Tylophorine Analogs Allosterically Regulates Heat Shock Cognate Protein 70 And Inhibits Hepatitis C Virus Replication.

Tylophorine analogs have been shown to exhibit diverse activities against cancer, inflammation, arthritis, and lupus in vivo. In this study, we demonstrated that two tylophorine analogs, DCB-3503 and rac-cryptopleurine, exhibit potent inhibitory activity against hepatitis C virus (HCV) replication in genotype 1b Con 1 isolate. The inhibition of HCV replication is at least partially mediated through cellular heat shock cognate protein 70 (Hsc70). Hsc70 associates with the HCV replication complex by primarily binding to the poly U/UC motifs in HCV RNA. The interaction of DCB-3503 and rac-cryptopleurine with Hsc70 promotes the ATP hydrolysis activity of Hsc70 in the presence of the 3' poly U/UC motif of HCV RNA. Regulating the ATPase activity of Hsc70 may be one of the mechanisms by which tylophorine analogs inhibit HCV replication. This study demonstrates the novel anti-HCV activity of tylophorine analogs. Our results also highlight the importance of Hsc70 in HCV replication.

Bifunctional inhibition of HIV-1 reverse transcriptase: a first step in designing a bifunctional triphosphate.

The onset of resistance to approved anti-AIDS drugs by HIV necessitates the search for novel inhibitors of HIV-1 reverse transcriptase (RT). Developing single molecular agents concurrently occupying the nucleoside and nonnucleoside binding sites in RT is an intriguing idea but the proof of concept has so far been elusive. As a first step, we describe molecular modeling to guide focused chemical syntheses of conjugates having nucleoside (d4T) and nonnucleoside (TIBO) moieties tethered by a flexible polyethylene glycol (PEG) linker. A triphosphate of d4T-6PEG-TIBO conjugate was successfully synthesized that is recognized as a substrate by HIV-1 RT and incorporated into a double-stranded DNA.

Blocking HIV-1 entry by a gp120 surface binding inhibitor.

We report the mode of action of a proteomimetic compound that binds to the exterior surface of gp120 and blocks HIV-1 entry into cells. Using a one cycle time-of-addition study and antibody competition binding studies, we have determined that the compound blocks HIV-1 entry through modulation of key protein-protein interactions mediated by gp120. The compound exhibits anti-HIV-1 replication activities against several pseudotype viruses derived from primary isolates and the resistant strains isolated from existing drug candidates with equal potency. Together, these data provide evidence that the proteomimetic compound represents a novel class of HIV-1 viral entry inhibitor that functions through protein surface recognition in analogy to an antibody.

Quantitation of paclitaxel and its two major metabolites using a liquid chromatography-electrospray ionization tandem mass spectrometry.

A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the determination of paclitaxel (Taxol) and its two major metabolites in human plasma has been developed. Samples were prepared after liquid-liquid extraction and analyzed on a C(18) column interfaced with a Q-Trap tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile-water (0.05% formic acid) (65:35) at the flow rate of 0.25 mL/min. The analytes and internal standard docetaxel were both detected by use of multiple reaction monitoring mode. The method was linear in the concentration range of 0.5-500.0 ng/mL for paclitaxel, 6α-hydroxypaclitaxel and p-3'-hydroxypaclitaxel, respectively. The lower limit of quantification (LLOQ) was 0.5 ng/mL for paclitaxel, 6α-hydroxypaclitaxel and p-3'-hydroxypaclitaxel, respectively. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 8.18%. The accuracy determined at three concentrations was within ±10.8% in terms of relative error. The total run time was 7.0 min. This assay offers advantages in terms of expediency, and suitability for the analysis of paclitaxel and its metabolites in various biological fluids.

Analysis of deoxyribonucleotide pools in human cancer cell lines using a liquid chromatography coupled with tandem mass spectrometry technique.

