Characterization of thymidylate synthetase and dihydrofolate reductase from Plasmodium berghei

Pattanakitsakul S. and Ruenwongsa P. 1984. Characterization of thymidylate synthetase and dihydrofolate reductase from Plasmodium berghei. International Journal for Parasitology14: 513–520. Thymidylate synthetase (TS) and dihydrofolate reductase (DHFR) from Plasmodium berghei were copurified by Sephacryl S-300 and Sephadex G-200 column chromatography and found to have an apparent mol. wt of 132,000. Electrophoresis of the partially purified enzyme under non-denaturing conditions showed the comigration of TS and DHFR. The mol. wt of TS was estimated to be 65,000 on SDS-gel electrophoresis. Both enzymes exhibit a broad pH optimum in the range of 6.5–8.0. Urea, NaCl and KC1 inhibit TS but activate DHFR. For TS, the apparent K_m for dUMP and methylene-tetrahydrofolate have been found to be 71.4 and 312.5 μM, respectively. For DHFR, the apparent K_m for dihydrofolate and NADPH have been found to be 4.4 and 12.5 μM, respectively. Inhibition of DHFR by pyrimethamine, methotrexate and trimethoprim are competitive with dihydrofolate with K_is of 0.63, 0.5 and 1.88 nM, respectively. FdUMP inhibition of TS is competitive with dUMP with K_is of 0.05 μM, but inhibition by methotrexate is uncompetitive with dUMP and MTHF with K_ii of 103 and 23 μM, respectively.     

Structural tuning of acridones for developing anticancer agents targeting dihydrofolate reductase

Targeting dihydrofolate reductase, here, we report the tumor growth inhibitory activity of substituted acridones. The screening of the molecules over 60 cell line panel of human cancer cells identified (S)-oxiran-2-ylmethyl 9-oxo-9,10-dihydroacridine-4-carboxylate (19) with average GI₅₀ 0.3 μM. The specificity of the compound to CCRF-CEM, MOLT-4 and SR cell lines of leukemia and SW-620, SF268, LOXIMVI, ACHN and MCF7 cancerous cells exhibiting GI₅₀ in the nM range was observed. C6 Glioma cells treated with compound 19 showed differentiated cell morphology and cell cycle arrest in G2/M phase. The interactions of the compound with dihydrofolate reductase were ascertained with the help of enzyme immunoassays, molecular docking and molecular dynamic studies.     

Design and synthesis of novel enhanced water soluble hydroxyethyl analogs of combretastatin A-4

Thirteen hydroxyethyl- analogs of combretastatin A-4 (CA-4) that contain the 1-(1′-hydroxyethyl)-1-(3″,4″,5″-trimethoxyphenyl)-2-(substituted phenyl)ethene framework were synthesized. Molecular modeling studies at the DFT level showed that compound 3j adopts a ‘twisted’ conformation mimicking CA-4. The cytotoxicity of the novel compounds against the growth of murine B16 melanoma and L1210 lymphoma cells in culture was measured using an MTT assay. Three analogs 3f, 3h, and 3j were active. Of these, 3j, which has the same substituents as CA-4 and IC₅₀ values of 16.1 and 4.1 μM against B16 and L1210 cells, respectively, was selected for further biological evaluation. The activity of 3j was verified by the NCI 60 cell line screen. Compound 3j causes microtubule depolymerization in A-10 cells with an EC₅₀ of 21.2 μM. Analog 3j, which has excellent water solubility of 479 μM, had antitumor activity in a syngeneic L1210 murine model.     

A nonfunctional protein in human leukemia cells reacts with antiserum to dihydrofolate reductase from L1210 leukemia cells

Antiserum raised in chickens to dihydrofolate reductase purified from L1210 leukemia cells by affinity chromatography inhibited the catalytic activity and the binding of methotrexate by the enzyme. Lysates of human chronic myelogenous leukemia cells, which had neither catalytic activity for dihydrofolate reductase nor binding of methotrexate, blocked the inhibiting effect of the antiserum on the function of the enzyme in L1210 cell lysates. In double immunodiffusion, these human leukemia cell lysates formed a single precipitin line against the antiserum. These findings indicate that nonfunctional dihydrofolate reductase in human leukemia cells share an antigenic determinant(s) with a functional form of the enzyme from L1210 murine leukemia cells.     

