS and the KOD series exhibited growth inhibitory effects against the human prostate cancer cell line PC3 at submicromolar concentrations.

Research paper thumbnail of O258 Telavancin inhibits peptidoglycan biosynthesis through preferential targeting of transglycosylation: evidence for a multivalent interaction between telavancin and lipid II

International Journal of Antimicrobial Agents, 2007

Objectives: Alveolar echinococcosis is one of the most pathogenic parasitic zoonoses in central E... more Objectives: Alveolar echinococcosis is one of the most pathogenic parasitic zoonoses in central Europe, caused after oral uptake of eggs of Echinococcus multilocularis, shed by infected foxes. At present, there seems to be a trend towards increased parasite density in central Europe and spread in western Europe, although it cannot be decided whether E. multilocularis recently has extended its habitat or whether the parasite has simply remained undetected until now in marginal regions. We analysed red fox data from an area close to the westernmost margin of E. multilocularis habitat in Europe, Belgium and a neighbouring province (Limburg) in The Netherlands (NL), with the aim of studying the emergence of the parasite in this area. Methods: A total of 1202 foxes has been analysed (1018 in Belgium, 184 in NL) with 179 infected (164 in Belgium, 15 in NL) using mucosal scrapings. Spatial coordinates of the locations of infected and uninfected foxes have been determined by GPS. In addition, additional data in southern Europe were analysed using GIS and modeling to study changes in distrinution and prevalence. Results: The large scale spatial distribution of the prevalence of infection among sampled foxes has been modeled as an ellipsoidal gradient, demonstrating increasing prevalence in southeastern direction that means towards the endemic area in central Europe. Using this gradient, the spatial pattern of E. multilocularis infection in Belgium and a contiguous region in NL could be shown to have a continuous distribution across national borders. Part of the Belgian data allowed investigation of the temporal changes in spatial distribution of E. multilocularis, revealing spreading into northwestern direction. A mathematical model describing the parasite population dynamics both in time and in space was fitted to the worm burdens of foxes sampled between 1996 and 2006 in NL. We found a strong indication that the parasite's reproduction number R 0 is greater than 1 and that the parasite is spreading to a wider region in Limburg. Based on the R 0 derived from the mathematical model of the parasite's transmission, we explore the effect of public health measures aimed to eradicate the infection. Conclusion: Increased infection pressure of E. multilocularis in northwestern Europe is most likely to occur at present. In Belgium human alveolar echinococcosis had been absent in the past, but 3 human cases have been reported in 2004.

Research paper thumbnail of Clinical Results with a B Cell Activating Anti-CD73 Antibody for the Immunotherapy of COVID-19

ABSTRACTRobust polyclonal humoral immune responses have the potential to generate a diverse set o... more ABSTRACTRobust polyclonal humoral immune responses have the potential to generate a diverse set of antibodies to neutralize and eliminate viruses such as SARS-CoV-2 and protect against transmission, re-infection and the evolution of variants that evade immunity. CD73 is present on subsets of human B and T cells where it plays a role in lymphocyte activation and migration. CD73 also functions as an ectoenzyme that converts AMP into immunosuppressive adenosine. We have developed a humanized anti-CD73 antibody, mupadolimab (CPI-006), that blocks CD73 enzymatic activity and activates CD73POS B cells, thereby inducing differentiation into plasmablasts, immunoglobulin class switching, and antibody secretion independent of the adenosine modulatory activity. These effects suggest mupadolimab may enhance the magnitude, diversity, and duration of anti-viral responses in patients with COVID-19. This hypothesis was tested in a dose escalation phase 1 trial in 29 hospitalized patients with COVID...

Research paper thumbnail of A Selective Small Molecule Factor VIIa Inhibitor, CRA-027483, Suppresses Acute Inflammation in Mice

Blood

Factor VIIa (FVIIa) is a well known member of the extrinsic coagulation cascade and therefore an ... more Factor VIIa (FVIIa) is a well known member of the extrinsic coagulation cascade and therefore an attractive therapeutic target for thrombosis. CRA-027483 is a potent, selective, reversible small molecule inhibitor of human FVIIa that displays anti-thrombotic activities in vitro and in vivo. In addition to the well-characterized role of the FVIIa/tissue factor (TF) complex in coagulation, FVIIa/TF complex has more recently been implicated in inflammation via intracellular signaling initiated by the cleavage of protease activated receptors (PARs) by the complex. Therefore, we assessed the ability of CRA-027483 to inhibit acute inflammation using a mouse model of endotoxemia. The compound was delivered subcutaneously one hour prior to LPS challenge and serum or plasma was harvested at varying time points post-challenge. Soluble mediators of coagulation and inflammation induced by LPS were measured by ELISA. Additionally, prothrombin time (PT) and compound exposure were determined. CRA-...