Endogenous ribonucleotides and deoxyribonucleotides play a critical role in cell function, and determination of their levels is of fundamental importance in understanding key cellular processes involved in energy metabolism and molecular and biochemical signaling pathways. In this study, we determined the respective ribonucleotide and deoxyribonucleotide pool sizes in different human cell lines using a simple sample preparation method and LC/MS/MS. This assay was used to determine alterations in deoxyribonucleotide pools in human pancreatic PANC1 cells in response to hypoxia and to treatment with either hydroxyurea or aphidicolin. The levels of all deoxyribonucleotide metabolites decreased with hypoxia treatment, except for dUMP, which increased by two-fold. This LC/MS/MS assay is simple, fast, and sensitive, and it represents a significant advance over previously published methodologies.

Synthesis of 4'-Ethynyl-2'-deoxy-4'-thioribonucleosides and Discovery of a Highly Potent and Less Toxic NRTI.

The synthesis of 4'-ethynyl-2'-deoxy-4'-thioribonucleosides was carried out utilizing an electrophilic glycosidation in which 4-ethynyl-4-thiofuranoid glycal 16 served as a glycosyl donor. Electrophilic glycosidation between 16 and the silylated nucleobases (N4-acetylcytosine, N6-benzoyladenine and N2-acetyl-O6-diphenylcarbamoylguanine) was carried out in the presence of N-iodosuccinimide (NIS) leading to the exclusive formation of the desired ß-anomers 29, 33 and 36. Anti-HIV studies demonstrated that these 4'-thio nucleosides were less cytotoxic to T-lymphocyte (i.e. MT-4 cells) than the corresponding 4'-ethynyl derivatives of 2'-deoxycytidine (44), 2'-deoxyadenosine (45) and 2'-deoxyguanosine (46). Comparison of the selectivity indices (SI) was made between 4'-thionucleosides (32, 41 and 43) and the corresponding 4'-oxygen analogues 44-46 by using the reported CC50 and EC50 values. In the case of cytosine and adenine nucleosides, comparable SI values were obtained: 32 (545) and 45 (458); 41 (>230) and 45 (1,630). In contrast, 4'-ethynyl-2'-deoxy-4'-thioguanosine 43 was found to possess a SI value of >18,200, which is twenty times better than that of 46 (933).

Determinants of individual variation in intracellular accumulation of anti-HIV nucleoside analog metabolites.

Individual variation in response to antiretroviral therapy is well-known, but it is not clear if demographic characteristics such as gender, age, and ethnicity are responsible for the variation. To optimize anti-HIV therapy and guide antiretroviral drug discovery, determinants that cause variable responses to therapy need to be evaluated. We investigated the determinants of intracellular concentrations of nucleoside analogs using peripheral blood mononuclear cells from 40 healthy donors. We observed individual differences in the concentrations of the intracellular nucleoside analogs; the mean concentrations of the triphosphate metabolite of ethynylstavudine (4'-Ed4T), zidovudine (AZT), and lamivudine (3TC) were 0.71 pmol/10(6) cells (minimum and maximum, 0.10 and 3.00 pmol/10(6) cells, respectively), 0.88 pmol/10(6) cells (minimum and maximum, 0.10 and 15.18 pmol/10(6) cells, respectively), and 1.70 pmol/10(6) cells (minimum and maximum, 0.20 and 7.73 pmol/10(6) cells, respectively). Gender and ethnicity had no effect on the concentration of 4'-Ed4T and 3TC metabolites. There was a trend for moderation of the concentrations of AZT metabolites by gender (P = 0.17 for gender·metabolite concentration). We observed variability in the activity and expression of cellular kinases. There was no statistically significant correlation between thymidine kinase 1 (TK-1) activity or expression and thymidine analog metabolite concentrations. The correlation between the activity of deoxycytidine kinase (dCK) and the 3TC monophosphate metabolite concentration showed a trend toward significance (P = 0.1). We observed an inverse correlation between the multidrug-resistant protein 2 (MRP2) expression index and the concentrations of AZT monophosphate, AZT triphosphate, and total AZT metabolites. Our findings suggest that the observed variation in clinical response to nucleoside analogs may be due partly to the individual differences in the intracellular concentrations, which in turn may be affected by the cellular kinases involved in the phosphorylation pathway and ATP-binding cassette (ABC) transport proteins.