A fluorescence study of substrate and inhibitor binding to bovine liver dihydrofolate reductase

• 1.1. Covalent coupling of fluorescein to methotrexate (MTX) by a 5-carbon spacer yields a dihydrofolate reductase (DHFR) inhibitor (FMTX) with K_i = 11 nM.
• 2.2. FMTX shows a fluorescence quenching with respect to fluorescein which is relieved by binding to the enzyme.
• 3.3. The dissociation constants (K_d) of MTX, FMTX, NADPH and 7,8-dihydrofolate (DHF) from bovine liver DHFR have been determined by fluorometric titrations.
• 4.4. The K_d values for NADPH, MTX and FMTX from the complementary binary complexes (MTX·DHFR, FMTX·DHFR and NADPH·DHFR) were also obtained; these show a 2- to 4-fold decrease with respect to those obtained by titration of the free enzyme.
• 5.5. A competitive assay for MTX has been developed by exploiting the fluorescence enhancement of DHFR-bound FMTX. This assay may be useful for the routine determination of MTX in the concentration range from 10⁻⁹ to 10⁻⁷ M.

     

Novel synthesized SLC-0111 thiazole and thiadiazole analogues: Determination of their carbonic anhydrase inhibitory activity and molecular modeling studies

In the presented work, we report the design and synthesis of novel SLC-0111 thiazole and thiadiazole analogues (11a–d, 12a–d, 16a–c and 17a–d). A bioisosteric replacement approach was adopted to replace the 4-fluorophenyl tail of SLC-0111 with thiazole and thiadiazole ones, which were thereafter extended with lipophilic un/substituted phenyl moieties. All the newly synthesized SLC-0111 analogues were evaluated in vitro for their inhibitory activity towards a panel of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) isoforms (hCA I, II, IX and XII), using a stopped-flow CO₂ hydrase assay. All the examined isoforms were inhibited by the primary sulfonamide derivatives (11a–d and 12a–d) in variable degrees with the following K_I ranges: 162.6–7136 nM for hCA I, 9.0–833.6 nM for hCA II, 7.9–153.0 nM for hCA IX, and 9.4–94.0 nM for hCA XII. In particular, compounds 12b and 12d displayed 5.5-fold more potent inhibitory activity (K_Is = 8.3 and 7.9 nM, respectively) than SLC-0111 (K_I = 45 nM) towards hCA IX. Molecular docking study was carried out for 12d within the hCA IX (PDB 3IAI) active site, to justify its inhibitory activity.     

Trimethoprim binding to bacterial and mammalian dihydrofolate reductase: a comparison by proton and carbon-13 nuclear magnetic resonance

The binding of trimethoprim to dihydrofolate reductase from L1210 mouse lymphoma cells has been studied by measuring the changes in chemical shift of nuclei of the ligand that accompanying binding. The 6- and 2',6'-proton chemical shifts of bound trimethoprim have been determined by transfer of saturation experiments, and the 2-carbon chemical shift has been determined by using [2-13C]trimethoprim. The changes in proton chemical shift are substantially smaller than those accompanying binding to bacterial dihydrofolate reductase [Cayley, P. J., Albrand, J. P., Feeney, J., Robert, G. C. K., Piper, E. A., & Burgen, A. S. V. (1979) Biochemistry 18, 3886]. It is shown that this difference arises largely from the fact that trimethoprim adopts different conformations when bound to mammalian and to bacterial dihydrofolate reductase. The proton chemical shifts are interpreted in terms of ring-current contributions from the two aromatic rings of trimethoprim itself and the nearby aromatic amino acid residues of the enzyme. The latter have been located by using the refined crystallographic coordinates of the Lactobacillus casei and Escherichia coli reductases in their complexes with methotrexate [Bolin, J. T., Filman, D. J., Matthews, D. A. & Kraut, J. (1982) J. Biol. Chem. 257, 13650], under the assumption that, as indicated by the 13C chemical shifts, the diaminopyrimidine ring of trimethoprim binds in the same way as does the corresponding part of methotrexate. With use of these assumptions, the conformation of trimethoprim bound to the dihydrofolate reductases from L. casei, E. coli, and L1210 cells has been calculated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunologic heterogeneity of dihydrofolate reductase from methotrexate sensitive and resistant L1210 leukemia cells

