Mechanisms of folate metabolism-related substances affecting Staphylococcus aureus infection.

Staphylococcus aureus (S. aureus) is one of the critical clinical pathogens which can cause multiple diseases ranging from skin infections to fatal sepsis. S. aureus is generally considered to be an extracellular pathogen. However, more and more evidence has shown that S. aureus can survive inside various cells. Folate plays an essential role in multiple life activities, including the conversion of serine and glycine, the remethylation of homocysteine to methionine, and the de novo synthesis of purine /dTMP, et al. More and more studies reported that S. aureus intracellular infection requires the involvement of folate metabolism. This review focused on the mechanisms of folate metabolism and related substances affecting S. aureus infection. Loss of tetrahydrofolic acid (THF)-dependent dTMP directly inhibits the nucleotide synthesis pathway of the S. aureus due to pabA deficiency. Besides, trimethoprim-sulfamethoxazole (TMP/SMX), a potent antibiotic that treats S. aureus infections, interferes in the process of the folate mechanism and leads to the production of thymidine-dependent small-colony variants (TD-SCVs). In addition, S. aureus is resistant to lysostaphin in the presence of serine hydroxymethyltransferase (SHMT). We provide new insights for understanding the molecular pathogenesis of S. aureus infection.

Pretreatment HIV-1 drug resistance in Argentina: results from a surveillance study performed according to WHO-proposed new methodology in 2014-15.

BACKGROUND: In Argentina, current national guidelines recommend starting with NNRTI-based regimens. Recently, there have been some local reports regarding concerning levels of NNRTI-transmitted resistance, but surveillance has never been carried out at a national level. OBJECTIVES: To determine the prevalence of HIV drug resistance in people starting ART in Argentina using a WHO-proposed methodology. METHODS: This was a cross-sectional, nationally representative study. Twenty-five antiretroviral-dispensing sites throughout the country were randomly chosen to enrol at least 330 persons starting ART, to generate a point prevalence estimate of resistance-associated mutations (RAMs) with a 5% CI (for the total population and for those without antiretroviral exposure). All consecutive patients older than 18 years starting or restarting ART in the chosen clinics were eligible. Samples were processed with Trugene and analysed using the Stanford algorithm. RESULTS: Between August 2014 and March 2015, we obtained 330 samples from people starting ART. The mean ±â€ŠSD age was 35 ±â€Š11 years, 63.4% were male, 16.6% had prior antiretroviral exposure and the median (IQR) CD4 count was 275 cells/mm3 (106-461). The prevalence of RAMs found was 14% (±4%) for the whole population (3% NRTI-RAMs; 11% NNRTI-RAMs and 2% PI-RAMs) and 13% (±4%) for those without prior antiretroviral exposure (3%, 10% and 2%, respectively). The most common mutation was K103N. CONCLUSIONS: This surveillance study showed concerning levels of HIV drug resistance in Argentina, especially to NNRTIs. Due to this finding, Argentina's Ministry of Health guidelines will change, recommending performing a resistance test for everyone before starting ART. If this is taken up properly, it also might function as a continuing surveillance tool.

Purification and Activity Evaluation of a Novel Thrombopoietin Mimetic Peptide.

The emergence of thrombopoietin mimetic peptides presents a promising therapeutic strategy for addressing thrombocytopenia. This particular study aimed to establish a direct, expeditious, and efficient method for modifying and purifying a novel thrombopoietin mimetic peptide. Precursor proteins were subjected to modification utilizing three distinct fatty acids: C25H42O7N2, C39H66O15N4, and C41H70O15N4. Liquid chromatography analyses demonstrated that C41H70O15N4 yielded the most effective modification results. Mass spectrometry findings validated the correspondence between the theoretical and actual molecular weights of each sample. In vivo experiments conducted on normal mice showcased that the C41H70O15N4 modification group exhibited the highest platelet count, peaking at an impressive 5047 × 109/L. This count was approximately twice that of the peak platelet count observed in the dTMP group and four times higher than the control group. Pharmacokinetic investigations revealed that the C41H70O15N4 modification group displayed the lengthiest half-life among beagles, persisting for 128.5 h. This duration was approximately 28.5 times longer than that of the unmodified dTMP group. These findings underscore the effectiveness of the established C41H70O15N4 modification and purification method in preserving the biological activity of the thrombopoietin mimetic peptide. The novel thrombopoietin mimetic peptide showcased notable attributes of simplicity and cost-effectiveness, while also exhibiting a significant platelet-promoting effect and an extended half-life. Consequently, this novel peptide holds substantial significance for advancing the treatment of thrombocytopenia.

Microbicides for multidrug-resistant and multitropic HIV-1.