Identification of chemicals and their metabolites from PHY906, a Chinese medicine formulation, in the plasma of a patient treated with irinotecan and PHY906 using liquid chromatography/tandem mass spectrometry (LC/MS/MS).

Traditional Chinese Medicine (TCM) is increasingly being used in combination with Western medicine. In general, TCM is comprised of multiple components in sharp contrast to Western medicine, where a single active chemical is used. Presently, there are no well-established standards for most of the chemical compounds of TCM and their respective metabolites. Moreover, there are no formal analytical methods for the identification of these chemicals, especially in trace amounts. The ability to measure the pharmacokinetic behaviors of chemicals and their metabolites from these herbal formulations are critical in understanding of the action of TCM. This paper describes the use of LC/MS/MS along with enzyme treatments and n-octanol/water partition coefficient, to investigate the chemical components of PHY906 and their metabolites in the plasma of a patient with metastatic colorectal cancer (mCRC) treated with irinotecan and PHY906. The chemicals from an aqueous extract of PHY906 and the plasma from a patient was prepared and separated on an Agilent ZORBAX-SB C(18) column, and eluted with acetonitrile/0.05% (v/v) formic acid. From the PHY906 aqueous extract, a total of 57 compounds and 27 metabolites were identified and tentatively assigned structures based on their identified mass spectrometry, enzyme digestion and n-octanol/water partition coefficient. In contrast, analysis of patient plasma identified only 33 chemicals and new metabolites. These findings demonstrated that LC/MS/MS was and effective and reliable method for studying the parent chemicals of the Chinese herbal medicine PHY906 and their metabolites in a patient with metastatic colorectal cancer.

Reversal of TNP-470-induced endothelial cell growth arrest by guanine and guanine nucleosides.

The mechanism of action of TNP-470 [O-(chloroacetyl-carbamoyl) fumagillol], which potently and selectively inhibits the proliferation of endothelial cells, is incompletely understood. Previous studies have established its binding protein and the most distal effector of its growth arrest activity as methionine aminopeptidase 2 (MetAP-2) and p21(WAF1/CIP1), respectively. However, the mechanistic steps between these two effectors have not been identified. We have found that addition of exogenous guanine and guanine-containing nucleosides to culture medium will completely reverse the cytostatic effect of TNP-470 on both cultured bovine aortic and mouse pulmonary endothelial cells. Western blotting showed that supplementation with exogenous guanosine reverses the induction of p21(WAF1/CIP1) by TNP-470. This "rescue" by guanine/guanosine was abolished when the guanine salvage pathway of nucleotide biosynthesis was inhibited with Immucillin H, suggesting that TNP-470 might reduce de novo guanine synthesis in endothelial cells. However, an analysis of inosine 5'-monophosphate dehydrogenase, the rate-limiting enzyme in de novo guanine synthesis and target of the antiangiogenic drug mycophenolic acid, showed no TNP-470-induced changes. Curiously, quantitation of cellular nucleotides confirmed that GTP levels were not reduced after TNP-470 treatment. Addition of guanosine at the start of G(1) phase causes a doubling in GTP levels that persists to the G(1)/S phase transition, where commitment to TNP-470 growth arrest occurs. Thus, guanine rescue involves an augmentation of cellular GTP beyond physiological levels rather than a restoration of a drug-induced GTP deficit. Determining the mechanism whereby this causes restoration of endothelial cell proliferation is an ongoing investigation.

Discovery of a synthetic dual inhibitor of HIV and HCV infection based on a tetrabutoxy-calix[4]arene scaffold.

