Effectors of HIV-1 protease peptidolytic activity.

High concentrations of salts dramatically affect the interaction of small ligands with HIV-1 protease. For instance, the Km and kcat values for Abz-Thr-Ile-Nle-p-nitro-Phe-Gln-Arg-NH2 (S) increased 120-fold and 3-fold, respectively, as the NaCl concentration in the assay decreased from 4.0 to 0.5 M. The Kd value for the competitive inhibitor amprenavir increased 12-fold over this concentration range of NaCl. The bimolecular rate constant for association of enzyme with amprenavir was independent of NaCl concentration, whereas the dissociation rate constant decreased with increasing NaCl concentration. Polyanionic polymers such as heparin or poly A substituted for NaCl. For example, the value of kcat/Km for S was 0.18 microM(-1) x s(-1) when the enzyme (<10 nM) was assayed in the standard buffer supplemented with 5 mM NaCl. If 0.01% poly A were included, the value of kcat/Km increased to 8.6 microM(-1) x s(-1). A DNA oligomer (23-mer) with an hexachlorofluoresceinyl moiety linked to the 5' end was studied as a model polyanionic polymer. The enzyme bound HF23 (Kd < 1 nM) with concomitant quenching of the hexachlorofluoresceinyl fluorescence. The stoichiometry for binding was 3 mol of enzyme per mol of oligomer. The hydrolytic activity of the enzyme with this oligomer was similar to that observed with poly A or high salt concentration when the molar ratio of oligomer to enzyme was greater than one. The results presented herein demonstrate that polyanionic polymers substitute for salts as effectors of HIV protease.

The E1 helicase of human papillomavirus type 11 binds to the origin of replication with low sequence specificity.

Expression of the human papillomavirus type 11 E1 and E2 genes is necessary and sufficient to support viral DNA replication. The full-length E2 protein is a transcriptional modulator that also interacts with the E1 helicase to form an E1/E2 complex at the viral origin of replication. Previous studies indicated that efficient binding of this complex to the replication origin is site-specific and that the E2 homodimer was required for efficient E1 binding. Human papillomavirus type 11 E2 and E1 proteins have been purified and their cooperative binding to the HPV type 11 viral replication origin has been characterized. Low-affinity E1 binding to the HPV type 11 replication origin was demonstrated and found to be largely nonspecific. DNA binding by E1 does not require complex formation with E2 and appears to be independent of ATP binding or hydrolysis. E1 binding quantitatively increased with the addition of increasing amounts of E2 and mutations in the E2 binding site demonstrated that the E2BS site is required for E1 and E2 to specifically bind as a high-affinity complex at the replication origin. Analysis of the A/T-rich E1 binding site via mutation showed that it was nonessential for high-affinity E1/E2 complex formation. Thus, although the replication functions between the animal and the human papillomaviruses are well conserved, there are subtle differences in the DNA binding requirements for E1, which may portend mechanistic differences among the DNA replication systems of various papillomavirus types.

Expression and characterization of the HCV NS3 helicase domain.

The hepatitis C virus (HCV) NS3 protein has two distinct biochemical domains. The N-terminal 20 kDa has serine protease activity (see Chapter 31 ) and the C-terminal 50 kDa has both nucleoside triphosphatase (NTPase) and helicase activities (1-4).

Product release is the major contributor to kcat for the hepatitis C virus helicase-catalyzed strand separation of short duplex DNA.

Hepatitis C virus (HCV) helicase catalyzes the ATP-dependent strand separation of duplex RNA and DNA containing a 3' single-stranded tail. Equilibrium and velocity sedimentation centrifugation experiments demonstrated that the enzyme was monomeric in the presence of DNA and ATP analogues. Steady-state and pre-steady-state kinetics for helicase activity were monitored by the fluorescence changes associated with strand separation of F21:HF31 that was formed from a 5'-hexachlorofluorescein-tagged 31-mer (HF31) and a complementary 3'-fluorescein-tagged 21-mer (F21). kcat for this reaction was 0.12 s-1. The fluorescence change associated with strand separation of F21:HF31 by excess enzyme and ATP was a biphasic process. The time course of the early phase (duplex unwinding) suggested only a few base pairs ( approximately 2) were disrupted concertedly. The maximal value of the rate constant (keff) describing the late phase of the reaction (strand separation) was 0. 5 s-1, which was 4-fold greater than kcat. Release of HF31 from E. HF31 in the presence of ATP (0.21 s-1) was the major contributor to kcat. At saturating ATP and competitor DNA concentrations, the enzyme unwound 44% of F21:HF31 that was initially bound to the enzyme (low processivity). These results are consistent with a passive mechanism for strand separation of F21:HF31 by HCV helicase.

