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Biomedical subjects

A R Khomutov

Publications and source records attributed to A R Khomutov.

At least 19 recordsLinked to original sources
PubMed2002

Inhibition of enzymes of polyamine biosynthesis by substrate-like O-substituted hydroxylamines.

The biogenic amines spermine, spermidine, and putrescine are essential factors of cell growth and differentiation. To inhibit pyridoxal-5'-phosphate dependent ornithine decarboxylase and pyruvate dependent S-adenosylmethionine decarboxylase, key enzymes of polyamine biosynthesis, a system of substrate-like O-substituted hydroxylamines is suggested. The best of these compounds were active at nanomolar concentrations. High potency and specificity of this type of inhibitors are discussed in terms of structural similarity of E-I and E-S complexes.

Adenosylmethionine Decarboxylase↗
PubMed2001

Hydroxylamine-containing inhibitors of polyamine biosynthesis and impairment of colon cancer cell growth.

Polyamine synthesis (by the action of ornithine decarboxylase [ODC] and S-adenosylmethionine decarboxylase [SAMDC]) and polyamine content are high in colon cancer. In addition, colonic lumen is rich in polyamines synthesised by colonic microflora; for this reason, polyamine depletion in colon cancer may be a logical approach to impair growth of colon cancer cells. We evaluated highly specific and reportedly non-toxic hydroxylamine-containing inhibitors of ODC (1-aminooxy-3-aminopropane, APA) and SAMDC (S-(5'-deoxy-5'-adenosyl)-methylthioethyl-hydroxylamine, AMA) in human colon cancer cells (Caco-2 and HT-29) in culture. APA depleted ODC activity within 24 hr, more rapidly than did difluoromethylornithine. APA and AMA in combination (100 microM each) reduced ODC and SAMDC activities to undetectable levels within 24 hr and intracellular polyamines to 8-23% of control. The resulting growth arrest could be reversed only by twice as much spermidine as is physiologically present in the colonic lumen. In concentrations sufficient to deplete growth, APA and AMA were not toxic. Simultaneous treatment with APA, AMA, and 5-fluorouracil reduced colon cancer cell survival more potently than treatment with 5-fluorouracil alone. The hydroxylamine-containing ODC and SAMDC inhibitors APA and AMA are potent inhibitors of colon cancer cell proliferation and might be therapeutically promising in colon cancer.

Adenosylmethionine Decarboxylase↗
PubMed1998

[Acyclic nucleotide analogues based on phosphonic acids].

The synthesis of novel nucleotide analogues on the basis of ethylphosphonic acid was described. A rigid structural element, an amide or a double bond, was characteristics of the compounds synthesized. The antiviral and cytotoxic activities of these compounds were studied in cell cultures.

Amides↗
PubMed1996

Crystal structures and solution studies of oxime adducts of mitochondrial aspartate aminotransferase.

The interaction of mitochondrial aspartate aminotransferase with hydroxylamine and five derivatives (in which the hydroxyl hydrogen is replaced by the side chain of naturally occurring amino acids) was investigated by X-ray diffraction as well as by kinetic and spectral measurements with the enzyme in solution. The inhibitors react with pyridoxal 5'-phosphate in the enzyme active site, both in solution and in the crystalline state, in a reversible single-step reaction forming spectrally distinct oxime adducts. Dissociation constants determined in solution range from 10(-8) M to 10(-6) M depending on the nature of the side-chain group. The crystal structures of the adducts of mitochondrial aspartate aminotransferase with the monocarboxylic analogue of L-aspartate in the open and closed enzyme conformation were determined at 0.23-nm and 0.25-nm resolution, respectively. This inhibitor binds to both the open and closed crystal forms of the enzyme without disturbing the crystalline order. Small differences in the conformation of the cofactor pyridoxal phosphate were detected between the omega-carboxylate of the inhibitor and Arg292 of the neighbouring subunit is mainly responsible for the attainment of near-coplanarity of the aldimine bond with the pyridine ring in the oxime adducts. Studies with a fluorescent probe aimed to detect shifts in the open/closed conformational equilibrium of the enzyme in oxime complexes showed that the hydroxylamine-derived inhibitors, even those containing a carboxylate group, do not induce the 'domain closure' in solution. This is probably due to the absence of the alpha-carboxylate group in the monocarboxylic hydroxylamine-derived inhibitors, emphasizing that both carboxylates of the substrates L-Asp and L-Glu are essential for stabilizing the closed form of aspartate aminotransferase.

