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Regiospecific syntheses of gamma- and alpha-conjugates of methotrexate and poly(L-lysine) are described. The alpha- and gamma-t-butyl esters, respectively, of methotrexate were coupled to poly(L-lysine) with diphenylphosphoryl azide in N,N-dimethylformamide, the ester-protecting group was cleaved with 15% hydrogen bromide in acetic acid, and small molecules were removed by dialysis. Poly(L-lysine) of Mr = 1,500-8,000 and 8,000-30,000 was used to prepare six different conjugates, which were characterized by ultraviolet absorbance measurement and quantitative amino acid analysis. The degree of substitution varied from one methotrexate per 4.7 lysines to one methotrexate per 10.2 lysines. Dihydrofolate reductase inhibition in a cell-free assay was observed with alpha- and gamma-conjugates, but the latter had the greater affinity (only 3-fold less than that of methotrexate itself). The binding of the conjugates exhibited a slight pH dependence, with affinity being greater at pH 7.2 than at pH 8.5 for both alpha- and gamma-conjugates. Toxicity to cultured rat hepatoma cells (H35) was also greater for the gamma-conjugates, and showed some dependence on the chain-length and degree of substitution of the poly(L-lysine) carrier. Cells resistant to methotrexate by virtue of a transport defect (H35R0.3 line) retained their sensitivity to the gamma-conjugate, but less so to the alpha-conjugate. There was also some retention of sensitivity in a more highly resistant cell line (H35R10) with impaired methotrexate transport and a concomitant increase in dihydrofolate reductase activity. gamma-Conjugation was likewise more favorable in cytotoxicity assays against L1210 murine leukemia cells, and there was partial retention of activity against highly methotrexate-resistant lines (L1210/R71 and L1210/R81) with a transport defect and/or an elevation of dihydrofolate reductase content. In antitumor assays against intraperitoneal L1210 leukemia in mice, a gamma-conjugate with Mr = 8,000-30,000 and one methotrexate per 5.5 lysines produced a 35-75% increase in lifespan when administered intraperitoneally at single doses equivalent to 10-20 mg/kg of methotrexate. A similar increase in lifespan with methotrexate alone on the single-dose regimen required 50-150 mg/kg. An alpha-conjugate of similar Mr and degree of substitution was inactive at nontoxic doses, as were other gamma-conjugates of lower Mr and/or degree of substitution.(ABSTRACT TRUNCATED AT 400 WORDS)

The kinetics of inhibitor binding to highly purified recombinant human dihydrofolate reductase (rHDHFR) have been examined. Methotrexate (MTX) binds rapidly (kon = 1.0 x 10(8) M-1 s-1) and tightly (koff/kon = 210 pM) to the preformed complex of rHDHFR with NADPH. The initial association reaction between rHDHFR.NADPH and MTX is followed by an isomerization of the resulting complex (kiso = 0.4 s-1) leading to a new conformer in which MTX is bound even more tightly (Ki = 3.4 pM). Similar results have been obtained with a major metabolite of MTX having four additional glutamate residues for which Ki = 1.4 pM. 7-HydroxyMTX, another major metabolite of MTX, is a weak inhibitor of rHDHFR (Ki = 8.9 nM), and a polyglutamate form of this metabolite is an equally weak inhibitor (Ki = 9.9 nM), so that the addition of glutamate residues to MTX or 7-hydroxyMTX has little effect on their binding. It follows that the significance of MTX polyglutamate formation relates to other roles such as increasing the cytotoxicity of MTX by prolonging intracellular retention of the drug. Another antifolate, trimethoprim, binds tightly to dihydrofolate reductases from bacterial sources, but weakly to rHDHFR in the ternary complex (KD = 0.5 microM). Although the association step is rapid (kon = 0.4 x 10(8) M-1 s-1), the dissociation rate is also rapid (koff = 15 s-1). Furthermore, there is no isomerization of the ternary complex of trimethoprim with rHDHFR, in contrast to the known isomerization of complexes of trimethoprim with bacterial dihydrofolate reductases.

Chicken liver dihydrofolate reductase is rapidly and stoichiometrically inactivated by a substituted 4,6-diaminodihydrotriazine containing a terminal benzenesulfonylfluoride (DTBSF). The substrate dihydrofolate largely prevents the enzyme inhibition by DTBSF, whereas NADPH had no effect, indicating that the inhibitor is bound at or near the folate site. Using radiolabeled inhibitor between 1.0 and 1.2 mol was incorporated/mol of enzyme (Mr = 21,651), following treatment with 8 M urea at 75 degrees C. Digestion of the maleylated, radiolabeled inhibitor-enzyme complex with trypsin and subsequent gel filtration on Sephadex G-50 SF yielded a single major peak of radioactivity. The covalently modified limited tryptic peptide was subsequently purified to homogeneity using high performance liquid chromatography. The radiolabeled tryptic peptide had the following sequence: Asn-Glu-Tyr (DTBS)-Lys-Tyr-Phe-Gln-Arg (residues 29-36). Automated Edman degradation of this peptide revealed that the radioactivity derived from the inhibitor was released at Step 3, identifying tyrosine-31 as the specific site of covalent attachment of the affinity label.

