(1/1594) Activation of stimulus-specific serine esterases (proteases) in the initiation of platelet secretion. I. Demonstration with organophosphorus inhibitors.

The effect of organophosphorus inhibitors of serine esterases (proteases) on secretion from washed rabbit platelets was examined. Five noncytotoxic stimuli were employed: collagen, thrombin, heterologous anti-platelet antibody (in the absence of complement), rabbit C3 bound to zymosan, and platelet activating factor derived from antigen-stimulated, IgE-sensitized rabbit basophils. Diisoprophyl phosphofluoridate, three series of p-nitrophenyl ethyl phosphonates, and a series of cyclohexyl phenylalkylphosphonofluridates were all found to be inhibitory to the platelet secretion. These are irreversible inhibitors of serine proteases but in this system were only inhibitory if added to the platelets concurrently with the stimuli. Pretreatment of either the platelets or the stimuli with the inhibitors followed by washing, was without effect on the subsequent reaction. This suggested the involvement of stimulus-activatable serine proteases in the secretory process. The concept was supported by finding that nonphosphorylating phosphonates or hydrolyzed phosphonates or phosphonofluoridates were without inhibitory action. The effect of a series of phosphonates or phosphonoflouridates in inhibiting each stimulus exhibited a unique activity-structure profile. The demonstration of such unique profiles with four series of inhibitors for each of the five stimuli was interpreted as demonstrating that a specific activatable serine protease was involved in the platelet secretory response to each stimulus.  (+info)

(2/1594) Comparative study of the anti-human cytomegalovirus activities and toxicities of a tetrahydrofuran phosphonate analogue of guanosine and cidofovir.

Cidofovir is the first nucleoside monophosphate analogue currently being used for the treatment of human cytomegalovirus (HCMV) retinitis in individuals with AIDS. Unfortunately, the period of therapy with the use of this compound may be limited due to the possible emergence of serious irreversible nephrotoxic effects. New drugs with improved toxicity profiles are needed. The goal of this study was to investigate the anticytomegaloviral properties and drug-induced toxicity of a novel phosphonate analogue, namely, (-)-2-(R)-dihydroxyphosphinoyl-5-(S)-(guanin-9'-yl-methyl) tetrahydrofuran (compound 1), in comparison with those of cidofovir. The inhibitory activities of both compounds on HCMV propagation in vitro were similar against the AD 169 and Towne strains, with 50% inhibitory concentrations ranging from 0.02 to 0.17 microgram/ml for cidofovir and < 0.05 to 0.09 microgram/ml for compound 1. A clinical HCMV isolate that was resistant to ganciclovir and that had a known mutation within the UL54 DNA polymerase gene and a cidofovir-resistant laboratory strain derived from strain AD 169 remained sensitive to compound 1, whereas their susceptibilities to ganciclovir and cidofovir were reduced by 33- and 10-fold, respectively. Both compound 1 and cidofovir exhibited equal potencies in an experimentally induced murine cytomegalovirus (MCMV) infection in mice, with a prevention or prolongation of mean day to death at dosages of 1.0, 3.2, and 10.0 mg/kg of body weight/day. In cytotoxicity experiments, compound 1 was found to be generally more toxic than cidofovir in cell lines Hs68, HFF, and 3T3-L1 (which are permissive for HCMV or MCMV replication) but less toxic than cidofovir in MRC-5 cells (which are permissive for HCMV replication). Drug-induced toxic side effects were noticed for both compounds in rats and guinea pigs in a 5-day repeated-dose study. In guinea pigs, a greater weight loss was noticed with cidofovir than with compound 1 at dosages of 3.0 and 10.0 mg/kg/day. An opposite effect was detected in rats, which were treated with the compounds at relatively high dosages (up to 100 mg/kg/day). Compound 1 and cidofovir were nephrotoxic in both rats and guinea pigs, with the epithelium lining the proximal convoluted tubules in the renal cortex being the primary target site. The incidence and the severity of the lesions were found to be dose dependent. The lesions observed were characterized by cytoplasm degeneration and nuclear modifications such as karyomegaly, the presence of pseudoinclusions, apoptosis, and degenerative changes. In the guinea pig model, a greater incidence and severity of lesions were observed for cidofovir than for compound 1 (P < 0.001) with a drug regimen of 10 mg/kg/day.  (+info)

(3/1594) Impact of 9-(2-phosphonylmethoxyethyl)adenine on (deoxy)ribonucleotide metabolism and nucleic acid synthesis in tumor cells.