Research paper thumbnail of Preliminary Clinical Data from a Phase 1 Trial with CPI-818, a Selective ITK Inhibitor That Preferentially Blocks the Growth of T Lymphoma Cells

Blood

Introduction ITK is a tyrosine kinase critical to T cell receptor (TCR) signaling. Overexpression... more Introduction ITK is a tyrosine kinase critical to T cell receptor (TCR) signaling. Overexpression of this gene has been reported in cutaneous T-cell lymphoma (CTCL) and peripheral T-cell lymphoma (PTCL). Genomic analyses have demonstrated the contribution of aberrant TCR signaling in the pathogenesis of T-cell lymphomas (TCL). RLK, a closely related kinase, is co-expressed with ITK in T and NK cells, and is partially functionally redundant with ITK signaling. In NK cells, ITK has been shown to be involved in FcγRIII signaling and antibody-dependent cellular cytotoxicity (ADCC). However, the relative contribution of ITK vs RLK in ADCC is not well understood. Thus, selective inhibition of ITK, but not other signal transduction components such as RLK, may be an effective strategy to treat TCL while preserving normal T and NK cell functions. CPI-818 is an orally bioavailable, covalent inhibitor of ITK with >100-fold selectivity over RLK and BTK. It was well tolerated and exhibited an...

Research paper thumbnail of Abstract 1313: CPI-818: A selective inhibitor of interleukin-2-inducible T-cell kinase (ITK) that inhibits T-cell receptor signaling, promotes Th1 skewing, and achieves objective tumor responses when administered to dogs with T cell lymphomas

Experimental and Molecular Therapeutics

Research paper thumbnail of A Novel Approach to Serine Protease Inhibition: Kinetic Characterization of Inhibitors Whose Potencies and Selectivities Are Dramatically Enhanced by Zinc(II)

Biochemistry Usa, 2000

Serine proteases play a role in a variety of disease states and thus are attractive targets for t... more Serine proteases play a role in a variety of disease states and thus are attractive targets for therapeutic intervention. We report the kinetic characterization of a class of serine protease inhibitors whose potencies and selectivities are dramatically enhanced in the presence of Zn(II). The structural basis for Zn(II)-mediated inhibition of trypsin-like proteases has recently been reported [Katz, B. A., Clark, J. M., Finer-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Moore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612]. A case study of the kinetic behavior of human tryptase inhibitors is provided to illustrate the general phenomenon of Zn(II)-mediated inhibition. Tryptase, Zn(II), and the inhibitor form a ternary complex which exhibits classic tight-binding inhibition. The half-life for release of inhibitor from the tryptase-Zn(II)-inhibitor complex has been measured for a number of inhibitors. Consistent with tight-binding behavior, potent tryptase inhibitors are characterized by extremely slow rates of dissociation from the ternary complex with half-lives on the order of hours. A model of human serum, designed to reproduce physiological levels of Zn(II), has been employed to evaluate the performance of Zn(II)-potentiated tryptase inhibitors under physiological conditions. We demonstrate that Zn(II)-mediated inhibition can be achieved at physiological Zn(II) levels.

Research paper thumbnail of Expression and characterization of recombinant gamma-tryptase

Protein Expression and Purification, Oct 1, 2006

Research paper thumbnail of The Biosynthetic Gene Cluster for a Monocyclic �-Lactam Antibiotic, Nocardicin A

Research paper thumbnail of Expression and characterization of recombinant ?-tryptase

Protein Express Purif, 2006

Research paper thumbnail of Cathepsin cysteine protease inhibitors

Research paper thumbnail of Small molecule-mediated inhibition of Factor VIIa catalytic activity blocks fVIIa:TF cellular signaling and chemokine secretion in human tumor cell lines

In addition to its role in coagulation, Factor VIIa (fVIIa) binding to TF has been demonstrated t... more In addition to its role in coagulation, Factor VIIa (fVIIa) binding to TF has been demonstrated to activate a variety of intracellular signaling pathways including MAPK, AKT, and Src. This activity has been shown to require TF binding as well as fVIIa protease activity. TF overexpression in tumor epithelial cells and tumor vasculature has been linked to prognosis. However a precise role for fVIIa:TF signaling in tumor cell growth and survival has not yet been established. We have demonstrated that fVIIa is able to activate MAPK, AKT, and early-response gene induction in the BxPC3 pancreatic human tumor cell line at physiologic (0.1-10nM) concentrations. TF expression in the tumor cell line is required for this intracellular signaling. The chemically-inactivated form of fVIIa, fVIIai, is unable to activate these signaling pathways. In addition, preincubation of fVIIa with potent, fVIIa-selective, reversible small molecule inhibitors of fVIIa protease activity inhibited the activation...

