(1/161) Photochemical studies and ultraviolet sensitization of Escherichia coli thymidylate kinase by various halogenated substrate analogs.

The effect of 5-iodo-2'-deoxyuridine monophosphate (IdUMP), various 5-halogenated-5'-azido-2', 5' -dideoxyuridine derivatives, 2'-deoxy-6-azauridine (AzdUrd), and its halogenated analogs on the ultraviolet sensitization of Escherichia coli thymidylate kinase has been investigated. Only those compounds iodinated in position 5 enhance the rate of ultraviolet inactivation of this enzyme. However, 5'-azido nucleosides with iodo, bromo, chloro, or fluoro substituents in position 5 neither protect nor sensitize thymidylate kinase to ultraviolet inactivation. Thymidine 5'-monophosphate partially protects the enzyme against ultraviolet inactivation either in the presence or absence of ultraviolet-sensitizing iodinated analogs. Magnesium ion does not enhance the ultraviolet inactivation of thymidylate kinase by 5-iodinated nucleoside analogs. The kinatic data support an active site-directed enhancement of the enzyme to ultraviolet inactivation by 5-iodo-2'-deoxyuridine monophosphate, since the concentration of IdUMP required to attain 50% maximal enhancement is 0.24 mM which is in good agreement with its Ki of 0.18 mM. When either [125I]IdUMP or [2-14C]IdUMP was irradiated with the enzyme, both radioactivities were associated with the enzyme, however only with the 14C analog was the amount bound at half-saturation essentially equal to the amount required to inactivate the enzyme by 50%. These data support the hypothesis that the active entity in the enhancement by IdUMP of thymidylate kinase inactivation during ultraviolet irradiation is the uridylate free radical which is formed photochemically from IdUMP. Photochemical studies of 6-azauracil (AzUra), 2'-deoxy-6-azauridine, and 5-iodo-2'-deoxy-6-azauridine (IAzdUrd) were performed. Photolysis of IAzdUrd in the presence of a hydrogen donor yields AzdUrd which upon further photolysis yields the photohydrate. The photohydrate of AzdUrd when incubated in the dark at pH 5.2 is 90% converted back to AzdUrd, whereas the photohydrate of AzUra is only partially (20%) converted to AzUra. The rate of deiodination of IAzdUrd is 2.1-fold greater than that of IdUMP. Although the Ki of IdUMP and IAzdUrd is similar, the increased photosensitivity of the aza analog accounts for the much greater enhancement of ultraviolet inactivation of thymidylate kinase. The ability of a compound to enhance the ultraviolet inactivation of deoxythymidylate kinase is correlated with the potential of the compound to produce a free radical rather than a photohydrate when the enzyme-substrate analog complex is irradiated.  (+info)

(2/161) Synthesis, characterization and preclinical formulation of a dual-action phenyl phosphate derivative of bromo-methoxy zidovudine (compound WHI-07) with potent anti-HIV and spermicidal activities.