Following exposure to 9-(2-phosphonylmethoxyethyl)adenine (an inhibitor of the cellular DNA polymerases alpha, delta and epsilon), human erythroleukemia K562, human T-lymphoid CEM and murine leukemia L1210 cells markedly accumulated in the S phase of the cell cycle. In contrast to DNA replication, RNA synthesis (transcription) and protein synthesis (mRNA translation) were not affected by 9-(2-phosphonylmethoxyethyl)-adenine. The ribonucleoside triphosphate pools were slightly elevated, while the intracellular levels of all four deoxyribonucleoside triphosphates were 1.5-4-fold increased in 9-(2-phosphonylmethoxyethyl)adenine-treated K562, CEM and L1210 cells. The effect of 9-(2-phosphonylmethoxyethyl)adenine on de novo (thymidylate synthase-mediated) and salvage (thymidine kinase-mediated) dTTP synthesis was investigated using radio-labelled nucleoside precursors. The amount of thymidylate synthase-derived dTTP in the acid soluble pool was 2-4-fold higher in PMEA-treated than in untreated K562 cells, which is in accord with the 3-4-fold expansion of the global dTTP level in the presence of 9-(2-phosphonylmethoxyethyl)adenine. Strikingly, 2-derived dTTP accumulated to a much higher extent (i.e. 16-40-fold) in the soluble dTTP pool upon 9-(2-phosphonylmethoxyethyl)adenine treatment. In keeping with this finding, a markedly increased thymidine kinase activity could be demonstrated in extracts of 9-(2-phosphonylmethoxyethyl)adenine-treated K562 cell cultures. Also, in the presence of 200 microM 9-(2-phosphonylmethoxyethyl)adenine, 14-fold less thymidylate synthase-derived but only 3-fold less thymidine kinase-derived dTTP was incorporated into the DNA of the K562 cells. These data show that thymidine incorporation may be inappropriate as a cell proliferation marker in the presence of DNA synthesis inhibitors such as 9-(2-phosphonylmethoxyethyl)adenine. Our findings indicate that 9-(2-phosphonylmethoxyethyl)adenine causes a peculiar pattern of (deoxy)ribonucleotide metabolism deregulation in drug-treated tumor cells, as a result of the metabolic block imposed by the drug on the S phase of the cell cycle.  (+info)

(4/1594) Early short-term 9-[2-(R)-(phosphonomethoxy)propyl]adenine treatment favorably alters the subsequent disease course in simian immunodeficiency virus-infected newborn Rhesus macaques.

Simian immunodeficiency virus (SIV) infection of newborn macaques is a useful animal model of human pediatric AIDS to study disease pathogenesis and to develop intervention strategies aimed at delaying disease. In the present study, we demonstrate that very early events of infection greatly determine the ultimate disease course, as short-term antiviral drug administration during the initial viremia stage significantly delayed the onset of AIDS. Fourteen newborn macaques were inoculated orally with uncloned, highly virulent SIVmac251. The four untreated control animals showed persistently high virus levels and poor antiviral immune responses; they developed fatal immunodeficiency within 15 weeks. In contrast, SIV-infected newborn macaques which were started on 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) treatment at 5 days of age and continued for either 14 or 60 days showed reduced virus levels and enhanced antiviral immune responses. This short-term PMPA treatment did not induce detectable emergence of SIV mutants with reduced in vitro susceptibility to PMPA. Although viremia increased in most animals after PMPA treatment was withdrawn, all animals remained disease-free for at least 6 months. Our data suggest that short-term treatment with a potent antiviral drug regimen during the initial viremia will significantly prolong AIDS-free survival for HIV-infected infants and adults.  (+info)

(5/1594) 9-[2-(Phosphonomethoxy)propyl]adenine (PMPA) therapy prolongs survival of infant macaques inoculated with simian immunodeficiency virus with reduced susceptibility to PMPA.

Simian immunodeficiency virus (SIV) infection of newborn rhesus macaques is a useful animal model of human immunodeficiency virus infection for the study of the emergence and clinical implications of drug-resistant viral mutants. We previously demonstrated that SIV-infected infant macaques receiving prolonged treatment with 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) developed viral mutants with fivefold reduced susceptibility to PMPA in vitro and that the development of these mutants was associated with the development of a K65R mutation and additional compensatory mutations in reverse transcriptase (RT). To study directly the virulence and clinical implications of these SIV mutants, two uncloned SIVmac isolates with similar fivefold reduced in vitro susceptibilities to PMPA but distinct RT genotypes, SIVmac055 (K65R, N69T, R82K A158S,S211N) and SIVmac385 (K65R, N69S, I118V), were each inoculated intravenously into six newborn rhesus macaques; 3 weeks later, three animals of each group were started on PMPA treatment. All six untreated animals developed persistently high levels of viremia and fatal immunodeficiency within 4 months. In contrast, the six PMPA-treated animals, despite having persistently high virus levels, survived significantly longer: 5 to 9 months for the three SIVmac055-infected infants and > or = 21 months for the three SIVmac385-infected infants. Virus from only one untreated animal demonstrated reversion to wild-type susceptibility and loss of the K65R mutation. In several other animals, additional RT mutations, including K64R and Y115F, were detected, but the biological role of these mutations is unclear since they did not affect the in vitro susceptibility of the virus to PMPA. In conclusion, this study demonstrates that although SIVmac mutants with the PMPA-selected K65R mutation in RT were highly virulent, PMPA treatment still offered strong therapeutic benefits. These results suggest that the potential emergence of HIV mutants with reduced susceptibility to PMPA in patients during prolonged PMPA therapy may not eliminate its therapeutic benefits.  (+info)

