Publications (79)236.87 Total impact
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Article: Structure and function of S-adenosylhomocysteine hydrolase
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ABSTRACT: In mammals, S-adenosylhomocysteine hydrolase (AdoHcyase) is the only known enzyme to catalyze the breakdown of S-adenosylhomocysteine (AdoHcy) to homocysteine and adenosine. AdoHcy is the product of all adenosylmethionine (AdoMet)-dependent biological transmethylations. These reactions have a wide range of products, and are common in all facets of biometabolism. As a product inhibitor, elevated levels of AdoHcy suppress AdoMet-dependent transmethylations. Thus, AdoHcyase is a regulator of biological transmethylation in general. The three-dimensional structure of AdoHcyase complexed with reduced nicotinamide adenine dinucleotide phosphate (NADH) and the inhibitor (1′R, 2′S, 3′R)-9-(2′,3′-dihyroxycyclopenten-1-yl)adenine (DHCeA) was solved by a combination of the crystallographic direct methods program, SnB, to determine the selenium atom substructure and by treating the multiwavelength anomalous diffraction data as a special case of multiple isomorphous replacement. The enzyme architecture resembles that observed for NAD-dependent dehydrogenases, with the catalytic domain and the cofactor binding domain each containing a modified Rossmann fold. The two domains form a deep active site cleft containing the cofactor and bound inhibitor molecule. A comparison of the inhibitor complex of the human enzyme and the structure of the rat enzyme, solved without inhibitor, suggests that a 17° rigid body movement of the catalytic domain occurs upon inhibitor/substrate binding.Cell Biochemistry and Biophysics 04/2012; 33(2):101-125. · 3.74 Impact Factor -
Article: Factors that restrict the cell permeation of cyclic prodrugs of an opioid peptide, part 3: Synthesis of analogs designed to have improved stability to oxidative metabolism.
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ABSTRACT: Previously, our laboratory reported that cyclic peptide prodrugs of the opioid peptide H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE) are metabolized by cytochrome P450 (CYP450) enzymes, which limits their systemic exposure after oral dosing to animals. In an attempt to design more metabolically stable cyclic prodrugs of DADLE, we synthesized analogs of DADLE cyclized with a coumarinic acid linker (CA; CA-DADLE), which contained modifications in the amino acid residues known to be susceptible to CYP450 oxidation. Metabolic stability and metabolite identification studies of CA-DADLE and its analogs were then compared using rat liver microsomes (RLM), guinea pig liver microsomes (GPLM), and human liver microsomes (HLM), as well as recombinant human recombinant cytochrome P450 3A4 (hCYP3A4). Similar to the results observed for CA-DADLE, incubation of its analogs with RLM, GPLM, and HLM resulted in monohydroxylation of an amino acid side chain on these cyclic prodrugs. When CA-DADLE was incubated with hCYP3A4, similar oxidative metabolism of the peptide was observed. In contrast, incubation of the CA-DADLE analogs with hCYP3A4 showed that these amino-acid-modified analogs are not substrates for this CYP450 isozyme. These results suggest that the amino-acid-modified analogs of CA-DADLE prepared in this study could be stable to metabolic oxidation by CYP3A4 expressed in human intestinal mucosal cells.Journal of Pharmaceutical Sciences 03/2012; 101(9):3486-99. · 3.06 Impact Factor -
Article: Factors that restrict the cell permeation of cyclic prodrugs of an opioid peptide, part 4: Characterization of the biopharmaceutical and physicochemical properties of two new cyclic prodrugs designed to be stable to oxidative metabolism by cytochrome P-450 enzymes in the intestinal mucosa.
