Novel benzimidazole phosphonates as potential inhibitors of protein prenylation.

Benzimidazole carboxyphosphonates and bisphosphonates have been prepared and evaluated for their activity as inhibitors of protein prenylation or isoprenoid biosynthesis. The nature of the phosphonate head group was found to dictate enzyme specificity. The lead carboxyphosphonate inhibits geranylgeranyl transferase II while its corresponding bisphosphonate analogue potently inhibits farnesyl diphosphate synthase. The most active inhibitors effectively disrupted protein prenylation in human multiple myeloma cells.

Exploiting the S4-S5 Specificity of Human Neutrophil Proteinase 3 to Improve the Potency of Peptidyl Di(chlorophenyl)-phosphonate Ester Inhibitors: A Kinetic and Molecular Modeling Analysis.

The neutrophilic serine protease proteinase 3 (PR3) is involved in inflammation and immune response and thus appears as a therapeutic target for a variety of infectious and inflammatory diseases. Here we combined kinetic and molecular docking studies to increase the potency of peptidyl-diphenyl phosphonate PR3 inhibitors. Occupancy of the S1 subsite of PR3 by a nVal residue and of the S4-S5 subsites by a biotinylated Val residue as obtained in biotin-VYDnVP(O-C6H4-4-Cl)2 enhanced the second-order inhibition constant kobs/[I] toward PR3 by more than 10 times ( kobs/[I] = 73000 ± 5000 M-1 s-1) as compared to the best phosphonate PR3 inhibitor previously reported. This inhibitor shows no significant inhibitory activity toward human neutrophil elastase and resists proteolytic degradation in sputa from cystic fibrosis patients. It also inhibits macaque PR3 but not the PR3 from rodents and can thus be used for in vivo assays in a primate model of inflammation.

The effect of PON1 enhancers on reducing acetylcholinesterase inhibition following organophosphate anticholinesterase exposure in rats.

Novel nucleophiles, a series of substituted phenoxyalkyl pyridinium oximes, have been previously shown by our laboratories to enhance in vitro paraoxonase 1 (PON1)-mediated degradation of a sarin surrogate (nitrophenyl isopropyl methylphosphonate, NIMP) and a VX surrogate (nitrophenyl ethyl methylphosphonate, NEMP). Five of the most efficacious of these nucleophiles were tested in rats for their ability to reduce the level of acetylcholinesterase (AChE) inhibition in brain and peripheral tissues following exposure to NIMP or NEMP. Following simultaneous administration of a nucleophile plus surrogate (at 3 dosages yielding about 10-50% AChE inhibition in the brain at 15 min), all five nucleophiles reduced the AChE inhibition in the brain at all 3 dosages, and reduced peripheral AChE inhibition at the lowest dosage. Protective effects were seen for only a short period of time, i.e., 15 min. Even though these nucleophiles are oximes, they are not effective AChE reactivators so it is unlikely that the resultant decreases in AChE inhibition are from appreciable AChE reactivation. It is likely that the protective effects seen are, at least in part, the result of enhancement of PON1-mediated surrogate degradation, an unprecedented mechanism of therapy that has the potential to be developed into a nerve agent countermeasure.

Prothoracicotropic hormone induces tyrosine phosphorylation in prothoracic glands of the silkworm, Bombyx mori.

In the present study, we investigated the tyrosine phosphorylation of Bombyx mori prothoracic glands using phosphotyrosine-specific antibodies and Western blot analysis. Results showed that prothoracicotropic hormone (PTTH) stimulates a rapid increase in tyrosine phosphorylation of at least 2 proteins in prothoracic glands, one of which was identified as extracellular signal-regulated kinase (ERK). The phosphorylation of another 120-kDa protein showed dose- and time-dependent stimulation by PTTH in vitro. In vitro activation of tyrosine phosphorylation was also verified by in vivo experiments: injection of PTTH into day-6 last-instar larvae greatly increased tyrosine phosphorylation. Treatment of prothoracic glands with the protein tyrosine phosphatase inhibitor, sodium orthovanadate, also resulted in tyrosine phosphorylation of several proteins and increased ecdysteroidogenesis. The PTTH-stimulated phosphorylation of the 120-kDa protein was markedly attenuated by genistein, a broad-spectrum tyrosine kinase inhibitor, but not by HNMPA-(AM)(3) , a specific inhibitor of insulin receptor tyrosine kinase. PP2, a more-selective inhibitor of the Src-family tyrosine kinases, partially inhibited PTTH-stimulated tyrosine phosphorylation, but not ecdysteroidogenesis. This result implies the possibility that in addition to ERK, the phosphorylation of the 120-kDa protein, which is not Src-family tyrosine kinase, is likely also involved in PTTH-stimulated ecdysteroidogenesis in B. mori.