(6/1594) Phase I study of combination therapy with intravenous cidofovir and oral ganciclovir for cytomegalovirus retinitis in patients with AIDS.

Ganciclovir and cidofovir, two antiviral agents used in the treatment of cytomegalovirus (CMV) retinitis, have a synergistic effect inhibiting CMV replication in vitro. In a phase I study, seven patients with AIDS-related CMV retinitis were treated with cidofovir (5 mg/kg intravenously every 2 weeks) combined with ganciclovir (1 g orally three times a day). During a median of 5.5 months (range, 1-12 months) of combined therapy, only one patient had retinitis progression, and only two of 28 blood cultures (specimens of which were obtained on a monthly basis) yielded CMV. Dose-limiting adverse ocular effects (anterior uveitis [two patients] and hypotony [two patients]) occurred in three of seven patients. The results suggest that combination therapy with intravenous cidofovir and oral ganciclovir (a regimen that does not require indwelling central venous catheter access) might enhance clinical efficacy. Less frequent administration of cidofovir in combination with oral ganciclovir should be prospectively studied to determine if the incidence of treatment-associated toxicity might be reduced.  (+info)

(7/1594) Actions of 3-[2-phosphonomethyl[1,1-biphenyl]-3-yl]alanine (PMBA) on cloned glycine receptors.

1. PMBA is a novel antagonist of strychnine-sensitive glycine receptors in the rat spinal cord, however, its mode of action is unknown. The actions of PMBA on rat glycine receptor alpha1 and alpha2 homomers in Xenopus oocytes were studied under two-electrode voltage-clamp. 2. Co-application of PMBA and glycine to both alpha1 and alpha2 homomers yielded inward currents which decayed to a steady-state. Responses rose slowly to the same steady-state amplitude following a 2 min pre-incubation in PMBA. Strychnine, but not picrotoxinin, showed similar antagonism to PMBA. The potency of PMBA was independent of membrane potential between -100 and 0 mV. 3. When tested against EC50 concentrations of glycine, PMBA was almost equally potent on alpha1 (IC50, 406+/-41 nM: Hill coefficient, 1.5+/-0.2) and alpha2 (IC50, 539+/-56 nM; Hill coefficient, 1.4+/-0.2) homomers. 4. PMBA (1-I0 microM) and strychnine (200 nM) reduced the potency of glycine and the amplitude of the maximal agonist response of alpha1 and alpha2 homomers. In 10 microM PMBA, two distinct classes of glycine response were observed on alpha2, only a single class of responses were observed on alpha1. 5. There are similarities in PMBA and strychnine antagonism, although these compounds are structurally distinct. The possibility that PMBA interacts at two binding sites which differ in alpha1 and alpha2 subunits is discussed. PMBA may provide a lead structure for novel antagonists with which to investigate structural differences in glycine receptor at alpha1 and alpha2 subunits.  (+info)

(8/1594) Fluorescein monophosphates as fluorogenic substrates for protein tyrosine phosphatases.

A series of novel fluorescein monophosphates aimed as substrates for protein tyrosine phosphatases (PTPs) were synthesized and evaluated against fluorescein diphosphate (FDP), the currently used fluorescent substrate for PTPs. In contrast to FDP, which is dephosphorylated to monophosphate and then to fluorescein in a sequential reaction, these monophosphates are dephosphorylated in a single step. This eliminates the complication in assaying PTPs due to the cleavage of the second phosphate group. The kinetic studies of these substrates with PTPs were performed and Michaelis-Menten parameters were obtained. These designed substrates have Km 0.03-0. 35 mM, kcat/Km of 3-100 mM-1 s-1 with CD45 and PTP1B. The results showed that the substrates with negative charge groups on the fluorescein have higher affinities for PTP1B, which are consistent with other observations. In this series, fluorescein monosulfate monophosphate (FMSP) was the best substrate observed. Since FMSP showed large increases in both absorption and fluorescence upon dephosphorylation by PTPs at pH>6.0, it is one of the most sensitive, stable and high affinity substrates reported for PTPs.  (+info)