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ABSTRACT: The biopharmaceutical and physicochemical properties of two new cyclic prodrugs (CA-[cychexalanine (Cha(4)), D-Leu(5) ]-Enkephalin (Enk) and coumarinic acid (CA)-[Cha(4), D-Ala(5)]-Enk) of opioid peptides that were designed to be stable to oxidative metabolism by cytochrome P-450 enzymes in the intestinal mucosa are described in this paper. Two-dimensional nuclear magnetic resonance studies and molecular dynamics simulations showed that these cyclic prodrugs exhibit unique solution conformations (i.e., type I β-turns), which are favorable for transcellular permeation. The calculated molecular surface areas and cLog P values confirmed that these new cyclic prodrugs are more lipophilic than linear opioid peptides and, thus, they should exhibit better transcellular permeation characteristics. However, Caco-2 cell permeation studies showed that the cyclic prodrugs were substrates for apically polarized efflux transporters (e.g., P-glycoprotein, which significantly limited their transcellular permeation). Permeability studies using an in situ rat intestinal perfusion model confirmed the poor intestinal permeation characteristics of CA-[Cha(4), D-Leu(5) ]-Enk and CA-[Cha(4), D-Ala(5)]-Enk as well as the stability of these two new cyclic prodrugs of opioid peptides to oxidative metabolism. In conclusion, these data clearly show that oral absorption of cyclic prodrugs of opioid peptides can only be achieved by designing molecules devoid of substrate activity for both cytochrome P-450 enzymes and efflux transporters in the intestinal mucosa.Journal of Pharmaceutical Sciences 02/2012; 101(9):3500-10. · 3.06 Impact Factor -
Article: Hidalgo, I. J., Raub, T. J., and Borchardt, R. T.: Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability, Gastroenterology, 96, 736-749, 1989--the backstory.
The AAPS Journal 05/2011; 13(3):323-7. · 5.09 Impact Factor -
Article: Comparative kinetics of cofactor association and dissociation for the human and trypanosomal S-adenosylhomocysteine hydrolases. 3. Role of lysyl and tyrosyl residues of the C-terminal extension.
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ABSTRACT: On the basis of the available X-ray structures of S-adenosylhomocysteine hydrolases (SAHHs), free energy simulations employing the MM-GBSA approach were applied to predict residues important to the differential cofactor binding properties of human and trypanosomal SAHHs (Hs-SAHH and Tc-SAHH), within 5 Å of the cofactor NAD(+)/NADH binding site. Among the 38 residues in this region, only four are different between the two enzymes. Surprisingly, the four nonidentical residues make no major contribution to differential cofactor binding between Hs-SAHH and Tc-SAHH. On the other hand, four pairs of identical residues are shown by free energy simulations to differentiate cofactor binding between Hs-SAHH and Tc-SAHH. Experimental mutagenesis was performed to test these predictions for a lysine residue and a tyrosine residue of the C-terminal extension that penetrates a partner subunit to form part of the cofactor binding site. The K431A mutant of Tc-SAHH (TcK431A) loses its cofactor binding affinity but retains the wild type's tetrameric structure, while the corresponding mutant of Hs-SAHH (HsK426A) loses both cofactor affinity and tetrameric structure [Ault-Riche, D. B., et al. (1994) J. Biol. Chem. 269, 31472-31478]. The tyrosine mutants HsY430A and TcY435A alter the NAD(+) association and dissociation kinetics, with HsY430A increasing the cofactor equilibrium dissociation constant from approximately 10 nM (Hs-SAHH) to ∼800 nM and TcY435A increasing the cofactor equilibrium dissociation constant from approximately 100 nM (Tc-SAHH) to ∼1 mM. Both changes result from larger increases in the off rate combined with smaller decreases in the on rate. These investigations demonstrate that computational free energy decomposition may be used to guide experimental studies by suggesting sensitive sites for mutagenesis. Our finding that identical residues in two orthologous proteins may give significantly different binding free energy contributions strongly suggests that comparative studies of homologous proteins should investigate not only different residues but also identical residues in these proteins.Biochemistry 09/2010; 49(38):8434-41. · 3.42 Impact Factor -
Article: Absorption barriers in the rat intestinal mucosa: 2. Application of physiologically based mathematical models to quantify mechanisms of drug permeation and metabolism.