Transient and steady-state kinetics have been examined for dihydrofolate reductase (DHFR) from a number of sources. Rates of hydride transfer at pH 7.65 cover a wide range, from 7 s−1for DHFR from a strain of Lactobacillus casei(LCDHFR1) to 3000 s−1for recombinant human DHFR (rHDHFR). In all cases as the pH is increased from 7 to 10, Vmaxfor the steady-state reaction decreases, and DVmax, the primary isotope effect, increases. This indicates a decrease in the rate of hydride transfer with increasing pH. The cross-over points, at which rates of product release and hydride transfer become equal, were calculated to occur at DVmax= 2.34. The higher the rate of hydride transfer at pH 7.65, the higher the pH of the cross-over point. For LCDHFR1 the low rate of hydride transfer results in this process being partially rate-limiting for the steady-state reaction even at pH 5, with a cross-over point at about pH 7.

Dihydrofolate reductase from Candida albicanswas purified 31,000-fold and characterized. In addition, the C. albicansdihydrofolate reductase gene was cloned into a plasmid vector and expressed in Escherichia coli, and the enzyme was purified from this source. Both preparations showed a single proteinstaining band with a molecular weight of about 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymes were stable and had an isoelectric point of pH 7.1 on gel isoelectric focusing. Kinetic characterization showed that the enzymes from each source had similar turnover numbers (about 11,000 min−1) and Kmvalues for NADPH and dihydrofolate of 3–4 µM. Like other eukaryotic dihydrofolate reductases, the C. albicansenzyme exhibited weak binding affinity for the antibacterial agent trimethoprim (Ki= 4 βM), but further characterization showed that the inhibitor binding profile of the yeast and mammalian enzymes differed. Methotrexate was a tight binding inhibitor of human but not C.albicansdihydrofolate reductase; the latter had a relatively high methotrexate Kiof 150 pM. The yeast and vertebrate enzymes also differed in their interactions with KCl and urea. These two agents activate vertebrate dihydrofolate reductases but inhibited the C. albicansenzyme. The sequence of the first 36 amino-terminal amino acids of the yeast enzyme was also determined. This portion of the C. albicansenzyme was more similar to human than to E. colidihydrofolate reductases (50% and 30% identity, respectively). Some key amino acid residues in the C. albicanssequence, such as E-30 (human enzyme numbering), were “vertebrate-like” whereas others, such as I-31, were not. These results indicate that there are physical and kinetic differences between the eukaryotic mammalian and yeast enzymes.

The transient state kinetics of catalysis for dihydrofolate reductase (DHFR) from several enzyme sources including highly purified recombinant human enzyme (rHDHFR) have been examined. Like DHFR from Escherichia coli, the enzyme from Lactobacillus casei, and isoenzyme 2 from Streptococcus faeciumexhibit a slow increase in activity upon addition of substrates to enzyme. No slow hysteresis of this type was detected with recombinant human DHFR (rHDHFR) or DHFR from chicken or bovine liver or L1210 mouse leukemia cells (MDHFR). In contrast, both rHDHFR and MDHFR exhibited a very rapid decrease in activity (t½= 30 and 20 ms, respectively) during a phase that occurred after the first turnover of the enzyme but before establishment of the steady state. This intermediate phase was not observed for the bacterial enzymes or the avian enzyme, nor was it observed with a mutant of rHDHFR in which Phe-31 has been replaced by leucine. For rHDHFR the intermediate phase is not a consequence of product inhibition, substrate depletion, or enzyme instability. It may therefore be concluded that this unusual transient state kinetic behavior results from the existence of two conformers of the enzyme, one of which has a higher turnover number than the other with the equilibrium shifting in favor of the less active conformer during the course of catalysis. The equilibrium is particularly favorable for the less active conformer when NADP is present in the active site of rHDHFR, whereas bound tetrahydrofolate favors the more active conformer. The more active conformer has a 6-fold higher Kmfor dihydrofolate than does the less active conformer. The existence of these conformers is likely to produce cooperative behavior by rHDHFR in vivo.