A potential anti-HIV and HCV drug candidate is highly desirable as coinfection has become a worldwide public health challenge. A potent compound based on a tetrabutoxy-calix[4]arene scaffold that possesses dual inhibition for both HIV and HCV is described. Structural activity relationship studies demonstrate the effects of lower-rim alkylation in maintaining cone conformation and upper-rim interacting head groups on the calix[4]arene play key roles for its potent dual antiviral activities.

A novel class of meso-tetrakis-porphyrin derivatives exhibits potent activities against hepatitis C virus genotype 1b replicons in vitro.

Recent years have seen the rapid advancement of new therapeutic agents against hepatitis C virus (HCV) in response to the need for treatment that is unmet by interferon (IFN)-based therapies. Most antiviral drugs discovered to date are small molecules that modulate viral enzyme activities. In the search for highly selective protein-binding molecules capable of disrupting the viral life cycle, we have identified a class of anionic tetraphenylporphyrins as potent and specific inhibitors of the HCV replicons. Based on the structure-activity relationship studies reported herein, meso-tetrakis-(3,5-dicarboxy-4,4'-biphenyl) porphyrin was found to be the most potent inhibitor of HCV genotype 1b (Con1) replicon systems but was less effective against the genotype 2a (JFH-1) replicon. This compound induced a reduction of viral RNA and protein levels when acting in the low nanomolar range. Moreover, the compound could suppress replicon rebound in drug-treated cells and exhibited additive to synergistic effects when combined with protease inhibitor BILN 2061 or with IFN-alpha-2a. Our results demonstrate the potential use of tetracarboxyphenylporphyrins as potent anti-HCV agents.

Synthesis and anti-HIV activity of 4'-C-ethynyl-2'-deoxy-4'-thio-nucleosides.

Synthesis of 4'-C-ethynyl-2'-deoxy-4'-thionucleosides was carried out based on electrophilic glycosidation using 4-C-ethynyl-4-thiofuranoid glycal. The glycal 15 was prepared as follows: oxidative cleavage of 6 with Pb(OAc)(4) forming the aldehyde 7, aldol reaction of 7 and subsequent silylation to furnish 8, conversion of the formyl group of 8 into an ethynyl group, and finally beta-elimination of the resulting 14 with t-BuLi. The glycosyl donor 16 was prepared by silyl-protection of 15. Electrophilic glycosidation was performed between silylated N(4)-acetylcytosine and 16 in the presence of N-iodosuccinimide. Radical-mediated removal of the introduced iodine atom followed by deprotection gave 4'-C-ethynyl-2'-deoxy-4'-thiocytidine (18).

Quantitation of Irinotecan and its two major metabolites using a liquid chromatography-electrospray ionization tandem mass spectrometric.

A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the determination of Irinotecan (CPT-11) and its metabolites in human plasma has been developed. Samples were prepared after protein precipitation and analyzed on a C(18) column interfaced with a Q-Trap tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile-water (0.05% formic acid), using gradient procedure. The analytes and internal standard camptothecin were both detected by use of multiple reaction monitoring mode. The method was linear in the concentration range of 10.0-2000.0ng/ml for CPT-11 and 0.5-200.0ng/ml for 7-ethyl-10-hydroxycamptothecin (SN-38), respectively. The lower limit of quantification (LLOQ) was 10ng/ml for CPT-11 and 0.5ng/ml for SN-38. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 10.6%. The accuracy determined at three concentrations was within +/-11.4% in terms of relative error. Due to the unavailability of standard for 7-ethyl-10-O-glucuronyl-camptothecin (SN-38G) and the importance of knowing the concentration of this metabolite, we developed a method for analysis SN-38G by taking advantage of the quantitive conversion of SN-38G to SN-38 using glucuronidase. This enzymatic method of identification and quantitation of gluconated compound can be widely used when the standard for phase II glucuronide metabolites are not available.

Impact of novel human immunodeficiency virus type 1 reverse transcriptase mutations P119S and T165A on 4'-ethynylthymidine analog resistance profile.