Amino Acid Sequence↗
PubMed1996

Aminooxy-analogues of spermidine: new partial agonists and antagonists at the polyamine site of the rat hippocampal NMDA receptor complex.

The amino-1-oxy- and amino-8-oxy-analogues of spermidine (1-O-SPD and 8-O-SPD) were tested in vitro with rat hippocampal membranes as potential modulators of the N-methyl-D-aspartate (NMDA) receptor complex via the polyamine regulatory site. In the presence of 1 microM glutamate and glycine, the binding of the NMDA channel ligand [3H]MK-801 was stimulated by 8-O-SPD (EC50 = 50 microM); 1-O-SPD was without significant influence at concentrations up to 1 mM. Addition of 2 and 4 microM of the polyamine agonist spermine eliminated the stimulatory property of 8-O-SPD, whereas 1-O-SPD was inhibitory under these conditions (IC50 = 274 and 481 microM, respectively). At higher concentrations of spermine, both compounds were inhibitory. Inhibition of [3H]MK-801 binding by the inverse polyamine agonists 1,10-diaminodecane, 1,12-diaminododecane, and arcaine was attenuated by 1 mM 1-O-SPD. The data are compatible with the notion that 8-O-SPD is a partial polyamine agonist and that 1-O-SPD is an antagonist without intrinsic activity.

Animals↗
PubMed1996

[New aminooxy analogs of biogenic polyamines].

A series of structural analogs of putrescine, spermidine, and spermine with the aminomethylene fragment substituted by the aminooxy group was suggested. The synthesis of the new aminooxy analogs of spermine was described. Biochemical aspects of the activity of the aminooxy analogs of polyamines were discussed in respect of their selective inhibition of normal and leukemic cells.

Animals↗
PubMed1995

Aminooxy analogues of spermidine evidence the divergent roles of the charged amino nitrogens in the cellular physiology of spermidine.

Two recently devised spermidine analogues, N-[2-aminooxyethyl]-1,4-diaminobutane (AOEPU) and 1-aminooxy-3-N-[3-aminopropyl]-aminopropane (APAPA), were used to elucidate the role of charge distribution in the functions of spermidine in cultured baby hamster kidney cells. The drugs did not affect cell proliferation nor did they relieve the growth-arrest but potentiated the metabolic disturbances caused by DL-alpha-difluoromethyl-ornithine (DFMO). Neither drug affected spermidine uptake but both competed with putrescine uptake. Neither drug could replace spermidine in the control of S-adenosylmethionine decarboxylase and accumulation of the reaction product. APAPA prevented spermine synthesis and showed that modest putrescine synthesis take place in the presence of DFMO. AOEPU, but not APAPA, interfered with cellular constituents resulting in enzymatic formation, accumulation and excretion to culture medium of UV-absorbing catabolites.

Animals↗
PubMed1995

The reduction of thymine residues in DNA by the combined action of UV light and hypophosphite.

UV irradiation (lambda = 254 nm) of thymine and uracil in aqueous solution containing salts of phosphinic acid (hypophosphites) results in the formation of the corresponding dihydropyrimidine derivatives. The peculiarities of this new photochemical reaction consist of a specificity towards 2,4-dioxophyrimidines, a high quantum yield and a neutral pH optimum. The quantum yield of photoconversion of thymine at pH 7.0 is equal to 1.9 x 10(-2) in 1 M NaH2PO2; it is diminished to 8.5 x 10(-3) in 0.1 M NaH2PO2. The same decrease in quantum yield with concentration is also found for uracil and uridine; quantum yields of their transformations in 0.1 M NaH2PO2 at pH 7.0 are 6.6 x 10(-2) and 1.5 x 10(-1) respectively. The mild conditions and high quantum yields characteristic of the photoinduced reaction above open up the possibility of obtaining DNA molecules which contain among pyrimidine photoproducts mainly dihydropyrimidine residues. A correlation between various types of thymine modifications in UV-irradiated double-stranded DNA (dimers and dihydrothymidine residues) and the amplitude of the intense negative band in the circular dichroism (CD) spectra specific for DNA liquid crystalline dispersions has been established. A possible application of this base-specific modification to the investigation of nucleic acids is considered.

Circular Dichroism↗
PubMed1993

Application of oxime formation in a radiometric assay of aminooxy compounds.