In vitro uptake, anabolism, and cellular retention of 1843U89 and other benzoquinazoline inhibitors of thymidylate synthase.

1843U89 is a potent inhibitor (Ki = 0.09 nM) of thymidylate synthase (TS; EC 2.1.1.45) that is in clinical trial for the treatment of solid tumors. Although it is an excellent substrate for the folate anabolizing enzyme folylpolyglutamate synthetase (FPGS), 1843U89 differs from other folate-based inhibitors of TS (e.g., CBC3717, D1694, and LY231514), in that the parent compound is as potent an enzyme inhibitor as its polyglutamated analogues. As reported (D.S. Duch et at., Cancer Res., 53:810-818, 1993), 1843U89 is 10-80-fold more cytotoxic than the close structural analogue 1031U89, which is an equipotent inhibitor of TS but is a less efficient substrate for FPGS. This correlation between substrate efficiency for FPGS and cytotoxicity suggests that polyglutamation of 1843U89 contributes to its cytotoxicity. In the current study, we measured intracellular levels of polyglutamated anabolites of 1031U89, 1843U89, and three other benzoquinazoline inhibitors of TS as well as anabolites of D1694 in HCT-8 ileocecal carcinoma cells. Each TS inhibitor was anabolized to polyglutamated analogues with one to five added glutamyl residues after exposure for 24 h to IC90 concentrations (those that inhibit growth by 90% after 72 h of constant exposure). D1694, which requires polyglutamation for potent enzyme inhibition as well as for cytotoxicity, was anabolized mostly to penta- and hexaglutamates, whereas approximately 80% of intracellular 1843U89 was the diglutamate analogue. The substrate efficiency of the benzoquinazolines for FPGS was predictive of the extent of intracellular anabolism. The diglutamate analogue of 1843U89 was only 1/100 as efficient a substrate for further glutamation as was 1843U89 itself. The efficient anabolism to the diglutamate analogue and the lack of dependence on further polyglutamation for enzyme inhibition or cytotoxicity provide a rationale for the reported 1843U89 sensitivity of cells with impaired FPGS activity. As part of an investigation of the effects of polyglutamation, we measured the retention of intracellular 1843U89 and D1694 anabolites after 24 h of exposure to 20 nM of each compound. After 48 h in drug-free medium, 7% of intracellular 1843U89 (mostly diglutamate analogue) and 36% of D1694 (mostly penta- and hexaglutamates) remained in the cells. Because prolonged retention (associated with tissue storage of polyglutamates) can contribute to clinical toxicities, 1843U89 may present fewer long-term toxicities than D1694.

Enzymatic synthesis of folate and antifolate polyglutamates with Escherichia coli folylpolyglutamate synthetase.

Escherichia coli folylpolyglutamate synthetase was used to synthesize micromole quantities of polyglutamyl conjugates of folic acid, methotrexate, and other analogs of folic acid. The products of the enzymatic reactions were purified by semipreparative C18 HPLC. The position of each amide linkage (gamma or alpha carboxyl) in the polyglutamated products was determined by limited and exhaustive hydrolyses with hog kidney folylpolyglutamate hydrolase and with yeast carboxypeptidase Y. Under standard reaction conditions, the E. coli enzyme added up to five glutamyl residues to each monoglutamated substrate, primarily at the gamma carboxyl position. Thus, an enzyme which naturally adds only two glutamates to naturally occurring folates can be used synthetically to make higher polyglutamates of a wide range of synthetic substrates. The products of the reactions are valuable tools for the study of the metabolism of antifolate drugs as well as metabolic reactions involving folate cofactors.