Dihydrofolate reductase from L1210 leukemia cells which are sensitive and resistant to methotrexate has the same physical and kinetic properties and immunoreactivity with a guinea pig antiserum raised to the enzyme purified from the methotrexate resistant strain. However, a chicken antiserum to dihydrofolate reductase from methotrexate sensitive L1210 cells has greater affinity for the homologous enzyme than for the enzyme from the MTX resistant cells indicating that there is some antigenic difference in these molecules.     

Intracellular phosphate and its possible role as an exchange anion for active transport of methotrexate in L1210 cells

L1210 cells transport P_i in the absence of added Na⁺. Uptake shows saturation kinetics (K_t = 1.7 mM), is temperature-dependent, and can be reduced 80% by high levels of unlabeled P_i, and thus has the characteristics of a carrier-mediated process. This transport process is also inhibited by methotrexate. The methotrexate-sensitive component constitutes half of total P_i uptake, and is reduced by 50% at a concentration of methotrexate (2 μM) that is comparable to its K_t (1.5 μM) for transport into the cells. An impermeable fluorescent analog of methotrexate and an irreversible inhibitor of the methotrexate transport system (carbodiimide-activated methotrexate) also inhibit this same P_i uptake component. It is concluded that methotrexate and P_i can be transported by the same carrier system. The basis for this shared uptake is suggested to be that the methotrexate carrier protein facilitates the obligatory exchange of extracellular folate compounds for intracellular divalent anions, and that a primary exchange anion is P_i. A principal energy source for active transport of methotrexate might then be the concentration gradient for P_i that is maintained by the Na⁺-dependent, P_i transport system of these cells.     

Dihydrofolate reductase inhibition effect of 5-substituted pyrido[2,3-d]pyrimidines: Synthesis,antitumor activity and molecular modeling study

A new series of pyrido[2,3-d]pyrimidines 3–18 bearing substitution at C-5 position was synthesized. All compounds were tested for their in vitro antitumor activity against five human cancer cell lines namely; hepatocellular carcinoma (HePG2), breast carcinoma (MCF-7), human prostate carcinoma (PC3), colorectal carcinoma (HCT-116), and cervical carcinoma (Hela) using doxorubicin as a positive control. Compounds 3, 4, 9, 11, 13, 14, 15 and 17 exhibited the highest antitumor activity against the tested cell lines and were selected to screen their enzymatic inhibition against dihydrofolate reductase enzyme (DHFR) compared with the reference drug methotrexate (MTX), to explain the probable mechanism of action of the observed anticancer activity. Compound 11 displayed the highest inhibitory activity (IC₅₀ = 6.5 µM) among the tested compounds in comparison with MTX (IC₅₀ = 5.57 µM). Also, compounds 13 and 14 showed high inhibitory activity against DHFR with IC₅₀ values 7.1 and 8.7 µM, respectively. Comparative molecular modeling study was performed between DHFR inhibitors 11, 13 and 14 of the highest activity and 10 of the lowest activity among the eight inhibitors against MTX. Docking studies into the active site of DHFR domain showed good agreement with the obtained biological results. Finally, compound 11 was found to be best antitumor, DHFR inhibitor, and it induced the process of apoptosis at Pre-G phase and cell cycle arrest at G2/M phase in MCF-7 cells.     

Antiplasmodial and antitumor activity of dihydroartemisinin analogs derived via the aza-Michael addition reaction

A series of dihydroartemisinin derivatives were synthesized via an aza-Michael addition reaction to a dihydroartemisinin-based acrylate and were evaluated for antiplasmodial and antitumor activity. The target compounds showed excellent antiplasmodial activity, with dihydroartemisinin derivatives 5, 7, 9 and 13 exhibiting IC₅₀ values of ?10 nM against both D10 and Dd2 strains of Plasmodium falciparum. Derivative 4d was the most active against the HeLa cancer cell line, with an IC₅₀ of 0.37 μM and the highest tumor specificity.     

