A series of S-alkylated derivatives of homocysteine were synthesized and characterized as inhibitors of human recombinant betaine-homocysteine S-methyltransferase (BHMT). Some of these compounds inhibit BHMT with IC50 values in the nanomolar range. BHMT is very sensitive to the structure of substituents on the sulfur atom of homocysteine. The S-carboxybutyl and S-carboxypentyl derivatives make the most potent inhibitors, and an additional sulfur atom in the alkyl chain is well tolerated. The respective (R,S)-5-(3-amino-3-carboxy-propylsulfanyl)-pentanoic, (R,S)-6-(3-amino-3-carboxy-propylsulfanyl)-hexanoic, and (R,S)-2-amino-4-(2-carboxymethylsulfanyl-ethylsulfanyl)-butyric acids are very potent inhibitors and are the strongest ever reported. We determined that (R,S)-5-(3-amino-3-carboxy-propylsulfanyl)-pentanoic acid displays competitive inhibition with respect to betaine binding with a Kappi of 12 nM. Some of these compounds are currently being tested in mice to study the influence of BHMT on the metabolism of sulfur amino acids in vivo.

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Flemingovo nam 2 166 10 Prague 6 Czech Republic

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Finkelstein JD, Harris BJ, Kyle WE. Methionine metabolism in mammals: kinetic study of betaine-homocysteine methyltransferase. Arch. Biochem. Biophys. 1972;153:320–324. PubMed

Millian NS, Garrow TA. Human betaine-homocysteine methyltransferase is a zinc metalloenzyme. Arch. Biochem. Biophys. 1998;356:93–98. PubMed

Breksa AP, III, Garrow TA. Recombinant human liver betaine-homocysteine S-methyltransferase: identification of three cysteine residues critical for zinc binding. Biochemistry. 1999;38:13991–13998. PubMed

Evans JC, Huddler DP, Jiracek J, Castro C, Millian NS, Garrow TA, Ludwig ML. Betaine-homocysteine methyltransferase. Zinc in a distorted barrel. Structure. 2002;10:1159–1071. PubMed

Gonzalez B, Pajares MA, Martinez-Ripoll M, Blundell TL, Sanz-Aparicio J. Crystal structure of rat liver betaine homocysteine S-methyltransferase reveals new oligomerization features and conformational changes upon substrate binding. J. Mol.Biol. 2004;338:771–782. PubMed

Peariso K, Goulding CW, Huang S, Matthews RG, Penner-Hahn JE. Characterization of the zinc binding site in methionine synthase enzymes of Escherichia coli: The role of zinc in the methylation of homocysteine. J. Am. Chem. Soc. 1998;120:8410–8416.

Peariso K, Zhou ZS, Smith AE, Matthews RG, Penner-Hahn JE. Characterization of the zinc sites in cobalamin-independent and cobalamin-dependent methionine synthase using zinc and selenium X-ray absorption spectroscopy. Biochemistry. 2001;40:987–993. PubMed

Szegedi SS, Garrow TA. Oligomerization is required for betaine-homocysteine S-methyltransferase function. Arch. Biochem. Biophys. 2004;426:32–42. PubMed

McKeever MP, Weir DG, Molloy A, Scott JM. Betaine-homocysteine methyltransferase: organ distribution in man, pig and rat and subcellular distribution in the rat. Clin. Sci. (Colch. ) 1991;81:551–556. PubMed

Kempson SA, Montrose MH. Osmotic regulation of renal betaine transport: transcription and beyond. Pflugers Arch. 2004;449:227–234. PubMed

Wettstein M, Weik C, Holneicher C, Häussinger D. Betaine as an osmolyte in rat liver: metabolism and cell-to-cell interactions. Hepatology. 1998;27:787–793. PubMed

Haussinger D. Neural control of hepatic osmolytes and parenchymal cell hydration. Anat. Rec. A Discov. Mol. Cell Evol. Biol. 2004;280:893–900. PubMed

Delgado-Reyes CV, Garrow TA. High sodium chloride intake decreases betaine-homocysteine methyltransferase expression in guinea pig liver and kidney. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2005;288:R182–R187. PubMed

Refsum H, Ueland PM, Nygard O, Vollset SE. Homocysteine and cardiovascular disease. Annu. Rev. Med. 1998;49:31–62. PubMed

Homocysteine Studies Collaboration. Homocysteine and risk of ischemic heart disease and stroke: a meta-analysis. JAMA. 2002;288:2015–2022. PubMed

Ray JG. Meta-analysis of hyperhomocysteinemia as a risk factor for venous thromboem bolic disease. Arch. Intern. Med. 1998;158:2101–2106. PubMed

Vollset SE, Refsum H, Irgens LM, Emblem BM, Tverdal A, Gjessing HK, Monsen AL, Ueland PM. Plasma total homocysteine, pregnancy complications, and adverse pregnancy outcomes: the Hordaland homocysteine study. Am. J. Clin. Nutr. 2000;71:962–968. PubMed

Ray JG, Laskin CA. Folic acid and homocyst(e)ine metabolic defects and the risk of placental abruption, pre-eclampsia and spontaneous pregnancy loss: A systematic review. Placenta. 1999;20:519–529. PubMed

Bottiglieri T. Folate, vitamin B-12, and neuropsychiatric disorders. Nutr. Rev. 1996;54:382–390. PubMed

Seshadri S, Beiser A, Selhub J, Jacques PF, Rosenberg IH, D'Agostino RB, Wilson PW, Wolf PA. Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. N. Engl. J. Med. 2002;346:476–483. PubMed