A radiometric method for objectively screening large numbers of compounds against Pneumocystis carinii in vitro.

A relatively simple method is reported for accurately quantitating the incorporation of [3H]para aminobenzoic acid (pABA) into the folates of Pneumocystis carinii cultured in vitro, and the subsequent development of a highly sensitive and reproducible 96-well microtitre plate drug screening system. Incorporation of [3H]pABA under optimized conditions has been utilized as a selective indicator of the in vitro viability of P. carinii against which the inhibitory effects of potential drugs were quantified. The anti-Pneumocystis agents pentamidine, sulfamethoxazole, 566C80 and piritrexim gave median inhibitory concentration values of 7.3, 0.1, 1.4 and approximately 100 microM, respectively in this assay. The results suggest that this 96-well plate P. carinii [3H]pABA-incorporation system is suitable as a rapid high throughput primary in vitro screen for detecting compounds with anti-Pneumocystis activity.

Microculture screening assay for primary in vitro evaluation of drugs against Pneumocystis carinii.

Pneumocystis carinii inoculated into 96-well filtration plate assemblies was shown to synthesize radiolabeled folates de novo from [para-3H]aminobenzoic acid ([3H]pABA). At the end of each incubation with [3H]pABA, a vacuum manifold was used to remove the medium and wash P. carinii. The membrane at the base of each well was dried and punched out, and the level of 3H retained was determined by direct scintillation counting. High-pressure liquid chromatography analysis of duplicate filters confirmed that direct counting of 3H retained on membranes (after correction for unmetabolized [3H]pABA) was an accurate reflection of total [3H]pABA incorporation by P. carinii. Greater than 95% of the 3H recovered was shown to be present as polyglutamated species. After digestion with rat plasma folic acid gamma-glutamyl hydrolase, para-aminobenzoylglutamate, N10-formyltetrahydrofolate, and tetrahydrofolate were identified as the major 3H-labeled components. para-Aminobenzoylglutamate was presumed to have arisen from folylpolyglutamates synthesized by P. carinii and was therefore included in the calculation of total [3H]pABA incorporation. P. carinii incorporation of [3H]pABA under optimal conditions was used as a selective measure of in vitro viability against which the inhibitory effects of some antipneumocystis agents (pentamidine, sulfamethoxazole, 566C80, and piritrexim) were quantitated. The concentrations of pentamidine, sulfamethoxazole, 566C80, and piritrexim required for 50% inhibition in this assay were 7.3, 0.1, 1.4, and approximately 100 microM, respectively. The results suggest that this 96-well [3H]pABA incorporation assay has considerable potential for objective in vitro drug screening against P. carinii.

Studies on the mechanism of antitumor action of 2-desamino-2-methyl-5,8-dideazaisofolic acid.

The new folate analogue, 2-desamino-2-methyl-5,8-dideazaisofolic acid, 2c, was synthesized and evaluated using a variety of biochemical and antitumor assays. For purposes of comparison, its 2-desamino, 2b, and 2-amino, 2a, counterparts, as well as N10-propargly-5,8-dideazafolic acid, 1a, and the corresponding 2-desamino, 1b, and 2-desamino-2-methyl, 1c, modifications were included in these studies. Compound 2c was found to be a potent inhibitor of the growth of L1210 and MCF-7 cells in culture, being only 2-fold and 5-fold less effective than 1c, respectively. However, although analogue 2c was 189-fold less inhibitory toward L1210 thymidylate synthase (TS) than 1c, its cytotoxicity was reversed completely by thymidine alone which suggests that the compound behaves as a TS inhibitor in cells. Enzymatically synthesized polyglutamates of 2c were substantially more inhibitory toward human TS than the parent compound. Compound 2c was the most efficient substrate for mammalian folyl-polyglutamate synthetase of the compounds studied having a Vmax/Km nearly 12-fold larger than 1c. Both 1c and 2c were effective inhibitors of the uptake of [3H]methotrexate into MOLT-4 cells, implying that each is efficiently transported into tumor cells. These results suggest that a weak inhibitor of TS in vitro can be a potent cytotoxic agent if it can readily gain entry into target cells and be converted to polyglutamated metabolites.