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ABSTRACT: The absorption of drug molecules is often investigated using in vitro or in situ models of the intestinal mucosa; however, few studies have quantified the kinetics that limit absorption. The objective of this study was to quantify kinetic rates of rat intestinal absorption, metabolism, and efflux using nonlinear mixed effects modeling. A multicompartment model accurately described the absorption and distribution of atenolol and verapamil as well as the metabolism of verapamil and distribution of the metabolite, norverapamil. The accurate description of atenolol data required inclusion of an intermediate compartment in addition to paracellular clearance, whereas verapamil and norverapamil were modeled in the absence of paracellular clearance. The absorption of verapamil was well characterized by linear kinetics, whereas the formation and distribution of norverapamil were well characterized by Michaelis-Menten kinetics. The model identified EDTA as a modulator of physical barriers, ketoconazole as an inhibitor of cytochrome P450 3A and P-glycoprotein (P-gp), and PSC-833 and GF-120918 as specific P-gp inhibitors. These results demonstrate the utility of a physiologically based model to characterize (i) the drug distribution across the in situ perfused rat intestine and (ii) the effect of chemical modulators in this biological system.Journal of Pharmaceutical Sciences 10/2009; 99(2):999-1015. · 3.06 Impact Factor -
Article: Absorption barriers in the rat intestinal mucosa: 1. Application of an in situ perfusion model to simultaneously assess drug permeation and metabolism.
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ABSTRACT: Modulation of intestinal drug absorption barriers can have a profound impact on the bioavailability of orally administered compounds. With its commonality of use as an absorption model, it is valuable to assess the role of such barriers in the rat intestinal mucosa. In the present study, atenolol and verapamil were concomitantly delivered in the in situ perfused rat intestine in the presence or absence of inhibitors to simultaneously assess the function and modulation of passive diffusion barriers, cytochrome P450 (CYP)3A metabolism and P-glycoprotein (P-gp) efflux. A high performance liquid chromatography-tandem mass spectrometry method measured atenolol, verapamil and the CYP3A-mediated metabolite, norverapamil, with linearity (r(2) > 0.99), precision (CV <or=7.5%) and accuracy (+/-17%). Absorption of parent drug was independent of verapamil concentration; however the formation and disposition of norverapamil were concentration-dependent and saturable. Norverapamil formation decreased (up to 80%) in the presence of CYP3A inhibitors and the fraction of norverapamil observed in the plasma was increased (4.5- to 7.2-fold) in the presence of P-gp inhibitors. These results suggest that in this model of the rat intestinal mucosa, atenolol serves as a marker for diffusion barriers whereas norverapamil formation and disposition are markers of CYP3A and P-gp, respectively.Journal of Pharmaceutical Sciences 09/2009; 99(2):982-98. · 3.06 Impact Factor -
Article: Absorption barriers in the rat intestinal mucosa. 3: Effects of polyethoxylated solubilizing agents on drug permeation and metabolism.
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ABSTRACT: Modern drug discovery chemical libraries contain a large number of molecular entities exhibiting low aqueous solubility, often necessitating the inclusion of solubilizing agents in preclinical models of absorption or metabolism. The objective of the present study was to investigate the effects of several commonly used polyethoxylated solubilizing agents on P450 (CYP) 3A and P-glycoprotein (P-gp) in the rat intestinal mucosa. Atenolol and verapamil were administered in the in situ perfused rat intestine or incubated with rat intestinal microsomes in the presence or absence of polyethylene glycol (PEG) 400 (2% or 20%, v/v) D-alpha-tocopheryl polyethylene glycol-1000 succinate (TPGS; 100 microg/mL), Cremophor EL (47.5 microg/mL) or polysorbate (Tween) 80 (25 microg/mL). Effects on the absorption of unchanged drug were minimal, with the exception of Tween 80 which caused a 5.0-fold increase in paracellular absorption. Rat intestinal CYP3A was significantly inhibited by PEG-400 and in situ, exceeded inhibition observed with ketoconazole. Cremophor and TPGS increased the fraction of norverapamil in the plasma, consistent with excipient-mediated inhibition of P-gp. These results suggest that caution be exercised when these solubilizing agents are included in preclinical oral dosing solutions as the perturbation of drug absorption barriers may heighten the risk of incorrectly classifying drug candidate PK-parameters.Journal of Pharmaceutical Sciences 08/2009; 99(2):1016-27. · 3.06 Impact Factor -
Article: Evaluation of NAD(H) analogues as selective inhibitors for Trypanosoma cruzi S-adenosylhomocysteine hydrolase.