Chauveau P, Chadefaux B, Coude M, Aupetit J, Hannedouche T, Kamoun P, Jungers P. Hyperhomocysteinemia, a risk factor for atherosclerosis in chronic uremic patients. Kidney Int. Suppl. 1993;41:S72–S77. PubMed

Finkelstein JD, Martin JJ. Methionine metabolism in mammals. Distribution of homocysteine between competing pathways. J. Biol. Chem. 1984;259:9508–9513. PubMed

Mato JM, Corrales FJ, Lu SC, Avila MA. S-adenosylmethionine: a control switch that regulates liver function. Faseb J. 2002;16:15–26. PubMed

Duranton B, Freund JN, Galluser M, Schleiffer R, Gosse F, Bergmann C, Hasselmann R, Raul F. Promotion of intestinal carcinogenesis by dietary methionine. Carcinogenesis. 1999;20:493–497. PubMed

Pavillard V, Nicolaou A, Double JA, Phillips RM. Methionine dependence of tumours: A biochemical strategy for optimizing paclitaxel chemosensitivity in vitro. Biochem. Pharmacol. 2006;71:772–778. PubMed

Mosharov E, Cranford MR, Banerjee R. The quantitatively important relationship between homocysteine metabolism and glutathione synthesis by the transsulfuration pathway and its regulation by redox changes. Biochemistry. 2000;39:13005–13011. PubMed

Awad WM, Jr, Whitney PL, Skiba WE, Mangum JH, Wells MS. Evidence for direct methyl transfer in betaine: homocysteine S-methyl- transferase. J. Biol. Chem. 1983;258:12790–12792. PubMed

Castro C, Gratson AA, Evans JC, Jiracek J, Collinsova M, Ludwig ML, Garrow TA. Dissecting the catalytic mechanism of betaine-homocysteine S-methyltransferase by use of intrinsic tryptophan fluorescence and site-directed mutagenesis. Biochemistry. 2004;43:5341–5351. PubMed

Lee KH, Cava M, Amiri P, Ottoboni T, Lindquist RN. Betaine:homocysteine methyltransferase from rat liver: Purification and inhibition by a boronic acid substrate analog. Arch. Biochem. Biophys. 1992;292:77–86. PubMed

Collinsova M, Castro C, Garrow TA, Yiotakis A, Dive V, Jiracek J. Combining combinatorial chemistry and affinity chromatography: highly selective inhibitors of human betaine:homocysteine S-methyltransferase. Chem. Biol. 2003;10:113–122. PubMed

Koval D, Kasicka V, Jiracek J, Collinsova M. Separation of diastereomers of phosphinic pseudopeptides by capillary zone electrophoresis and reverse phase high-performance liquid chromatography. J. Sep. Sci. 2003;26:653–660.

Koval D, Kasicka V, Jiracek J, Collinsova M. Physicochemical characterization of phosphinic pseudopeptides by capillary zone electrophoresis in highly acid background electrolytes. Electrophoresis. 2003;24:774–781. PubMed

Koval D, Kasicka V, Jiracek J, Collinsova M, Garrow TA. Determination of dissociation constant of phosphinate group in phosphinic pseudopeptides by capillary zone electrophoresis. J. Chromatogr. B. 2002;770:145–154. PubMed

Koval D, Kasicka V, Jiracek J, Collinsova M, Garrow TA. Analysis and characterization of phosphinic pseudopeptides by capillary zone electrophoresis. Electrophoresis. 2002;23:215–222. PubMed

Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72:248–254. PubMed

Farrington GK, Kumar A, Wedler FC. Design and synthesis of phosphonate inhibitors of glutamine synthetase. J. Med. Chem. 1987;30:2062–2067. PubMed

Viornery C, Pechy P, Boegli M, Aronsson BO, Descouts P, Gratzel M. Synthesis of new polyphosphonic acids. Phosphorus Sulfur Silicon Relat. Elem. 2002;177:231–241.

Rejman D, Masojidkova M, De Clercq E, Rosenberg I. 2 '-C-alkoxy and 2 '-C-aryloxy derivatives of N-(2-phosphonomethoxyethyl)purines and -pyrimidines: Synthesis and biological activity. Nucleosides Nucleotides & Nucleic Acids. 2001;20:1497–1522. PubMed

Arbuzov BA, Vinogradova VS, Novoselskaja AD. Reaction of N-hydroxymethylchloroacetamide with triethyl phosphite and some dialkyl chlorophosphites. J. Gen. Chem. USSR (Engl. Transl. ) 1967;37:2061–2065.

Garrow TA. Purification, kinetic properties, and cDNA cloning of mammalian betaine-homocysteine methyltransferase. J. Biol. Chem. 1996;271:22831–22838. PubMed

Todhunter JA. Reversible Enzyme Inhibition. In: Purich DL, editor. Enzyme Kinetics and Mechanism. New York: Academic Press; 1979. pp. 383–411.

Min K-L, Steghens J-P, Henry R, Doutheau A, Collombel C. N-Dibenzylhospho-N'-3-(2,6-dichlorophenyl)propyl-guanidine is a bisubstrate-analog for creatine kinase. Biochim. Biophys. Acta. 1997;1342:83–89. PubMed

Segel IH. Enzyme Kinetics. Behaviour and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. New York: John Wiley & Sons, Inc.; 1993.

Dixon M. The graphical determination of Km and Ki. Biochem. J. 1972;129:197–202. PubMed PMC

Structure-activity study of new inhibitors of human betaine-homocysteine S-methyltransferase