In vivo and in vitro metabolism of 5-deazaacyclotetrahydrofolate, an acyclic tetrahydrofolate analogue.

5-Deazaacyclotetrahydrofolate is a cytotoxic tetrahydrofolate analogue which inhibits glycinamide ribonucleotide transformylase (Kelley et al., J. Med. Chem., 33: 561-567, 1990). Cultured mouse L-cells and human MCF-7 and MOLT-4 cells concentrated the drug several hundred-fold after 24 h of continuous exposure to a cytotoxic level (100-200 nM) of radiolabeled drug. High performance liquid chromatography analysis revealed that each cell type metabolized greater than or equal to 80% of the internalized drug to polyglutamated forms, which are more potent glycinamide ribonucleotide transformylase inhibitors. In L-cells, 45% of the polyglutamated metabolites were also N-formylated. The pharmacokinetics and distribution of [14C]-deazaacyclotetrahydrofolate were studied in C57BL/6 male mice. Its plasma half-life was 2.15 h. Radiolabel was concentrated to well above plasma level in the kidney, pancreas, and liver. Metabolism was examined in tumor-bearing and in normal mice. Twenty-four h after a single i.p. injection (50 mg/kg), drug equivalents were 0.6 nmol/g (83% polyglutamated) in colon-38 adenocarcinoma carried s.c., 2.4 nmol/g (100% polyglutamated) in ascitic P388 cells, and 3.7 nmol/g (76% polyglutamated and approximately 20% formylated) in mouse liver. Elimination was mostly in the urine as unmetabolized drug. Feces contained 5-deazaacyclotetrahydropteroate (parent compound less glutamate). In conclusion, 5-deazaacyclotetrahydrofolate was shown to be concentrated to well above the extracellular level and metabolized to more active polyglutamated forms by transformed cells grown in culture and in mice.

Synthesis and biological activity of an acyclic analogue of 5,6,7,8-tetrahydrofolic acid, N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5- pyrimidinyl)propyl]amino]-benzoyl]-L-glutamic acid.

The synthesis and biological evaluation of N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]amino]- benzoyl]-L-glutamic acid (1) (5-DACTHF, 543U76), an acyclic analogue of 5,6,7,8-tetrahydrofolic acid (THFA), are described. The key intermediate, hemiaminal 8, was prepared in four stages from 3-chloropropionaldehyde diethyl acetal. Reaction of 8 with dimethyl N-(4-aminobenzoyl)-L-glutamate gave the 2,4-bis(acetylamino) derivative 11, which was hydrolyzed with 1 N sodium hydroxide to give 1; the glycine analogue 16 was prepared in a similar manner. The N-methyl analogue 2 and N-formyl analogue 3 were prepared from 11 and 1, respectively. Compounds 1-3 inhibited growth of Detroit 98 and L cells in cell culture, with IC50s ranging from 2 to 0.018 microM. Cell culture toxicity reversal studies and enzyme inhibition tests showed that 1 was cytotoxic but not by the mechanism of the dihydrofolate reductase inhibitor aminopterin. Compound 1 and its polyglutamylated homologues inhibited glycinamide ribonucleotide transformylase (GAR-TFase) and aminoimidazole ribonucleotide transformylase (AICAR-TFase), the folate-dependent enzymes in de novo purine biosynthesis; and 1 was an effective substrate for mammalian folyl-polyglutamate synthetase. The compound inhibited (IC50 = 20 nM) the conversion of [14C]formate to [14C]-formylglycinamide ribonucleotide by MOLT-4 cells in culture. These data suggest that the site of action of 1 is inhibition of purine de novo biosynthesis. Moderate activity was observed against P388 leukemia in vivo.

alpha-Carboxyl-linked glutamates in the folylpolyglutamates of Escherichia coli.