Affinity purification and glucose specificity of aldose reductase from bovine lens

Aldose reductase, a possible key enzyme of sugar-cataract formation in diabetes, has been purified from bovine lens by a five-step procedure including affinity chromatography with Mātrex gel red A. The enzyme was purified 12,600-fold and was apparently homogeneous by polyacrylamide gel electrophoresis. The glucose specificity of the purified enzyme was studied with d-glucose anomers and d-glucitol as substrates. The ratios of the reduction rate of α-d-glucose to that of β-d-glucose at 10, 13, and 20 mm were 1.90, 1.76, and 1.72, respectively. These values were in good agreement with the ratios (1.92, 1.81, and 1.66) calculated on the basis of the rate constants reported for d-glucose mutarotation equilibrium (J. M. Los, L. B. Simpson, and K. Wiesner, 1956, J. Amer. Chem. Soc.78, 1564–1568) and the assumption that aldose reductase acts on the aldehyde form of d-glucose. In addition, the composition of d-glucose produced from d-glucitol in the reverse reaction was 63% α anomer and 37% β anomer, which also agreed well with the values, 65 and 35%, respectively, calculated from the rate constants in reactions from the aldehyde form to both the α anomer and the β anomer. It was suggested from these kinetic analyses that aldose reductase acts on the aldehyde form of d-glucose (K_m = 0.66 μm) but not on either the α or the β anomer of d-glucose.     

cis-Dichloroplatinum (II) complexes with aminomethylnicotinate and -isonicotinate ligands

6-Aminomethylnicotinic acid (1a) and 2-aminomethylisonicotinic acid (1b) were each reacted with K₂PtCl₄ in aqueous 1 M HCl to give the corresponding N,N-chelated cis-dichloroplatinum(II) complexes 2. These were converted into amides 3 via their mixed anhydrides by treating them first with ethyl chloroformate and then with the respective 1° or 2° amine. The analogous 6-aminomethylnicotinic acid ester complexes 7 were obtained by reaction of the preformed ligands with K₂PtCl₄.     

Turnover of dihydrofolate reductase in rapidly dividing cells

The turnover of dihydrofolate reductase has been studied in rapidly dividing cells of mouse lymphoma L1210 and Lactobacillus casei. Cells in culture were exposed to [¹⁴C]leucine for 24 hr and the subsequent decrease in radioactivity of the enzyme was followed as a function of time. The L1210 enzyme was isolated in pure form by subjecting the cell sonicate to affinity chromatography on amethopterin-Sepharose. The L. casei cells were processed by a multistep procedure which yielded the pure enzyme in both of its principal forms: (I), without TPNH; and (II), containing an equimolal amount of noncovalently bound TPNH. The half-lives ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2></mtext>}}$) of dihydrofolate reductase in the amethopterin-sensitive L1210 cells (L1210/S) and in the cells of a partially resistant subline (L1210/R2), characterized by an 8-fold increase in enzyme level, were 18 and 19 hr. When these cells were grown in the presence of sublethal concentrations of amethopterin, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values were increased to 39 and 90 hr. These results suggest that the transient increase in dihydrofolate reductase activity, observed when cells are exposed to amethopterin, is due largely to a decreased susceptibility of the enzyme-inhibitor complex to degradation. Bound TPNH also increases the half-life of dihydrofolate reductase as shown by the fact that forms (I) and (II) of the L. casei enzyme had $\text{t}{}_{\mathrm{<mtext>x1</mtext><mtext>2</mtext>}}$ values of approximately 3 and 9 hr.     