Research paper thumbnail of Efficacy of telavancin, a lipoglycopeptide antibiotic, in experimental models of Gram-positive infection

Expert review of anti-infective therapy, 2014

Telavancin is a parenteral lipoglycopeptide antibiotic with a dual mechanism of action contributi... more Telavancin is a parenteral lipoglycopeptide antibiotic with a dual mechanism of action contributing to bactericidal activity against multidrug-resistant Gram-positive pathogens. It has been approved for the treatment of complicated skin and skin structure infections due to susceptible Gram-positive bacteria and hospital-acquired/ventilator-associated bacterial pneumonia due to Staphylococcus aureus when other alternatives are unsuitable. Telavancin has been demonstrated to be efficacious in multiple animal models of soft tissue, cardiac, systemic, lung, bone, brain and device-associated infections involving clinically relevant Gram-positive pathogens, including methicillin-resistant S. aureus, glycopeptide-intermediate S. aureus, heterogeneous vancomycin-intermediate S. aureus and daptomycin non-susceptible methicillin-resistant S. aureus. The AUC0-24h/MIC ratio is the primary pharmacodynamically-linked pharmacokinetic parameter. The preclinical data for telavancin supports further ...

Research paper thumbnail of Expression and characterization of recombinant γ-tryptase

Protein Expression and Purification, 2006

Tryptases are trypsin-like serine proteases whose expression is restricted to cells of hematopoie... more Tryptases are trypsin-like serine proteases whose expression is restricted to cells of hematopoietic origin, notably mast cells. γ-Tryptase, a recently described member of the family also known as transmembrane tryptase (TMT), is a membrane-bound serine protease found in the secretory granules or on the surface of degranulated mast cells. The 321 amino acid protein contains an 18 amino acid propeptide

Research paper thumbnail of Impact of telavancin on prothrombin time and activated partial thromboplastin time as determined using point-of-care coagulometers

Journal of Thrombosis and Thrombolysis, 2013

Telavancin is approved in the United States, Canada, and Europe (At the time of submission, the t... more Telavancin is approved in the United States, Canada, and Europe (At the time of submission, the telavancin European marketing authorization for nosocomial pneumonia was suspended until Theravance provides evidence of a new European Medicines Agency approved supplier) as an antibiotic to treat certain Gram-positive bacterial skin infections. Telavancin has been shown to prolong plasmatic prothrombin (PT) and activated partial thromboplastin (aPTT) clotting times in clinical diagnostic lab-based assays. In this study, we evaluated the potential for telavancin to prolong whole blood PT/International Normalized Ratio (INR) and aPTT tests on point-of-care (POC) instruments. Whole blood collected from 8 healthy subjects was supplemented with telavancin to final concentrations of 0, 10, 20, and 100 μg/ml. Final concentrations were selected to match trough, twice trough, and peak plasma levels following the approved 10 mg/kg dose. Four widely employed POC coagulation instruments were chosen to be representative of the POC platforms currently in use.. These systems were the Roche Coaguchek XS, the Abbott iSTAT, the ITC Hemochron SIG+, and the Alere INRatio2 POC devices. The PT/INR measured by the Coaguchek XS showed the greatest sensitivity to the presence of telavancin. The PT/INR measured by the Hemochron SIG+ and iSTAT were sensitive to telavancin but to a lesser extent. The INRatio2 was the least sensitive to the presence of telavancin when testing the whole blood PT/INR. Only the Hemochron SIG+ device was capable of measuring aPTT and showed a concentration-dependent increase in aPTT. This study supports the current recommendation that PT and aPTT monitoring be conducted immediately to the next dose of telavancin when coagulation parameters are tested using POC instrumentation.