In a systematic effort to develop a microbicide contraceptive capable of preventing transmission of human immunodeficiency virus (HIV), as well as providing fertility control, we have previously identified novel phenyl phosphate derivatives of zidovudine (ZDV) with 5-halo 6-alkoxy substitutions in the thymine ring and halo substitution in the phenyl moiety respectively. Here, we describe the synthesis, characterization, and successful preclinical formulation of our lead compound, 5-bromo-6-methoxy-3'-azidothymidine-5'-(p-bromophenyl) methoxyalaninyl phosphate (WHI-07), which exhibits potent anti-HIV and sperm immobilizing activities. The anti-HIV activity of WHI-07 was tested by measuring viral p24 antigen production and reverse transcriptase activity as markers of viral replication in HIV-1 infected human peripheral blood mononuclear cells (PBMC). WHI-07 inhibited replication of HIV in a concentration-dependent fashion with nanomolar IC50 values. The effects of WHI-07 on human sperm motion kinematics were analysed by computer-assisted sperm analysis (CASA), and its effects on sperm membrane integrity were examined by confocal laser scanning microscopy (CLSM), and high-resolution low-voltage scanning electron microscopy (HR-LVSEM). WHI-07 caused cessation of sperm motility in a concentration- and time-dependent fashion. The in-vitro cytotoxicities of WHI-07 and nonoxynol-9 (N-9) were compared using normal human ectocervical and endocervical epithelial cells by the MTT cell viability assay. Unlike N-9, WHI-07 had no effect upon sperm plasma and acrosomal membrane integrity. N-9 was cytotoxic to normal human ectocervical and endocervical cells at spermicidal doses, whereas WHI-07 was selectively spermicidal. The in-vivo vaginal absorption and vaginal toxicity of 2% gel-microemulsion of WHI-07 was studied in the rabbit model. The sperm immobilizing activity of WHI-07 was 18-fold more potent than that of N-9. Over a 10 day period, there was no irritation or local toxicity to the vaginal epithelia or systemic absorption of WHI-07. Therefore, as a potent anti-HIV agent with spermicidal activity, and lack of mucosal toxicity, WHI-07 may have the clinical potential to become the active ingredient of a vaginal contraceptive for women who are at high risk for acquiring HIV by heterosexual vaginal transmission.  (+info)

(3/161) Impact of polyglutamation on sensitivity to raltitrexed and methotrexate in relation to drug-induced inhibition of de novo thymidylate and purine biosynthesis in CCRF-CEM cell lines.

The aim of this study was to investigate the influence of folylpolyglutamyl synthetase (FPGS) activity on the cellular pharmacology of the classical antifolates raltitrexed and methotrexate (MTX) using two human leukemia cell lines, CCRF-CEM and CCRF-CEM:RC2Tomudex. Cell growth inhibition and drug-induced inhibition of de novo thymidylate and purine biosynthesis were used as measures of the cellular effects of the drugs. CCRF-CEM:RC2Tomudex cells had <11% of the FPGS activity of CCRF-CEM cells, whereas MTX uptake and TS activity were equivalent. In CCRF-CEM:RC2Tomudex cells, MTX polyglutamate formation was undetectable after exposure to 1 microM [3H]MTX for 24 h. After exposure to 0.1 microM raltitrexed, levels of total intracellular raltitrexed-derived material in CCRF-CEM:RC2Tomudex cells were 30- to 50-fold lower than in the CCRF-CEM cell line. CCRF-CEM: RC2Tomudex cells were >1000-fold resistant to raltitrexed and 6-fold resistant to lometrexol but sensitive to MTX and nolatrexed when exposed to these antifolates for 96 h. After 6 h of exposure, CCRF-CEM cells retained sensitivity to MTX and raltitrexed but were less sensitive to lometrexol-mediated growth inhibition. In contrast, CCRF-CEM: RC2Tomudex cells were markedly insensitive to raltitrexed, lometrexol, and to a lesser degree, MTX. Simultaneous measurement of de novo thymidylate and purine biosynthesis revealed 90% inhibition of TS activity by 100 nM MTX in both cell lines, whereas inhibition of de novo purine synthesis was only observed in CCRF-CEM cells, and only after exposure to 1000 nM MTX. Ten nM raltitrexed induced >90% inhibition of TS activity in CCRF-CEM cells, whereas in CCRF-CEM:RC2Tomudex cells, there was no evidence of inhibition after exposure to 1000 nM raltitrexed. These studies demonstrate that polyglutamation is a critical determinant of the cellular pharmacology of both raltitrexed and MTX, markedly influencing potency in the case of raltitrexed and locus of action in the case of MTX.  (+info)

(4/161) Structural requirements for potent human spermicidal activity of dual-function aryl phosphate derivative of bromo-methoxy zidovudine (compound WHI-07).