Transport and metabolism of N-δ-chloroacetyl-l-ornithine by Saccharomyces cerevisiae

The general amino acid transport system of Saccharomyces cerevisiae functions in the uptake of neutral, basic, and acidic amino acids (M. Grenson, C. Hou, and M. Crabeel, 1970,J. Bacteriol. 103, 770–777; J. Rytka, 1975,J. Bacteriol.121, 562–570; C. Darte and M. Grenson, 1975,Biochem. Biophys. Res. Commun.67, 1028–1033). We have previously demonstrated that this transport system can be inhibited by the amino acid, N-δ-chloroacetyl-l-ornithine (NCAO) (F. S., Larimore and R.J. Roon, 1978,Biochemistry17, 431–436). In the present study radiolabeled NCAO was synthesized and its transport and metabolism studied. Under initial rate conditions: (a) NCAO was transported by the general amino acid transport system with a K_m of 52 μm, a V of 32 nmol/min/mg cells, and a pH optimum of 5.0; (b) the V for NCAO transport in gap mutants, which lack the general amino acid transport system, was approximately 1% of that observed with wild-type cells; (c) the V for NCAO in cells deprived of glucose was less than 5% of that observed when glucose was present. NCAO was transiently concentrated more than 1000-fold by yeast cells when glucose served as an energy source. The internal pool of NCAO was metabolized by the yeast cells and the products were excreted. When 100 μm [¹⁴C]NCAO was incubated with a yeast cell suspension for 8 h, more than 95% of the compound was converted into two ninhydrin-negative excretory products. The effect of NCAO on the growth of yeast cells was determined. Wild-type strains did not grow when 1 mm NCAO was present in the medium. The growth of gap mutants was not inhibited by 1 mm NCAO.     

Improving the catalytic efficiency of aldo-keto reductase KmAKR towards t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate via semi-rational design

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is an important chiral diol synthon of atorvastatin calcium. Previously, we constructed a variant KmAKR-W297H (M1) of Kluyveromyces marxianus aldo-keto reductase (KmAKR, designated as M0), possessing excellent diastereoselectivity but moderate activity towards t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1). In this work, KmAKR-W297H/Y296W/K29H (M3) was developed via semi-rational design. It exhibited much improved catalytic efficiency towards (5R)-1. The K_m values of M3 for NADPH and (5R)-1 were 0.15 mmol/L and 1.41 mmol/L, and the maximal reaction rate v_max was 55.56 μmol/min/mg. Compared with M1, the catalytic efficiency k_cat/K_m of M3 was increased 2.64-fold. Coupled with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration, M3 took 3.5 h to completely reduce (5R)-1 at up to 100.0 g/L, producing 237.4 mmol/L (3R,5R)-2 in d.e._P value above 99.5%. The space-time yield (STY) of M3-catalyzed (3R,5R)-2 synthesis was 372.8 g/L/d.     

Cholinergic and neuroprotective drugs for the treatment of Alzheimer and neuronal vascular diseases. II. Synthesis, biological assessment, and molecular modelling of new tacrine analogues from highly substituted 2-aminopyridine-3-carbonitriles

The synthesis, biological assessment, and molecular modelling of new tacrine analogues 11-22 is described. Compounds 11-22 have been obtained by Friedländer-type reaction of 2-aminopyridine-3-carbonitriles 1-10 with cyclohexanone or 1-benzyl-4-piperidone. The biological evaluation showed that some of these molecules were good AChE inhibitors, in the nanomolar range, and quite selective regarding the inhibition of BuChE, the most potent being 5-amino-2-(dimethylamino)-6,7,8,9-tetrahydrobenzo[1,8-b]-naphthyridine-3-carbonitrile (11) [IC₅₀ (EeAChE: 14 nM); IC₅₀ (eqBuChE: 5.2 ??M]. Kinetic studies on the easily available and potent anticholinesterasic compound 5-amino-2-(methoxy)-6,7,8,9-tetrahydrobenzo[1,8-b]-naphthyridine-3-carbonitrile (16) [IC₅₀ (EeAChE: 64 nM); IC₅₀ (eqBuChE: 9.6 ??M] showed that this compound is a mixed-type inhibitor (K_i = 69.2 nM) of EeAChE. Molecular modelling on inhibitor 16 confirms that this compound, as expected and similarly to tacrine, binds at the catalytic active site of EeAChE. The neuroprotective profile of molecules 11-22 has been investigated in SH-SY5Y neuroblastoma cells stressed with a mixture of oligomycin-A/rotenone. Compound 16 was also able to rescue by 50% cell death induced by okadaic acid in SH-SY5Y cells. From these results we conclude that the neuroprotective profile of these molecules is moderate, the most potent being compounds 12 and 17 which reduced cell death by 29%. Compound 16 does not affect ACh- nor K⁺-induced calcium signals in bovine chromaffin cells. Consequently, tacrine analogues 11-22 can be considered attractive therapeutic molecules on two key pharmacological targets playing key roles in the progression of Alzheimer, that is, cholinergic dysfunction and oxidative stress, as well as in neuronal cerebrovascular diseases.     