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ABSTRACT: S-Adenosylhomocysteine (AdoHcy) hydrolases (SAHHs) from human sources (Hs-SAHHs) bind the cofactor NAD(+) more tightly than several parasitic SAHHs by around 1000-fold. This property suggests the cofactor binding site of this essential enzyme as a potential anti-parasitic drug target, e.g., against SAHH from Trypansoma cruzi (Tc-SAHH). The on-rate and off-rate constants and the equilibrium dissociation constants were determined for NAD(+)/NADH analogues and suggested that NADH analogues were the most promising for selective inhibition of Tc-SAHH. None significantly inhibited Hs-SAHH while S-NADH and H-NADH (see Figure 1) reduced the catalytic activity of Tc-SAHH to < 10% in six minutes of exposure.Nucleosides Nucleotides & Nucleic Acids 05/2009; 28(5):473-84. · 0.90 Impact Factor -
Article: The rationale for targeting the NAD/NADH cofactor binding site of parasitic S-adenosyl-L-homocysteine hydrolase for the design of anti-parasitic drugs.
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ABSTRACT: Trypanosomal S-adenoyl-L-homocysteine hydrolase (Tc-SAHH), considered as a target for treatment of Chagas disease, has the same catalytic mechanism as human SAHH (Hs-SAHH) and both enzymes have very similar x-ray structures. Efforts toward the design of selective inhibitors against Tc-SAHH targeting the substrate binding site have not to date shown any significant promise. Systematic kinetic and thermodynamic studies on association and dissociation of cofactor NAD/H for Tc-SAHH and Hs-SAHH provide a rationale for the design of anti-parasitic drugs directed toward cofactor-binding sites. Analogues of NAD and their reduced forms show significant selective inactivation of Tc-SAHH, confirming that this design approach is rational.Nucleosides Nucleotides & Nucleic Acids 05/2009; 28(5):485-503. · 0.90 Impact Factor -
Article: A comparison of the effects of p-glycoprotein inhibitors on the blood-brain barrier permeation of cyclic prodrugs of an opioid peptide (DADLE).
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ABSTRACT: The objective of this study was to elucidate the role of P-glycoprotein (P-gp) in restricting the blood-brain barrier (BBB) permeation of cyclic prodrugs of the opioid peptide DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). The BBB permeation characteristics of these prodrugs and DADLE were determined using an in situ perfused rat brain model and in vitro cell culture model (MDCK-MDR1 cells) of the BBB. The activities of P-gp in these models were characterized using a known substrate (quinidine) and known inhibitors [cyclosporine A (CyA), GF-120918, PSC-833] of P-gp. Cyclic peptide prodrugs exhibited very poor permeation in both models. Inclusion of GF-120918, CyA, or PSC-833 in the brain perfusion medium or the cell culture medium significantly increased the permeation of these cyclic prodrugs. The order of potency of these P-gp inhibitors, as measured using the cyclic prodrugs as substrates, was, by in vitro MDCK-MDR1 cells: GF-120918 = CyA >or= PSC-833; and by in situ rat brain perfusion: GF-120918 > CyA = PSC-833. In conclusion, P-gp in the BBB is the major factor restricting the brain permeation of these cyclic prodrugs. MDCK-MDR1 cells can predict the order of potencies of the investigated P-gp inhibitors to enhance the rat BBB permeation of quinidine and the cyclic prodrugs.Journal of Pharmaceutical Sciences 10/2008; 98(6):2227-36. · 3.06 Impact Factor -
Article: Factors that restrict intestinal cell permeation of cyclic prodrugs of an opioid peptide (DADLE): Part II. Role of metabolic enzymes in the intestinal mucosa.