Folate cofactors in most cells contain polyglutamate side chains, which since the late 1940s have been assumed to be linked via their gamma-COOH groups. We report here an investigation of the structure of the polyglutamate chain attached to the folates of Escherichia coli. Folates were extracted from E. coli grown with [7-14C] p-aminobenzoate and cleaved to p-aminobenzoyl polyglutamates of varying chain lengths (pAB(Glu)n) by the method of Foo et al. (Foo, S. K., Cichowicz, D. J., and Shane, B. (1980) Anal. Biochem. 107, 109-115). The pAB(Glu)n derived from E. coli did not co-chromatograph with chemically synthesized pAB(gamma-Glu)n-Glu on several high performance liquid chromatography (HPLC) systems, except for the triglutamate which did elute with pAB(gamma-Glu)2-Glu. E. coli-derived pAB(Glu)3-8 were purified by HPLC on C18 columns eluted with acetonitrile/trifluoroacetic acid, and the structures were determined through mass spectrometry, chiral amino acid analysis, and peptidase digestion experiments. Molecular weight determinations on the methyl ester derivatives of E. coli-derived pAB(Glu)n by liquid secondary ion mass spectrometry and sequence analysis using collision-activated dissociation on a tandem mass spectrometer confirmed the structures as pAB(Glu)3-8. Chiral HPLC of hydrolyzed and dansylated E. coli-derived materials, on a beta-cyclodextrin column, identified the glutamate as the L-enantiomer. pAB(Glu)n were digested with carboxypeptidase Y, which specifically cleaved glutamates linked at their alpha-carboxyls; E. coli-derived pAB(Glu)4-8 (but not synthetic pAB(gamma-Glu1-6-Glu) were sequentially digested to pAB(gamma-Glu)2-Glu. Thus, in E. coli folylpolyglutamates, glutamate residues 4-8 were each linked to the polyglutamate chain at the alpha-carboxyl of the preceding glutamate.

A kinetic analysis of the inhibition of rat and bovine trypsins by naturally occurring protease inhibitors.

The dissociation constants (Ki) of the interaction of 10 naturally occurring inhibitors with rat anionic and bovine trypsins were determined employing three independent methods. Both enzymes bound very tightly (Ki less than 10(-9)) to bovine pancreatic, lima bean, and the Kunitz soybean inhibitors. With the exception of ovomucoid, rat trypsin bound more tightly than bovine trypsin to inhibitors derived from navy bean, lima bean, soybean (Bowman-Birk) and potato and to ovoinhibitor, leupeptin and antipain. These findings emphasize the caution that must be exercised in the interpretation of experiments involving the inhibition of trypsins from heterologous species of animals by naturally occurring inhibitors.

Co-purification and characterization of UDP-glucose 4-epimerase and UDP-N-acetylglucosamine 4-epimerase from porcine submaxillary glands.

UDP-glucose 4-epimerase and UDP-N-acetylglucosamine 4-epimerase have been co-purified about 9,000-fold from porcine submaxillary glands by affinity chromatography on UDP-hexanolamine-agarose. The homogeneous epimerase has apparent Mr = 88,000 and contains two subunit species with apparent Mr = 37,000 and 35,000, respectively. The two subunits, however, are indistinguishable as judged by peptide mapping. The purified enzyme catalyzes equally well the reversible reactions UDP-glucose in equilibrium UDP-galactose and UDP-N-acetylglucosamine in equilibrium UDP-N-acetylgalactosamine. At saturating substrate concentrations, the ratio of the rate of the former reaction to that of the latter is 1.13 in the forward direction and 0.44 in the backward direction. Both reactions have the same Keq = 0.38 and the same dependence on pH. Moreover, both activities are lost at about the same rate by heat denaturation of the epimerase or reaction with N-ethylmaleimide. Kinetic analysis reveals that the reactants for one reaction are competitive inhibitors of the other reaction, with the Ki values of the inhibitors essentially identical with their Km values as substrates. Taken together, these studies suggest that UDP-glucose 4-epimerase and UDP-N-acetylglucosamine 4-epimerase activities reside in a single enzyme.