2',3'-Didehydro-3'-deoxy-4'-ethynylthymidine (4'-Ed4T), a derivative of stavudine (d4T), has potent activity against human immunodeficiency virus and is much less inhibitory to mitochondrial DNA synthesis and cell growth than its progenitor, d4T. 4'-Ed4T triphosphate was a better reverse transcriptase (RT) inhibitor than d4T triphosphate, due to the additional binding of the 4'-ethynyl group at a presumed hydrophobic pocket in the RT active site. Previous in vitro selection for 4'-Ed4T-resistant viral strains revealed M184V and P119S/T165A/M184V mutations on days 26 and 81, respectively; M184V and P119S/T165A/M184V conferred 3- and 130-fold resistance to 4'-Ed4T, respectively. We investigated the relative contributions of these mutations, engineered into the strain NL4-3 background, to drug resistance, RT activity, and viral growth. Viral variants with single RT mutations (P119S or T165A) did not show resistance to 4'-Ed4T; however, M184V and P119S/T165A/M184V conferred three- and fivefold resistance, respectively, compared with that of the wild-type virus. The P119S/M184V and T165A/M184V variants showed about fourfold resistance to 4'-Ed4T. The differences in the growth kinetics of the variants were not more than threefold. The purified RT of mutants with the P119S/M184V and T165A/M184V mutations were inhibited by 4'-Ed4TTP with 8- to 13-fold less efficiency than wild-type RT. M184V may be the primary resistance-associated mutation of 4'-Ed4T, and P119S and T165A are secondary mutations. On the basis of our findings and the results of structural modeling, a virus with a high degree of resistance to 4'-Ed4T (e.g., more than 50-fold resistance) will be difficult to develop. The previously observed 130-fold resistance of the virus with P119S/T165A/M184V to 4'-Ed4T may be partly due to mutations both in the RT sequence and outside the RT sequence.

Comparative study of the persistence of anti-HIV activity of deoxynucleoside HIV reverse transcriptase inhibitors after removal from culture.

BACKGROUND: Most in vitro assays of drug potency may not adequately predict the performance in vivo. Methods to assess the persistence of antiviral activity of deoxynucleoside analogs, which require intracellular activation to the active metabolites that can persist in cells, will be important for designing dosages, combination regimens, and assessing treatment compliance. Using an HIV-IIIB/TZM-bl indicator cell culture system, we assessed the ability of an inhibitor to protect cells from infection and to delay viral rebound after removal of inhibitor from culture. RESULTS: The order of protection of cells from HIV-infection was 4'-Ed4T > LFD4C > DDI > D4T > 3TC > AZT > FTC > NVP. The fold-increase in EC50 to delay viral rebound was DDI < 4'-Ed4T < LFD4C < FTC < D4T < 3TC < NVP < AZT. The ranking of persistence of anti-HIV activity of the inhibitors based on the two-component assay was DDI > 4'-Ed4T > LFD4C > FTC = D4T > 3TC > NVP > AZT. CONCLUSION: The persistence ranking was derived from assays based on measures of single viral replication-cycle and cumulative inhibition at multiple time-points. Therefore, a better indicator of the pharmacodynamic property of an inhibitor. The persistence of anti-HIV activity assay may complement in vitro potency assays to better predict in vivo performance of nucleoside analogs.

Mechanism of inhibition of human immunodeficiency virus type 1 reverse transcriptase by a stavudine analogue, 4'-ethynyl stavudine triphosphate.