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ABSTRACT: The objective of this study was to determine the relative importance of metabolism by cytochrome P450 (CYP) enzymes versus efflux by P-glycoprotein (P-gp) in restricting the intestinal mucosal permeation of cyclic prodrugs (AOA-DADLE, CA-DADLE, OMCA-DADLE) of the opioid peptide DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). AOA-DADLE, CA-DADLE, and OMCA-DADLE were shown to be rapidly metabolized by rat liver microsomes and human CYP-3A4 and to a lesser extent by esterases. Using an in situ perfused rat ileum model, ketoconazole, a CYP 3A inhibitor, was shown to have no effect (AOA-DADLE) or a slight enhancing effect (OMCA-DADLE, twofold; CA-DADLE, threefold) on their intestinal mucosal permeation. In contrast, inclusion of PSC-833, a P-gp inhibitor, in the perfusate significantly enhanced (7-16-fold) the permeation of the three cyclic prodrugs. Since PSC-833 was found to be a weak inhibitor of CYP 3A4 and to have no inhibitory effects on esterases, phenol sulfotransferases, and glucuronyltransferases, it is suggested PSC-833 enhances intestinal mucosal permeation of these cyclic prodrugs by inhibiting their polarized efflux and not by inhibiting their metabolism. Furthermore, efflux transporters (e.g., P-gp), not metabolic enzymes (e.g., CYP 3A, esterases), restrict the permeation of peptide prodrugs across the rat intestinal mucosa.Journal of Pharmaceutical Sciences 07/2008; 98(1):349-61. · 3.06 Impact Factor -
Article: Factors that restrict the intestinal cell permeation of cyclic prodrugs of an opioid peptide (DADLE): Part I. Role of efflux transporters in the intestinal mucosa.
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ABSTRACT: The objective of this study was to elucidate the role of P-glycoprotein (P-gp) in restricting the intestinal mucosal permeation of cyclic prodrugs (AOA-DADLE, CA-DADLE, and OMCA-DADLE) of the opioid peptide DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). In the Caco-2 cell model, the high P(app,BL-to-AP)/P(app,AP-to-BL) ratios of AOA-DADLE, CA-DADLE, and OMCA-DADLE (71-117) were significantly decreased by including known P-gp inhibitors, GF-12098, cyclosporine (CyA), or PSC-833, in the incubation media, suggesting that P-gp is restricting the AP-to-BL permeation of these cyclic prodrugs. In the in situ perfused rat ileum model, AOA-DADLE, CA-DADLE, and OMCA-DADLE were shown to exhibit very low permeation into the mesenteric blood (P(B) = 0.40, 0.56 and 0.42 x 10(-7) cm/s, respectively). PSC-833 was found to increase significantly the P(B) values for all three prodrugs. In contrast, CyA and GF-12918 were either inactive or substantially less active than PSC-833 in increasing the P(B) values of these prodrugs. These data suggest that, while P-gp plays a role, other factors (e.g., substrate activity for other efflux transporters and/or for metabolic enzymes) may contribute to restricting the permeation of AOA-DADLE, CA-DADLE, and OMCA-DADLE across the rat intestinal mucosa.Journal of Pharmaceutical Sciences 07/2008; 98(1):337-48. · 3.06 Impact Factor -
Article: Synthesis of 5'-functionalized nucleosides: S-Adenosylhomocysteine analogues with the carbon-5' and sulfur atoms replaced by a vinyl or halovinyl unit.
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ABSTRACT: Adenosine and uridine analogues functionalized with alkenyl or fluoroalkenyl chain at C5' were prepared employing cross-metathesis, Negishi couplings, and Wittig reactions. Metathesis of the protected 5'-deoxy-5'-methyleneadenosine or uridine analogues with six-carbon amino acids (homoallylglycines) in the presence of Grubbs catalysts gave nucleoside analogues with the C5'-C6' double bond. Alternatively, the Pd-catalyzed cross-coupling between the protected 5'-deoxy-5'-(iodomethylene) nucleosides and suitable alkylzinc bromides also provided analogues with alkenyl unit. Stereoselective Pd-catalyzed monoalkylation of 5'-(bromofluoromethylene)-5'-deoxyadenosine with alkylzinc bromides afforded adenosylhomocysteine analogues with a 6'-(fluoro)vinyl motif. The vinylic adenine nucleosides produced time-dependent inactivation of the S-adenosyl-l-homocysteine hydrolases.Bioorganic & medicinal chemistry 06/2008; 16(10):5424-33. · 2.82 Impact Factor -
Article: Molecular dynamics simulations of domain motions of substrate-free S-adenosyl- L-homocysteine hydrolase in solution.