Research paper thumbnail of Practical Robust Fit of Enzyme Inhibition Data

Methods in Enzymology, 2004

Research paper thumbnail of Contribution of Multicentered Short Hydrogen Bond Arrays to Potency of Active Site-Directed Serine Protease Inhibitors

Journal of the American Chemical Society, 2002

We describe and compare the pH dependencies of the potencies and of the bound structures of two i... more We describe and compare the pH dependencies of the potencies and of the bound structures of two inhibitor isosteres that form multicentered short hydrogen bond arrays at the active sites of trypsin, thrombin, and urokinase type plasminogen activator (urokinase or uPA) over certain ranges of pH. Depending on the pH, short hydrogen bond arrays at the active site are mediated by two waters, one in the oxyanion hole (H(2)O(oxy)) and one on the other (S2) side of the inhibitor (H(2)O(S2)), by one water (H(2)O(oxy)), or by no water. The dramatic variation in the length of the active site hydrogen bonds as a function of pH, of inhibitor, and of enzyme, along with the involvement or absence of ordered water, produces a large structural manifold of active site hydrogen bond motifs. Diverse examples of multicentered and two-centered short hydrogen bond arrays, both at and away from the active site, recently discovered in several protein crystal systems, suggest that short hydrogen bonds in proteins may be more common than has been recognized. The short hydrogen bond arrays resemble one another with respect to ionic nature, highly polar environment, multitude of associated ordinary hydrogen bonds, and disparate pK(a) values of participating groups. Comparison of structures and K(i) values of trypsin complexes at pH values where the multicentered short hydrogen bond arrays mediating inhibitor binding are present or absent indicate that these arrays have a minor effect on inhibitor potency. These features suggest little covalent nature within the short hydrogen bonds, despite their extraordinary shortness (as short as 2.0 A).

Research paper thumbnail of Elaborate Manifold of Short Hydrogen Bond Arrays Mediating Binding of Active Site-directed Serine Protease Inhibitors

Journal of Molecular Biology, 2003

An extensive structural manifold of short hydrogen bond-mediated, active site-directed, serine pr... more An extensive structural manifold of short hydrogen bond-mediated, active site-directed, serine protease inhibition motifs is revealed in a set of over 300 crystal structures involving a large suite of small molecule inhibitors (2-(2-phenol)-indoles and 2-(2-phenol)-benzimidazoles) determined over a wide range of pH (3.5-11.4). The active site hydrogen-bonding mode was found to vary markedly with pH, with the steric and electronic properties of the inhibitor, and with the type of protease (trypsin, thrombin or urokinase type plasminogen activator (uPA)). The pH dependence of the active site hydrogen-bonding motif is often intricate, constituting a distinct fingerprint of each complex. Isosteric replacements or minor substitutions within the inhibitor that modulate the pK(a) of the phenol hydroxyl involved in short hydrogen bonding, or that affect steric interactions distal to the active site, can significantly shift the pH-dependent structural profile characteristic of the parent scaffold, or produce active site-binding motifs unique to the bound analog. Ionization equilibria at the active site associated with inhibitor binding are probed in a series of the protease-inhibitor complexes through analysis of the pH dependence of the structure and environment of the active site-binding groups involved in short hydrogen bond arrays. Structures determined at high pH (>11), suggest that the pK(a) of His57 is dramatically elevated, to a value as high as approximately 11 in certain complexes. K(i) values involving uPA and trypsin determined as a function of pH for a set of inhibitors show pronounced parabolic pH dependence, the pH for optimal inhibition governed by the pK(a) of the inhibitor phenol involved in short hydrogen bonds. Comparison of structures of trypsin, thrombin and uPA, each bound by the same inhibitor, highlights important structural variations in the S1 and active sites accessible for engineering notable selectivity into remarkably small molecules with low nanomolar K(i) values.

Research paper thumbnail of Dissecting and Designing Inhibitor Selectivity Determinants at the S1 Site Using an Artificial Ala190 Protease (Ala190 uPA)

Journal of Molecular Biology, 2004

A site-directed mutant of the serine protease urokinase-type plasminogen activator (uPA), was pro... more A site-directed mutant of the serine protease urokinase-type plasminogen activator (uPA), was produced to assess the contribution of the Ser190 side-chain to the affinity and selectivity of lead uPA inhibitors in the absence of other differences present in comparisons of natural proteases. Crystallography and enzymology involving WT and Ala190 uPA were used to calculate free energy binding contributions of hydrogen bonds involving the Ser190 hydroxyl group (O(gamma)(Ser190)) responsible for the remarkable selectivity of 6-halo-5-amidinoindole and 6-halo-5-amidinobenzimidazole inhibitors toward uPA and against natural Ala190 protease anti-targets. Crystal structures of uPA complexes of novel, active site-directed arylguanidine and 2-aminobenzimidazole inhibitors of WT uPA, together with associated K(i) values for WT and Ala190 uPA, also indicate a significant role of Ser190 in the binding of these classes of uPA inhibitors. Structures and associated K(i) values for a lead inhibitor (CA-11) bound to uPA and to five other proteases, as well as for other leads bound to multiple proteases, help reveal the features responsible for the potency (K(i)=11nM) and selectivity of the remarkably small inhibitor, CA-11. The 6-fluoro-5-amidinobenzimidzole, CA-11, is more than 1000-fold selective against natural Ala190 protease anti-targets, and more than 100-fold selective against other Ser190 anti-targets.