New potent cathepsin G phosphonate inhibitors.

Cathepsin G is an enzyme with dual chymotrypsin and trypsin-like specificity. As a leukocyte proteinase it is involved in the early stages of the immune response. In this work the synthesis and inhibitory activity of diaryl phosphonic-type irreversible cathepsin G inhibitors are described. Modification of the lead structure Z-Phg(P)(OPh)2 (k(obs)/I=91 M(-1)s(-1)) in phenyl ester moieties followed by incorporation of the basic functional group into the aromatic side chain yielded highly potent cathepsin G inhibitor Z-(4-guanidine)Phg(P)(OC6H4-4-S-Me)2 with the apparent second-order inhibition value at 15,600 M(-1)s(-1). Further elongation of the obtained compound by tripeptide resulted in the inhibitor Ac-Phe-Val-Thr-(4-guanidine)Phg(P)(OC6H4-4-S-Me)2 with the highest k(obs)/I value ever reported in literature (256,000 M(-1)s(-1)).

Metabolic activation of pradefovir by CYP3A4 and its potential as an inhibitor or inducer.

Metabolic activation of pradefovir to 9-(2-phosphonylmethoxyethyl)adenine (PMEA) was evaluated by using cDNA-expressed CYP isozymes in portal vein-cannulated rats following oral administration and in human liver microsomes. The enzyme induction potential of pradefovir was evaluated in rats following multiple oral dosing and in primary cultures of human hepatocytes. The results indicated that CYP3A4 is the only cDNA-expressed CYP isozyme catalyzing the conversion of pradefovir to PMEA. Pradefovir was converted to PMEA in human liver microsomes with a K(m) of 60 microM, a maximum rate of metabolism of 228 pmol/min/mg protein, and an intrinsic clearance of about 359 ml/min. Addition of ketoconazole and monoclonal antibody 3A4 significantly inhibits the conversion of pradefovir to PMEA in human liver microsomes, suggesting the predominant role of CYP3A4 in the metabolic activation of pradefovir. Pradefovir at 0.2, 2, and 20 microM was neither a direct inhibitor nor a mechanism-based inhibitor of CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1, and CYP1A2 in human liver microsomes. In rats, the liver was the site of metabolic activation of pradefovir, whereas the small intestine did not play a significant role in the metabolic conversion of pradefovir to PMEA. Daily oral dosing (300 mg/kg of body weight) to rats for 8 days showed that pradefovir was not an inducer of P450 enzymes in rats. Furthermore, pradefovir at 10 microg/ml was not an inducer of either CYP1A2 or CYP3A4/5 in primary cultures of human hepatocytes.

Lack of antagonism between abacavir, lamivudine, and tenofovir against wild-type and drug-resistant HIV-1.

Potential contributors to the high rate of virologic failure observed for tenofovir, abacavir, and lamivudine include a low genetic barrier to resistance for this regimen and antagonistic drug-drug interactions. To examine the second possibility, we tested combinations of abacavir, tenofovir, and lamivudine against wild-type and drug-resistant HIV-1 in vitro using peripheral blood mononuclear cells and MT-4 cells. Antagonistic interactions were not detected for any combination. If the systems examined accurately reflect the in vivo situation, antagonism does not substantially contribute to the poor efficacy of this triple combination.

Actions of sarin on fast-twitch and slow-twitch skeletal muscles of the cat and protective action by anticholinergic drugs.

1. The organophosphate cholinesterase inhibitor sarin has been studied for its effects on the contraction of fast-twitch (flexor hallucis longus, FHL) and slow-twitch (soleus) muscles in the cat.2. In both muscles lower doses (2.5 to 20 mug intra-arterially) potentiated, and higher doses (20 mug and over) depressed, twitches produced by indirect stimulation. Muscle action potentials became repetitive. There was no simple relationship between degree of inhibition of blood cholinesterase and effect on muscle twitch.3. The effects of the drug on repetitively stimulated muscles were dependent on both dose and frequency of stimulation. Doses of 2.5 to 10 mug increased the degree of fusion of low frequency tetani (10 Hz for FHL, 5 Hz for soleus) but depressed and caused non-maintenance of tetani at high frequencies (150 Hz and above for FHL, 60 Hz and above for soleus). Doses of 20 mug and above depressed and caused non-maintenance of tetani at all frequencies.4. The anticholinergic drug N-ethyl-2-pyrrolidylmethylcyclopentylphenyl glycollate (PMCG) injected intra-arterially protected both muscles from the effects of sarin on twitch but less so from effects on tetani. When given after sarin the drug reversed twitch potentiation and repetitive firing. In contrast, atropine had little effect on the responses to sarin.5. The effects of (+)-tubocurarine were compared with those of PMCG.6. The possible mode of action of PMCG is discussed.