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ABSTRACT: S-Adenosyl-L-homocysteine hydrolase (SAHH) is an enzyme regulating intracellular methylation reactions. The homotetrameric SAHH exists in an open conformation in absence of substrate, while enzyme:inhibitor complexes crystallize in the closed conformation, in which the ligands are engulfed by the protein due to an 18 degrees domain reorientation within each of the four subunits. We present a microscopic description of the structure and dynamics of the substrate-free, NAD(+)-bound SAHH in solution, based on a 15-ns molecular dynamics simulation in explicit solvent. In the trajectory, the four cofactor-binding domains formed a relatively rigid core with structure very similar to the crystal conformation. The four substrate-binding domains, located at the protein exterior, also retained internal structures similar to the crystal, while undergoing large amplitude rigid-body reorientations. The trajectory domain motions exhibited two interesting properties. First, within each subunit the domains fluctuated between open and closed conformations, while at the tetramer level 80% of the domain motions were perpendicular to the direction of the open-to-closed structural transition. Second, the domain reorientations in solution could be represented as a sum of two components, faster, with 20-50 ps correlation time and 3-4 degrees amplitude, and slower, with 8-23 ns correlation time and amplitude of 14-22 degrees . The faster motion is similar to the 1.5 cm(-1) frequency hinge-bending vibrations found in our recent normal mode analysis (Wang et al., Biochemistry 2005;44:7228-7239). The slower motion agrees with fluorescence anisotropy decay measurements, which detected a 10-20 ns domain reorientation of ca. 26 degrees amplitude in the substrate-free enzyme (Wang et al., Biochemistry 2006;45:7778-7786). Our simulations are thus in excellent agreement with experimental data. The simulations allow us to assign the observed nanosecond fluorescence anisotropy signal to fluctuations in domain orientations, and indicate that the microscopic mechanism of the motion involves rotational diffusion within a cone of 10-20 degrees . Overall, our simulation results complement the existing experimental data and provide important new insights into SAHH domain motions in solution, which play a crucial role in the catalytic mechanism of SAHH.Proteins Structure Function and Bioinformatics 05/2008; 71(1):131-43. · 3.39 Impact Factor -
Article: Comparative kinetics of cofactor association and dissociation for the human and trypanosomal S-adenosylhomocysteine hydrolases. 2. The role of helix 18 stability.
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ABSTRACT: The S-adenosyl- l-homocysteine (AdoHcy) hydrolases (SAHH) from Homo sapiens (Hs-SAHH) and from the parasite Trypanosoma cruzi (Tc-SAHH) are very similar in structure and catalytic properties but differ in the kinetics and thermodynamics of association and dissociation of the cofactor NAD (+). The binding of NAD (+) and NADH in SAHH appears structurally to be mediated by helix 18, formed by seven residues near the C-terminus of the adjacent subunit. Helix-propensity estimates indicate decreasing stability of helix 18 in the order Hs-SAHH > Tc-SAHH > Ld-SAHH (from Leishmania donovani) > Pf-SAHH (from Plasmodium falciparum), which would be consistent with the previous observations. Here we report the properties of Hs-18Pf-SAHH, the human enzyme with plasmodial helix 18, and Tc-18Hs-SAHH, the trypanosomal enzyme with human helix 18. Hs-18Tc-SAHH, the human enzyme with trypanosomal helix 18, was also prepared but differed insignificantly from Hs-SAHH. Association of NAD (+) with Hs-SAHH, Hs-18Pf-SAHH, Tc-18Hs-SAHH, and Tc-SAHH exhibited biphasic kinetics for all enzymes. A thermal maximum in rate, attributed to the onset of local structural alterations in or near the binding site, occurred at 35, 33, 30, and 15 degrees C, respectively. This order is consistent with some reversible changes within helix 18 but does require influence of other properties of the "host enzyme". Dissociation of NAD (+) from the same series of enzymes also exhibited biphasic kinetics with a transition to faster rates (a larger entropy of activation more than compensates for a larger enthalpy of activation) at temperatures of 41, 38, 36, and 29 degrees C, respectively. This order is also consistent with changes in helix 18 but again requiring influence of other properties of the "host enzyme". Global unfolding of all fully reconstituted holoenzymes occurred around 63 degrees C, confirming that the kinetic transition temperatures did not arise from a major disruption of the protein structure.Biochemistry 04/2008; 47(17):4983-91. · 3.42 Impact Factor -
Article: The antiviral drug ribavirin is a selective inhibitor of S-adenosyl-L-homocysteine hydrolase from Trypanosoma cruzi.