2',3'-Didehydro-3'-deoxy-4'-ethynylthymidine (4'-Ed4T), a recently discovered nucleoside reverse transcriptase (RT) inhibitor, exhibits 5- to 10-fold-higher activity against human immunodeficiency virus type 1 (HIV-1) and less cytotoxicity than does its parental compound d4T (stavudine). Using steady-state kinetic approaches, we have previously shown that (i) 4'-ethynyl-d4T triphosphate (4'-Ed4TTP) inhibits HIV-1 RT more efficiently than d4TTP does and (ii) its inhibition efficiency toward the RT M184V mutant is threefold less than that toward wild-type (wt) RT. In this study we used pre-steady-state kinetic approaches in an attempt to understand its mechanism of inhibition. With wt and the M184V mutant RTs, 4'-Ed4TTP has three- to fivefold-lower K(d) (dissociation constant) values than d4TTP, while d4TTP has up to eightfold-higher K(d) values than dTTP. Inhibition is more effective in DNA replication with RNA template than with DNA template. In general, the M184V mutant exhibits poorer binding for all three nucleoside triphosphates than does wt RT. The structural basis for the lower binding affinity of d4TTP than of dTTP could be the lack of hydrogen bonds from the missing 3'-hydroxyl group in d4TTP to the backbone amide of Y115 and also to the side chain of Q151. The structural basis for the higher binding affinity of 4'-Ed4TTP than of d4TTP could be the additional binding of the 4'-ethynyl group in a preformed hydrophobic pocket by A114, Y115, M184, F160, and part of D185.

Synthesis and anti-HIV activity of 4'-substituted 4'-thiothymidines: a new entry based on nucleophilic substitution of the 4'-acetoxy group.

Diacetoxylation of 1-(2,5-dideoxy-beta-L-glycero-pent-4-eno-4-thiofuranosyl)thymine (13) with Pb(OAc) 4 allowed introduction of an acetoxy leaving group to the 4'-position. Nucleophilic substitution of the resulting 4'-acetoxy derivative (14) with silicon reagents enabled us to prepare the 4'-phenylthio (17a), 4'-azido (18a), 4'-methoxy (20a), and 4'-allyl (21a) analogues of 4'-thiothymidine. 4'-Cyano ( 25a) and 4'-ethynyl (31) nucleosides were also synthesized from 3',5'-bis-O-TBDMS derivative (24). Among novel 4'-substituted 4'-thiothymidines, the 4'-azido (33), 4'-cyano (36), and 4'-ethynyl (37) derivatives were found to show potent inhibitory activity against HIV-1 and HIV-2. It is noteworthy that 36 and 37 were also inhibitory against replication of HIV variant resistant to 3TC (HIV-1 M184V), being as potent as against HIV-1 IIIB.

Intracellular metabolism and persistence of the anti-human immunodeficiency virus activity of 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine, a novel thymidine analog.

The therapeutic benefits of current antiretroviral therapy are limited by the evolution of drug-resistant virus and long-term toxicity. Novel antiretroviral compounds with activity against drug-resistant viruses are needed. 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine (4'-Ed4T), a novel thymidine analog, has potent anti-human immunodeficiency virus (HIV) activity, maintains considerable activity against multidrug-resistant HIV strains, and is less inhibitory to mitochondrial DNA synthesis in cell culture than its progenitor stavudine (D4T). We investigated the intracellular metabolism and anti-HIV activity of 4'-Ed4T. The profile of 4'-Ed4T metabolites was qualitatively similar to that for zidovudine (AZT), with the monophosphate metabolite as the major metabolite, in contrast to that for D4T, with relatively poor formation of total metabolites. The first phosphorylation step for 4'-Ed4T in cells was more efficient than that for D4T but less than that for AZT. The amount of 4'-Ed4T triphosphate (4'-Ed4TTP) was higher than that of AZTTP at 24 h in culture. There was a dose-dependent accumulation of 4'-Ed4T diphosphate and 4'-Ed4TTP on up-regulation of thymidylate kinase and 3-phosphoglycerate kinase expression in Tet-On RKO cells, respectively. The anti-HIV activity of 4'-Ed4T in cells persisted even after 48 h of drug removal from culture in comparison with AZT, D4T, and nevirapine (NVP). The order of increasing persistence of anti-HIV activity of these compounds after drug removal was 4'-Ed4T > D4T > AZT > NVP. In conclusion, with the persistence of 4'-Ed4TTP and persistent anti-HIV activity in cells, we anticipate less frequent dosing and fewer patient compliance issues than for D4T. 4'-Ed4T is a promising antiviral candidate for HIV type 1 chemotherapy.

