Relationship between Intracellular Concentration of S-Adenosylhomocysteine and Inhibition of Vaccinia Virus Replication and Inhibition of Murine L-929 Cell Growth

9-(trans-2',trans-3'-Dihydroxycyclopent-4'-enyl)-adenine (compound 1) and -3-deazaadenine (compound 2), which are specific inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase, were reported earlier by our laboratory (M. Hasobe, J. G. McKee, D. R. Borcherding, and R. T. Borchardt, Antimicrob. Agents Chemother. 31:1849-1851, 1987) to have anti-vaccinia virus activity with reduced murine L-929 cell toxicity compared with the prototype compound neplanocin A. In this study, we showed that the antiviral and cytotoxic effects of compounds 1 and 2 can be related to intracellular concentrations of AdoHey, which are elevated in cells treated with these inhibitors of AdoHcy hydrolase. For example, concentrations of analogs 1 and 2 that produce 50% inhibition of vaccinia virus replication caused only slight elevations in intracellular levels of AdoHcy (from 50 [controls] to 100 to 125 [drug-treated cells] pmol/mg of protein) and elevations in the ratios of AdoHcy/S-adenosylmethionine (from 0.05 to 0.1 [controls] to 0.15 to 0.19 [drug-treated cells]). In contrast to the extreme susceptibility of virus replication to slight elevations in intracellular AdoHcy, cell viability was quite tolerant to higher levels of this metabolite. For example, concentrations of analogs 1 and 2 that produced 50% inhibition of L-929 cell replication caused significant increases in intracellular levels of AdoHcy (to 825 to 950 pmol/mg of protein) and elevations in AdoHcy/S-adenosylmethionine ratios (approximately 1.3). These data make it possible to assign a therapeutic index of 7 to 8 to these compounds on the basis of the comparison of intracellular levels of AdoHcy that caused 50% inhibition of vaccinia virus replication with those that caused 50% inhibition of L-929 cell replication.This work was supported by a Public Health Service grant from the National Institutes of Health (GM-29332) and a grant from Glaxo, Inc

Probing the helical content of growth hormone-releasing factor analogs using electrospray ionization mass spectrometry

AbstractA series of growth hormone-releasing factor analogs have been studied by both circular dichroism and electrospray ionization mass spectrometry (ESI/MS). The peptides are 32 residues long and are known to adopt a random-coil structure in aqueous solution but become increasing helical as the proportion of organic solvent is increased. Deuterium exchange was observed as an increase in mass of the peptide, as measured by ESI/MS. Rates of exchange were measured and half-lives calculated for analogs containing amino acid substitutions designed to promote or discourage helix formation. Exchange was slower in peptides that are helical (as shown by circular dichroism) than in randomly coiled peptides. Solution conditions that favor helix formation also produced slower exchange rates. These studies suggest that ESI/MS can provide date about the extent and stability of helix formation

9-(trans-2',trans-3'-Dihydroxycyclopent-4'-Enyl)-Adenine and -3-Deazaadenine: Analogs of Neplanocin A Which Retain Potent Antiviral Activity but Exhibit Reduced Cytotoxicity

Two synthetic analogs of neplanocin A, which were shown in a separate study to be inhibitors of S-adenosylhomocysteine hydrolase and devoid of substrate activity with adenosine kinase, were found in this study to inhibit vaccinia virus replication in murine L929 cells but to have reduced cytotoxicity compared with that of the parent compound. These results confirm that S-adenosylhomocysteine hydrolase is the molecular target which mediates the antiviral effects of neplanocin A and that transformation by cellular adenosine kinase mediates its cytotoxic properties.This work was supported by Public Health Service research grant GM-29332 from the National Institutes of Health

Crystallization and preliminary X-ray analysis of human placental S-adenosylhomocysteine hydrolase

A recombinant form of human placental S-adenosylhomocysteine (AdoHcy) hydrolase expressed in E. coli, which was inactivated by a type-I mechanism-based inhibitor, has been crystallized using the hanging-drop vapour-diffusion technique. The crystals grow as fiat plates, with unit-cell dimensions a=96.2, b=173.6, c=142.9A, ct=fl=7=90 °. The crystals exhibit the symmetry of space group C222 and diffract to a minimum spacing of "-~ 2.0 A resolution at the Cornell High Energy Synchrotron Source. On the basis of density calculations two monomers of the tetrameric protein are estimated to be present in the asymmetric unit (Vm = 2.99 ,~3 Da-l). The selfrotation function clearly indicates the location of the noncrystallographic twofold axis.The authors would like to thank Steve Ealick and colleagues at CHESS for access to these facilities. This work is supported by a grant from the NIH (GM29332) to RTB

Evaluation of NAD(H) analogues as selective inhibitors for Trypanosoma cruzi S-Adenosylhomocysteine hydrolase

This is an Accepted Manuscript of an article published by Taylor & Francis in Nucleosides, Nucleotides and Nucleic Acids in May 2009, available online: http://www.tandfonline.com/10.1080/15257770903044572.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 (Fig. 1) reduced the catalytic activity of Tc-SAHH to <10% in six minutes of exposure

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

Based on 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 non-identical 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 et al., 1994 J Biol Chem, 269, 31472–8). 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 about 800 nM while TcY435A increases the cofactor equilibrium dissociation constant from approximately 100 nM (Tc-SAHH) to about 1 mM. Both changes result from larger increases in off-rate combined with smaller decreases in 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