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ABSTRACT: Ribavirin (1,2,4-triazole-3-carboxamide riboside) is a well-known antiviral drug. Ribavirin has also been reported to inhibit human S-adenosyl-L-homocysteine hydrolase (Hs-SAHH), which catalyzes the conversion of S-adenosyl-L-homocysteine to adenosine and homocysteine. We now report that ribavirin, which is structurally similar to adenosine, produces time-dependent inactivation of Hs-SAHH and Trypanosoma cruzi SAHH (Tc-SAHH). Ribavirin binds to the adenosine-binding site of the two SAHHs and reduces the NAD(+) cofactor to NADH. The reversible binding step of ribavirin to Hs-SAHH and Tc-SAHH has similar K(I) values (266 and 194 microM), but the slow inactivation step is 5-fold faster with Tc-SAHH. Ribavirin may provide a structural lead for design of more selective inhibitors of Tc-SAHH as potential anti-parasitic drugs.Bioorganic & Medicinal Chemistry 01/2008; 15(23):7281-7. · 2.92 Impact Factor -
Article: The effect of cosolutes on the isomerization of aspartic acid residues and conformational stability in a monoclonal antibody.
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ABSTRACT: The aspartate residue (Asp 32) located in the complementarity-determining region (CDR) of a recombinant humanized monoclonal antibody (MAb I) is highly susceptible to the isomerization reaction. The modification of Asp 32 residue due to the isomerization reaction results in a significant reduction in the binding affinity of MAb I to IgE. The binding of a MAb I therapeutic to IgE is important for its desired pharmacological effect. In earlier investigations, we demonstrated that the conformational flexibility and residue exposure are factors that are responsible for the observed reactivity of Asp 32 in MAb I. This report explores the role of cosolutes such as glycerol and sucrose in the modulation of Asp 32 reactivity in MAb I. These cosolutes are routinely incorporated in injectable pharmaceutical formulations. The reactivity of the Asp residue in MAb I in these different cosolute-based formulations was compared to its reactivity in a peptide model VDYDG comprising residues 29-33 of MAb I. The formulations of MAb I and VDYDG containing varying concentrations of glycerol and sucrose were incubated at 50 degrees C for a period of 5-7 days. The isomerization of the Asp residue in VDYDG and MAb I was monitored using rp-HPLC and hydrophobic interaction chromatography (HIC), respectively. Structural analysis of MAb I using differential scanning calorimetry (DSC) demonstrated that the structural stability of MAb I was increased in formulations containing glycerol and sucrose. However, the stability of Asp 32 in MAb I was significantly decreased in these formulations. This research suggests that a formulation approach that relies purely on enhancing the structural stability of proteins through addition of these cosolutes could result in problems associated with the chemical stability of these biomolecules.Journal of Pharmaceutical Sciences 08/2007; 96(7):1708-18. · 3.06 Impact Factor -
Article: Comparative kinetics of cofactor association and dissociation for the human and trypanosomal S-adenosylhomocysteine hydrolases. 1. Basic features of the association and dissociation processes.