WHI-07, a novel bromo-methoxy-substituted aryl phosphate derivative of zidovudine (ZDV), is a potent dual-function contraceptive agent. Although the bromo-methoxy functional groups in the thymine ring of its ZDV are very important for its sperm-immobilizing activity (SIA), the importance of the esterification of the phosphate group with an amino acid side chain and the identity of the para substituent in the aryl moiety remain unclear. In the present study, we have synthesized 23 new analogues of WHI-07 by replacing the alanine (Ala) side chain with different amino acids containing nonpolar side chains, namely tryptophan (Trp), proline (Pro), phenylalanine (Phe), leucine (Leu), methionine (Met), valine (Val), or glycine (Gly). The para substituents on the aryl moiety included bromo, chloro, fluoro, nitro, or methoxy groups. The SIA of each of the 23 WHI-07 analogues was evaluated by computer-assisted sperm analysis. The potential cytotoxicity of these compounds against normal human ectocervical and endocervical epithelial cells was evaluated using MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) cell viability assays. The replacement of the Ala side chain of WHI-07 with Val, Leu, or Phe led to a complete loss of SIA (EC(50) values > 500 microM), whereas replacement with Trp reduced the SIA by 4-fold. The presence of para substituents on the phenyl moiety led to significant alterations in SIA. The anti-human immunodeficiency virus (HIV) activity of Trp-containing WHI-07 analogues was also diminished. Our finding highlights the necessity of Ala side chain and the presence of electron-withdrawing para-bromo substituent on the phenyl moiety in addition to bromo-methoxy functionalization groups on the thymine ring in order for the phosphoramidate derivatives of ZDV to be effective dual-function spermicidal agents. Unlike the detergent-type microbicide, nonoxynol-9, which was cytotoxic to normal human ectocervical and endocervical epithelial cells (IC(50) values of 22 microM and 16 microM, respectively) at spermicidal concentrations (EC(50) = 81 microM), WHI-07 and its active analogues were selectively spermicidal without cytotoxicity against female genital tract epithelial cells. WHI-07 and its Trp analogues hold particular clinical promise for the development of novel, nondetergent-type prophylactic contraceptives for the prevention of heterosexual HIV/acquired immunodeficiency syndrome transmission.  (+info)

(5/161) Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate.

BACKGROUND: Thymidylate kinase (TMPK) is a nucleoside monophosphate kinase that catalyzes the reversible phosphoryltransfer between ATP and TMP to yield ADP and TDP. In addition to its vital role in supplying precursors for DNA synthesis, human TMPK has an important medical role participating in the activation of a number of anti-HIV prodrugs. RESULTS: Crystal structures of human TMPK in complex with TMP and ADP, TMP and the ATP analog AppNHp, TMP with ADP and the phosphoryl analog AlF(3), TDP and ADP, and the bisubstrate analog TP(5)A were determined. The conformations of the P-loop, the LID region, and the adenine-binding loop vary according to the nature of the complex. Substitution of ADP by AppNHp results in partial closure of the P-loop and the rotation of the TMP phosphate group to a catalytically unfavorable position, which rotates back in the AlF(3) complex to a position suitable for in-line attack. In the fully closed state observed in the TP(5)A and the TDP-ADP complexes, Asp15 interacts strongly with the 3'-hydroxyl group of TMP. CONCLUSIONS: The observed changes of nucleotide state and conformation and the corresponding protein structural changes are correlated with intermediates occurring along the reaction coordinate and show the sequence of events occurring during phosphate transfer. The low catalytic activity of human TMPK appears to be determined by structural changes required to achieve catalytic competence and it is suggested that a mechanism might exist to accelerate the activity.  (+info)

(6/161) A deoxyribonucleotidase in mitochondria: involvement in regulation of dNTP pools and possible link to genetic disease.