Comparison of the phosphorylation of 4'-ethynyl 2',3'-dihydro-3'-deoxythymidine with that of other anti-human immunodeficiency virus thymidine analogs.

Thymidine analogs, including 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxy-3'-deoxythymidine (D4T), are important antiretroviral agents. To exert antiretroviral activity, these analogs undergo a stepwise phosphorylation intracellularly to the active triphosphate metabolites. We previously reported that 4'-substituted D4T with an ethynyl group (i.e., 4'-ethynyl D4T) increased the anti-human immunodeficiency virus (HIV) activity and was active against multidrug-resistant HIV strains. 4'-Ethynyl D4T is a better substrate for phosphorylation by human thymidine kinase 1 than D4T is. In this report, we first studied the enzymes involved in the phosphorylation of 4'-ethynyl D4T from monophosphate to triphosphate metabolites. The 4'-ethynyl D4TMP is phosphorylated by recombinant human TMP kinase with a K(m) of 19 +/- 4 microM and a k(cat) of 0.007 +/- 0.001 s(-1); the relative efficiency is about 9 and 15% of those of D4TMP and AZTMP, respectively. Several enzymes from crude cellular extracts, including nucleoside diphosphate kinase, pyruvate kinase, creatine kinase, and 3-phosphoglycerate kinase, could phosphorylate 4'-ethynyl D4T-diphosphate. The relative phosphorylation efficiencies of 4'-ethynyl D4TDP were about 3 to 25% of those of D4TDP and were generally similar to those of AZTDP. In T-lymphoid cell lines, there was a preponderant accumulation of 4'-ethynyl D4TMP, suggesting that TMP kinase could be the rate-limiting enzyme in the metabolism of 4'-ethynyl D4T. Although the same enzymes are involved in the stepwise phosphorylation of thymidine analogs, their behaviors in phosphorylating metabolites of 4'-ethynyl D4T are different from those of D4T and AZT. Qualitatively, the metabolism of 4'-ethynyl D4T is more similar to that of AZT than to that of its progenitor, D4T.

Highly selective action of triphosphate metabolite of 4'-ethynyl D4T: a novel anti-HIV compound against HIV-1 RT.

2',3'-Didehydro-3'-deoxy-4'-ethynylthymidine (4'-Ed4T), is a recently discovered nucleoside reverse transcriptase inhibitor (NRTI) showing a 5- to 10-fold greater anti-human immunodeficiency virus type 1 (HIV-1) activity and less cellular and mitochondrial toxicity than its parental compound, stavudine (D4T). It is also active against a variety of NRTI-resistant HIV-1 mutants under non-cytotoxic concentrations. In this study, the effects of 4'-Ed4TTP, which is the triphosphate metabolite of 4'-Ed4T, on HIV-1 reverse transcriptase (RT) activity were investigated. We found that 4'-Ed4TTP was a substrate of HIV-1 RT serving as a DNA chain terminator, and it inhibited the DNA polymerase activity of RT more efficiently than D4TTP. The value of Ki(4'-Ed4TTP)/Km(dTTP) is 0.15 for DNA/RNA primer/template duplex (P/T), but 0.7 for DNA/DNA P/T, suggesting 4'-Ed4TTP inhibits RT more efficiently during RNA-dependent DNA synthesis than DNA-dependent DNA synthesis. 4'-Ed4TTP was also found to inhibit the 3TC (Lamivudine)-resistant RT mutant, M184V, with 3-fold less efficiency than the wild type (wt) RT. 4'-Ed4TTP showed much less inhibitory effects toward major host DNA polymerases. Overall, our results suggest that 4'-Ed4TTP is the active form for anti-HIV-1 activity via its inhibitory effect against RT.