A radiometric determination of monoaminooxy analogues of naturally occurring polyamines is described in which [2-14C]acetone is employed as reagent. The reagent is volatile while the oxime product is not allowing unreacted reagent to be removed and the oxime formation to be completed by lyophilization in vacuo. The residual radioactive compound is soluble in water and proportional to the reactive aminooxy content of the reaction mixture and can be quantified by liquid scintillation counting. The assay method is inexpensive and simple and has high specificity and flexibility in sample volume enabling reliable quantification of reactive aminooxy amines in biological extracts at concentrations exceeding 0.25 microM. Optimal pH values for oxime formation of five monoaminooxy analogues of polyamines with acetone were resolved. Reactions via reversible intermediates to irreversible oximes were sped up by removal of water. Complete oxime formation and stability was confirmed by 1H NMR studies. Tested drugs readily formed oximes with pyridoxal 5'-phosphate, too. Diaminooxy analogue of cadaverine formed a volatile oxime with acetone. The method was used to monitor the stability of aminooxy analogues of putrescine and spermidine during storage and under culture conditions and to establish their scant accumulation in, but fast catabolism by, cultured baby hamster kidney cells.

Amines↗
PubMed1992

Monitoring of the uptake and metabolism of aminooxy analogues of polyamines in cultured cells by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the determination of polyamines and their aminooxy analogues is described. Oxime derivatization with a ketone is used to protect the aminooxy group during post-column reaction with o-phthalaldehyde. The amount of the polyamines and of the oximes of their aminooxy analogues can be determined simultaneously in cultured cells and cell culture media. The limit of detection is 20-30 pmol, and the response of the fluorescence detection is linear up to 4 nmol. The separation of the aminooxy analogues from the naturally occurring polyamines can be varied by using different ketones for oxime formation. The method was used to measure the stability of aminooxy analogues of putrescine (1-aminooxy-3-aminopropane) and spermidine [N-(2-aminooxyethyl)-1,4-diaminobutane and 1-aminooxy-3-N-(3-aminopropyl)aminopropane] in cell culture media and the uptake into cultured baby hamster kidney (BHK21/C13) cells.

Animals↗
PubMed1991

Effect of magnesium ions on the inhibition of S-adenosylmethionine decarboxylase from Escherichia coli by [2-(amino-oxy)ethyl](5'-deoxyadenosin-5'-yl)(methyl)sulphonium .

[2-(Amino-oxy)ethyl](5'-deoxyadenosin-5'-yl)(methyl)sulphonium+ ++, the amino-oxy analogue of decarboxylated S-adenosylmethionine, is a potent irreversible inhibitor of Escherichia coli S-adenosylmethionine decarboxylase [Khomutov, Zavalova, Syrku, Artamonova & Khomutov (1983) Bioorg. Khim. 9, 130-131; Artamonova, Zavalova, Khomutov & Khomutov (1986) Bioorg. Khim. 12, 206-212]. We have shown that Mg2+ ions are required for the irreversible inhibition of the decarboxylase, and that S-adenosylmethionine protects against this inhibition.

Adenosylmethionine Decarboxylase↗
PubMed1991

Regulation of S-adenosylmethionine decarboxylase in L1210 leukemia cells. Studies using an irreversible inhibitor of the enzyme.

A potent irreversible inhibitor of S-adenosylmethionine (AdoMet) decarboxylase, S-(5'-adenosyl)-methylthio-2-aminooxyethane (AdoMeSaoe), was used to study the regulatory control of this key enzyme in the polyamine biosynthetic pathway. Treatment of L1210 cells with the inhibitor completely eradicated the growth-induced rise in AdoMet decarboxylase activity, resulting in a marked decrease in cellular content of spermidine and spermine. The putrescine content, on the other hand, was greatly elevated. Although no detectable AdoMet decarboxylase activity was found in the L1210 cells after treatment with AdoMeSaoe, the cells contained 50-fold higher amounts of AdoMet decarboxylase protein, compared to untreated cells during exponential growth. Part of this increase was shown to be due to elevated synthesis of the enzyme. This stimulation appeared to be related to the decrease in cellular spermidine and spermine content, since addition of either one of the polyamines counteracted the rise in AdoMet decarboxylase synthesis. The synthesis rate was determined by immunoprecipitation of labeled enzyme after a short pulse with [35S]methionine. In addition to a protein that co-migrated with pure rat AdoMet decarboxylase (Mr approximately 32,000), the antibody precipitated a somewhat larger labeled protein (Mr approximately 37,000) that most likely represents the proenzyme form. Treatment of the L1210 cells with AdoMetSaoe also gave rise to a marked stabilization of the decarboxylase which contributed to the increase in its cellular protein content. Addition of spermidine did not significantly affect this stabilization, whereas the addition of spermine reduced the half-life of the enzyme to almost that of the control cells.