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ABSTRACT: The S-adenosyl-l-homocysteine (AdoHcy) hydrolases catalyze the reversible conversion of AdoHcy to adenosine and homocysteine, making use of a catalytic cycle in which a tightly bound NAD+ oxidizes the 3-hydroxyl group of the substrate at the beginning of the cycle, activating the 4-CH bond for elimination of homocysteine, followed by Michael addition of water to the resulting intermediate and a final reduction by the tightly bound NADH to give adenosine. The equilibrium and kinetic properties of the association and dissociation of the cofactor NAD+ from the enzymes of Homo sapiens (Hs-SAHH) and Trypanosoma cruzi (Tc-SAHH) are qualitatively similar but quantitatively distinct. Both enzymes bind NAD+ in a complex scheme. The four active sites of the homotetrameric apoenzyme appear to divide into two numerically equal classes of active sites. One class of sites binds cofactor weakly and generates full activity very rapidly (in less than 1 min). The other class binds cofactor more strongly but generates activity only slowly (>30 min). In the case of Tc-SAHH, the final affinity for NAD+ is roughly micromolar and this affinity persists as the equilibrium affinity. In the case of Hs-SAHH, the slow-binding phase terminates in micromolar affinity also, but over a period of hours, the dissociation rate constant decreases until the final equilibrium affinity is in the nanomolar range. The slow binding of NAD+ by both enzymes exhibits saturation kinetics with respect to the cofactor concentration; however, binding to Hs-SAHH has a maximum rate constant around 0.06 s-1, while the rate constant for binding to Tc-SAHH levels out at 0.006 s-1. In contrast to the complex kinetics of association, both enzymes undergo dissociation of NAD+ from all four sites in a single first-order reaction. The equilibrium affinities of both Hs-SAHH and Tc-SAHH for NADH are in the nanomolar range. The dissociation rate constants and the slow-binding association rate constants for NAD+ show a complex temperature dependence with both enzymes; however, the cofactor always dissociates more rapidly from Tc-SAHH than from Hs-SAHH, the ratio being around 80-fold at 37 degrees C, and the cofactor binds more rapidly to Hs-SAHH than to Tc-SAHH above approximately 16 degrees C. These features present an opening for selective inhibition of Tc-SAHH over Hs-SAHH, demonstrated with the thioamide analogues of NAD+ and NADH. Both analogues bind to Hs-SAHH with approximately 40 nM affinities but much more weakly to Tc-SAHH (0.6-15 microM). Nevertheless, both analogues inactivated Tc-SAHH 60% (NAD+ analogue) or 100% (NADH analogue) within 30 min, while the degree of inhibition of Hs-SAHH approached 30% only after 12 h. The rate of loss of activity is equal to the rate of dissociation of the cofactor and thus 80-fold faster at 37 degrees C for Tc-SAHH.Biochemistry 05/2007; 46(19):5798-809. · 3.42 Impact Factor -
Article: Aspartate isomerization in the complementarity-determining regions of two closely related monoclonal antibodies.
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ABSTRACT: The aspartic acid residues (Asp) present in the complementarity-determining regions (CDRs) of the light chains of two recombinant monoclonal antibodies (MAbs), MAb I and MAb II, are highly susceptible to isomerization due to the presence of glycine residues (Gly) on their C-terminal ends. Asp isomerization in these MAbs leads to formation of the isoaspartate (IsoAsp) and the cyclic imide (Asu) variants of these MAbs. Both MAb I and MAb II, employed in this study, elicit their pharmacological responses through binding human IgE. The formation of the MAb variants as a result of Asp isomerization significantly reduces the binding affinities of these antibodies to IgE, thereby reducing their potencies. Here we report on significant differences in the susceptibility of the MAb I and the MAb II to Asp isomerization. The molecular basis for these differences in rates of Asp isomerization was elucidated. The effect of primary sequence on Asp isomerization was evaluated using pentapeptide models of the MAbs, which included the labile Asp residues and their neighboring amino acid residues. The separation of the parent MAbs and pentapeptides from their isomerization products was achieved using hydrophobic interaction chromatography (HIC) and rp-HPLC, respectively. Structural characterization of the MAbs was performed using differential scanning calorimetry (DSC), circular dichroism (CD), and X-ray crystallography. Our investigations demonstrate that the differences in the Asp isomerization rates between MAb I and MAb II can be attributed to structural factors including the conformational flexibility and the extent of solvent exposure of the labile Asp residue.Biochemistry 03/2007; 46(6):1534-44. · 3.42 Impact Factor
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1994–2012
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University of Kansas
- • Department of Pharmaceutical Chemistry
- • Department of Molecular Biosciences
Lawrence, KS, USA
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2000–2008
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Florida International University
- Department of Chemistry and Biochemistry
Miami, FL, USA
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2003
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Bristol-Myers Squibb
- Pharmaceutical Research Institute
New York City, NY, USA
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2002
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LEO Pharma
Copenhagen, Capital Region, Denmark
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1998
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Brigham Young University - Provo Main Campus
- Department of Chemistry and Biochemistry
Provo, UT, USA
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