Three cytosolic and one plasma membrane-bound 5'-nucleotidases have been cloned and characterized. Their various substrate specificities suggest widely different functions in nucleotide metabolism. We now describe a 5'-nucleotidase in mitochondria. The enzyme, named dNT-2, dephosphorylates specifically the 5'- and 2'(3')-phosphates of uracil and thymine deoxyribonucleotides. The cDNA of human dNT-2 codes for a 25.9-kDa polypeptide with a typical mitochondrial leader peptide, providing the structural basis for two-step processing during import into the mitochondrial matrix. The deduced amino acid sequence is 52% identical to that of a recently described cytosolic deoxyribonucleotidase (dNT-1). The two enzymes share many catalytic properties, but dNT-2 shows a narrower substrate specificity. Mitochondrial localization of dNT-2 was demonstrated by the mitochondrial fluorescence of 293 cells expressing a dNT-2-green fluorescent protein (GFP) fusion protein. 293 cells expressing fusion proteins without leader peptide or with dNT-1 showed a cytosolic fluorescence. During in vitro import into mitochondria, the preprotein lost the leader peptide. We suggest that dNT-2 protects mitochondrial DNA replication from overproduction of dTTP, in particular in resting cells. Mitochondrial toxicity of dTTP can be inferred from a severe inborn error of metabolism in which the loss of thymidine phosphorylase led to dTTP accumulation and aberrant mitochondrial DNA replication. We localized the gene for dNT-2 on chromosome 17p11.2 in the Smith-Magenis syndrome-critical region, raising the possibility that dNT-2 is involved in the etiology of this genetic disease.  (+info)

(7/161) Cyclosaligenyl-2',3'-didehydro-2',3'-dideoxythymidine monophosphate: efficient intracellular delivery of d4TMP.

Cyclosaligenyl-2',3'-didehydro-2', 3'-dideoxythymidine-5'-monophosphate (cycloSal-d4TMP) is a potent and selective inhibitor of human immunodeficiency virus replication in cell culture and differs from other nucleotide prodrug approaches in that it is designed to selectively deliver the nucleotide 5'-monophosphate by a controlled, chemically induced hydrolysis. Its antiviral efficacy in cell culture is at least as good as, if not superior to, that of d4T. CycloSal-d4TMP was found to lead to the efficient intracellular release of d4TMP in a variety of cell lines, including both wild-type CEM and thymidine kinase-deficient CEM/TK(-) cells. Under similar experimental conditions, exposure of CEM/TK(-) cells to d4T failed to result in significant d4TTP levels. The intracellular conversion of cycloSal-d4TMP proved to be both time and dose dependent. The half-life of d4TTP generated intracellularly from d4T- or cycloSal-d4TMP-treated CEM cells was approximately 3.5 h, and the intracellular ratios of d4TTP/d4TMP in cells exposed to cycloSal-d4TMP gradually increased from 1 to 3.4 upon prolonged incubation. Radiolabeled cycloSal-d4TMP could be separated as its two R(p) and S(p) diastereomers on high-performance liquid chromatography. The R(p) diastereomer of cycloSal-d4TMP was 3- to 7-fold more efficient in releasing d4TMP and generating d4TTP than the S(p) cycloSal-d4TMP diastereomer. This correlated well with the 5-fold more pronounced antiviral activity of the R(p) diastereomer versus the S(p) diastereomer. d4TMP is a poor substrate for the cytosolic 5'(3')-deoxyribonucleotidase (V(max)/K(m) for d4TMP: 0.08 of V(max)/K(m) for dTMP) and is only slowly hydrolyzed to d4T. This contributes to the efficient conversion of the prodrug of d4TTP.  (+info)

(8/161) Differential removal of thymidine nucleotide analogues from blocked DNA chains by human immunodeficiency virus reverse transcriptase in the presence of physiological concentrations of 2'-deoxynucleoside triphosphates.

Removal of 2',3'-didehydro-3'-deoxythymidine-5'-monophosphate (d4TMP) from a blocked DNA chain can occur through transfer of the chain-terminating residue to a nucleotide acceptor by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). ATP-dependent removal of either d4TMP or 3'-azido-3'-deoxythymidine-5'-monophosphate (AZTMP) is increased in AZT resistant HIV-1 RT (containing D67N/K70R/T215F/K219Q mutations). Removal of d4TMP is strongly inhibited by the next complementary deoxynucleoside triphosphate (50% inhibitory concentration [IC(50)] of approximately 0.5 microM), whereas removal of AZTMP is much less sensitive to this inhibition (IC(50) of >100 microM). This could explain the lack of cross-resistance by AZT-resistant HIV-1 to d4T in phenotypic drug susceptibility assays.  (+info)