Adenosylmethionine Decarboxylase↗
PubMed1990

Uptake of 3H-labeled 1-aminooxy-3-aminopropane by baby hamster kidney cells.

The uptake, catabolism, and release of H-labeled 1-aminooxy-3-aminopropane, a new putrescine analog shown to be a potent polyamine antimetabolite, into and from baby hamster kidney cells (BHK21/C13) were studied. The results show that [3H]-1-aminooxy-3-aminopropane (APA) is not concentrated in the cell, does not compete with polyamines for transport and reveals no difference in uptake between polyamine-depleted and control cells. After a 12-h culture, 60% of APA was recovered intact in the culture media. At this time point, only 30% of the intracellular radioactivity was intact APA, showing that the drug is catabolized in the cells. This intracellular ratio persisted throughout the 4-day culture period. The metabolites of APA were not characterized further. The results indicate that the drug is not recognized as a polyamine by the cells and does not replace or interfere with the polyamines in cellular functions. Thus, its potent affinity to ornithine decarboxylase and spermidine synthase is likely to be due to close structural similarity with the intermediates formed in these reactions. This has implications for the mechanisms involved.

Animals↗
PubMed1990

Aminooxy analogues of spermidine as inhibitors of spermine synthase and substrates of hepatic polyamine acetylating activity.

Aminooxy analogues of spermidine, 1-aminooxy-3-N-[3-aminopropyl]- aminopropane (AP-APA) and N-[2-aminooxyethyl]-1,4-diaminobutane (AOE-PU), were tested as substrates or inhibitors of the enzymes involved in methionine and polyamine metabolism. Both compounds were good competitive inhibitors and poor substrates of spermine synthase, good substrates of cytosolic polyamine acetyltransferase, inactivators of S-adenosylmethionine decarboxylase and inhibitors of ornithine decarboxylase. AP-APA and AOE-PU showed K1-values of 1.5 and 186 microM as inhibitors of purified spermine synthase, and Km-values of 1.4 and 2.1 mM as substrates of the crude hepatic polyamine acetyltransferase activity. AP-APA was more potent than AOE-PU in crude enzyme preparations. Neither drug had any significant effect at 1 mM concentration on the activities of spermidine synthase, methionine adenosyltransferase, S-adenosylhomocysteine hydrolase, and methylthioadenosine phosphorylase. The results suggest that compounds of this type are valuable tools in unraveling the physiology of polyamines.

Acetylation↗
PubMed1989

Cellular characterization of a new irreversible inhibitor of S-adenosylmethionine decarboxylase and its use in determining the relative abilities of individual polyamines to sustain growth and viability of L1210 cells.

S-(5'-Deoxy-5'-adenosyl)methylthioethylhydroxylamine (AMA) is an irreversible inhibitor of S-adenosylmethionine (AdoMet) decarboxylase, which is designed to bind covalently the pyruvate residue at the enzyme active site. In the present study the cellular effects of AMA were characterized for the first time in cultured L1210 leukaemia cells. At the approximate IC50 (concn. giving 50% inhibition; 100 microM), AMA decreased spermidine and spermine by more than 80% at 48 h while increasing putrescine more than 10-fold. As an indication of enzyme specificity, growth inhibition was fully prevented with exogenous spermidine. When compared with the irreversible inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine (DFMO), at similar growth-inhibitory concentrations, AMA was less cytotoxic, as determined by colony-formation efficiency. In combination with AMA, DFMO eliminated the rise in putrescine and decreased growth in an additive manner. The near-total depletion of intracellular polyamine pools achieved with the drug combination provided an opportunity to examine the relative abilities of individual polyamines to support growth and viability. Of the three exogenously supplied polyamines, only spermidine fully sustained cell growth and viability at control values during incubations totalling 120 h. By contrast, spermine supported growth at 23% of control and viability at 8%. Putrescine was similarly ineffective, supporting growth at 13% of control and viability at 7%. The data indicate that, in L1210 cells, spermidine is apparently the preferred polyamine in growth-related functions and is capable of fully supporting cell growth by itself. However, because spermine and putrescine can also support growth to some extent, maximum interference with growth and viability is best achieved by strategies which deplete all three polyamine pools.

Adenosylmethionine Decarboxylase↗