Identification of 6-acetyl-7-methyl-7,8-dihydropterin as a degradation product of 5,10-methenyl-5,6,7,8-tetrahydromethanopterin

During purification procedures and upon aerobic heating with alkali a green-yellow degradation fluorescent product (GY) was formed from 5,10-methenyl-5,6,7,8-tetrahydromethanopterin, an intermediate in the reduction of CO₂ to methane [J. T. Keltjens, L. Daniels, H. G. Janssen, and G. D. Vogels (1983)Eur. J. Biochem.130, 545–552]. GY was suggested to be a 6-(1-oxo)-7,8-dihydropterin. On the basis of the spectral properties and the results of degradation studies, it was now shown that the structure of GY is 6-acetyl-7-methyl-7,8-dihydropterin. This structure was confirmed by synthesis of the compound and other reference substances.     

Electrostatic control of enzyme reactions: The mechanism of inhibition of glucose oxidase by putrescine

The interaction of putrescine dihydrochloride with glucose oxidase is reported. At pH 7.65 glucose oxidase is strongly anionic (Z = ?80). The pK_a of an essential acidic group on the reduced form of the enzyme is extremely sensitive to ionic strength, as predicted by simple electrostatic theory [J. G. Voet, J. Coe, J. Epstein, V. Matossian, and T. Shipley (1981), Biochemistry, 20, 7182–7185]. Putrescine dihydrochloride was found to inhibit glucose oxidase at pH 7.65 at a constant ionic strength of 0.05. The kinetics do not obey simple competitive inhibition, however. The data can best be explained by a model in which change in the electrostatic potential of the enzyme on putrescine binding changes the observed pK_a of the essential acidic group. The pH dependence of putrescine inhibition supports this interpretation. At I = 0.05, 5 mM putrescine was found to change the pK_a of the essential acidic group from 7.6 to 7.1. The shift in the pK_a as a function of putrescine concentration at pH 7.7 and I = 0.05 also supports the model presented. The K_a for putrescine to the active form of the enzyme was calculated to be 4.2 mm.     

Studies on ribonucleoside-diphosphate reductase in permeable animal cells. II. Catalytic and regulatory properties of the enzyme in mouse L cells

Ribonucleoside-diphosphate reductase (EC 1.17.4.1) was studied in mouse L cells selectively permeabilized to small molecules by treatment with dextran sulfate (R. Kucera and H. Paulus, 1982, Arch. Biochem. Biophys. 214, 102–113). The reduction of CDP was almost completely dependent on added ATP or adenyl-5′-yl imidodiphosphate, and that of GDP on dTTP. The pattern of inhibition by deoxyribonucleoside triphosphates was similar to that observed by others in cell-free preparations except for a somewhat higher sensitivity to inhibition. The substrate saturation curves for CDP and GDP were hyperbolic with apparent K_m values of 0.05 and 0.24 mm, respectively. The maximum velocities for CDP and GDP reduction were close to the in vivo rate of DNA synthesis. Ribonucleotide reductase activity was not affected by the addition of ferric salts but was inhibited by the chelators bathophenanthroline sulfonate and thenoyltrifluoroacetone and also by hydroxyurea. EDTA caused a reversible stimulation of GDP reduction and an irreversible inhibition of CDP reduction; the latter could be partially reactivated by the addition of magnesium salts. Ribonucleotide reductase activity was inhibited by arsenite but only slightly stimulated by NADPH or dithiols; however, if the cells were first treated with 2,6-dichlorophenolindophenol, an almost complete dependence on NADPH was observed which could also be met by dithiothreitol or dihydrolipoic acid but not by reduced glutathione. This suggests that ribonucleotide reductase in dextran sulfate-treated L cells is relatively tightly coupled to an endogenous hydrogen donor system.