Research paper thumbnail of A novel serine protease inhibition motif involving a multi-centered short hydrogen bonding network at the active site

Journal of Molecular Biology, 2001

Research paper thumbnail of Optimization of Subsite Binding to the β5 Subunit of the Human 20S Proteasome Using Vinyl Sulfones and 2-Keto-1,3,4-oxadiazoles:  Syntheses and Cellular Properties of Potent, Selective Proteasome Inhibitors

Journal of Medicinal Chemistry, 2006

Beginning with the peptide sequence Cbz-Ile-Glu(OtBu)-Ala-Leu found in PSI (3), a series of vinyl... more Beginning with the peptide sequence Cbz-Ile-Glu(OtBu)-Ala-Leu found in PSI (3), a series of vinyl sulfones (VS) were synthesized for evaluation as inhibitors of the chymotrypsin-like activity of the 20S proteasome. Variations at the key P3 position confirmed the importance of a long side chain capped with a hydrophobic group for optimal potency, consistent with a model of binding to the S3 subsite. The tert-butyl glutamic ester initially used at P3 gave plasma unstable, insoluble compounds and was replaced with the better isostere, N-beta-neopentyl asparagine. The inhibitors were shortened by replacing the N-terminal Cbz-isoleucine with a p-tosyl group without loss of potency. Small l-amino acids were used at P2, where d-substitution was not tolerated. The resulting optimized P4-P3-P2 sequence was grafted onto a novel proteasome inhibitor warhead, 2-keto-1,3,4-oxadiazoles (KOD), to produce reversible, subnanomolar proteasome inhibitors that were 1000-fold selective versus cathepsin B (CatB), cathepsin S (CatS), and trypsin-like as well as PGPH-like proteasome activity. A number of compounds in both the VS and the KOD series exhibited growth inhibitory effects against the human prostate cancer cell line PC3 at submicromolar concentrations.

Research paper thumbnail of O258 Telavancin inhibits peptidoglycan biosynthesis through preferential targeting of transglycosylation: evidence for a multivalent interaction between telavancin and lipid II

International Journal of Antimicrobial Agents, 2007

Objectives: Alveolar echinococcosis is one of the most pathogenic parasitic zoonoses in central E... more Objectives: Alveolar echinococcosis is one of the most pathogenic parasitic zoonoses in central Europe, caused after oral uptake of eggs of Echinococcus multilocularis, shed by infected foxes. At present, there seems to be a trend towards increased parasite density in central Europe and spread in western Europe, although it cannot be decided whether E. multilocularis recently has extended its habitat or whether the parasite has simply remained undetected until now in marginal regions. We analysed red fox data from an area close to the westernmost margin of E. multilocularis habitat in Europe, Belgium and a neighbouring province (Limburg) in The Netherlands (NL), with the aim of studying the emergence of the parasite in this area. Methods: A total of 1202 foxes has been analysed (1018 in Belgium, 184 in NL) with 179 infected (164 in Belgium, 15 in NL) using mucosal scrapings. Spatial coordinates of the locations of infected and uninfected foxes have been determined by GPS. In addition, additional data in southern Europe were analysed using GIS and modeling to study changes in distrinution and prevalence. Results: The large scale spatial distribution of the prevalence of infection among sampled foxes has been modeled as an ellipsoidal gradient, demonstrating increasing prevalence in southeastern direction that means towards the endemic area in central Europe. Using this gradient, the spatial pattern of E. multilocularis infection in Belgium and a contiguous region in NL could be shown to have a continuous distribution across national borders. Part of the Belgian data allowed investigation of the temporal changes in spatial distribution of E. multilocularis, revealing spreading into northwestern direction. A mathematical model describing the parasite population dynamics both in time and in space was fitted to the worm burdens of foxes sampled between 1996 and 2006 in NL. We found a strong indication that the parasite's reproduction number R 0 is greater than 1 and that the parasite is spreading to a wider region in Limburg. Based on the R 0 derived from the mathematical model of the parasite's transmission, we explore the effect of public health measures aimed to eradicate the infection. Conclusion: Increased infection pressure of E. multilocularis in northwestern Europe is most likely to occur at present. In Belgium human alveolar echinococcosis had been absent in the past, but 3 human cases have been reported in 2004.