The most common mode of acquiring HIV-1 is via sexual transmission across the genital mucosa. Topical microbicides are a promising prevention strategy for the protection against HIV infection and may ultimately have an impact on the global AIDS pandemic. The effectiveness of a microbicide to prevent HIV-1 transmission will depend on the evolutionary and genital transmission dynamics of the viral subtypes, and sexual behavioral characteristics. Contemporary antiretroviral therapy has led to virological failure as a result of HIV-1 reverse transcriptase gene mutations. The transmission of these multidrug-resistant HIV-1 variants, and the superinfection with the same or distinct HIV-1 subtypes and recombination is a formidable hindrance inherent to global microbicide development. Consequently, mechanism-based microbicides targeting both the cell-free and cell-associated HIV-1 variants and subtypes can be expected to have superior clinical efficacy and safety profiles compared with polymeric anionic microbicides. This review describes the discovery of potent anti-HIV-1 agents against multidrug-resistant and multitropic HIV-1 variants with implications for global microbicide development. Stampidine and thiourea non-nucleoside reverse transcriptase inhibitors (NNRTIs) have demonstrated highly potent activity against clinically relevant multidrug-resistant and recombinant HIV-1 isolates spanning different subtypes across several continents. Extensive preclinical studies have shown that stampidine and a candidate thiourea NNRTI (HI-443) have clinical potential as a safe combination microbicide to inhibit, prevent or treat mucosal HIV-1 infections.

Rational drug design of multifunctional phosphoramidate substituted nucleoside analogs.

This review focuses on our approach to the study of the effect of a series of phosphoramidate substituted nucleoside analogs on model systems for cancer, HIV and fertility. This approach allowed the development of compound WHI-07, an arylphosphoramidate derivative of zidavudine. This compound is a multifunctional agent showing potent activity in the above mentioned model systems. Our rational drug design provided such a powerful derivative with all the necessary characteristic of a drug candidate. Importantly, we have experimental evidence that each of the groups associated with the molecular frame of WHI-07 imparts the multifunctional ability for this agent. In addition, we have also suggested a possible biological pathway for WHI-07 including various products with their therapeutic targets that are formed during the course of its metabolism inside the cell. We also propose which individual moieties in the structure of WHI-07 are responsible for the biological activity from the formation of these metabolites. A detailed structure-activity relationship is presented in the review in connection with various structural modifications of the agent. Application of this active agent in animal models shows the potential usefulness of this agent as a drug candidate. We further plan to utilize gene-chip technology to identify new targets and modes of action using microarrays to measure expression changes in thousands of gene products. In conclusion, we have demonstrated the power of multifunctional drug design to discover drugs to combat various diseases. We believe this is the future direction of the drug discovery process.

Antitumor activity of oxysterols. Effect of two water-soluble monophosphoric acid diesters of 7 beta-hydroxycholesterol on mastocytoma P815 in vivo.

Oxysterols, a family of naturally occurring products, have been shown to possess several biological activities. In particular, they are more toxic towards tumor cells than towards normal cells. In addition, they markedly modify immune cell responses. To carry out in vivo studies, we have synthesized phosphodiesters of 7 beta-hydroxycholesterol (JB69 and XA29). These water-soluble prodrugs have a similar toxicity to their parent compound under in vitro conditions. When administered intraperitoneally to mice bearing the P815 mastocytoma, they induced significant increases in life span. The results depend on the administration protocol. Under appropriate conditions, 20 to 40% of treated mice recover completely. This, together with their immunological effect, suggests that these oxysterols should be considered to be agents for immunochemotherapeutic investigations. By their ability to inhibit HMG CoA reductase, they may prevent the biosynthesis of prenyl groups whose coupling to oncogenes is responsible for the biological activity expression of the latter. Several indications are compatible with an effect on the cell membrane. Our recent studies have shown Protein Kinase C to be a target of oxysterols. On the basis of results obtained by our group and by others, we believe that oxysterols may form a new class of antitumor agents.

Effect of oxysterol derivatives on the time course development of hepatocarcinoma in transgenic mice.

Among their biological properties, several oxysterols display a stronger toxicity towards tumor cells than towards normal cells. Water-soluble phosphodiesters of 7 beta-hydroxycholesterol (JB69 and XA29), that were proved to retain a specific antitumoral activity in vitro, have been recently developed, allowing in vivo studies. They have been assayed on transgenic mice expressing the Large T antigen of SV40 in their liver and developing systematically hepatocarcinoma within 8 months. We show that JB69 and XA29 administered intraperitoneally into transgenic mice, before the onset of adenoma, may prevent or delay the tumor development. Consequently, oxysterol derivatives might constitute new efficient prodrugs against naturally occurring tumors.

Stampidine prevents mortality in an experimental mouse model of viral hemorrhagic fever caused by lassa virus.

BACKGROUND: The potential use of microorganisms as agents of biological warfare (BW) is a growing concern. Lassa virus, a member of the Arenavirus class of Hemorrhagic fever (HF) viruses has emerged as a worldwide concern among public health officials. The purpose of the present study was to further elucidate the antiviral activity spectrum of stampidine, a novel nucleoside analog with potent anti-viral activity against the immunodeficiency viruses HIV-1, HIV-2, and FIV, by examining its effects on survival of mice challenged with Lassa virus. METHODS: We examined the therapeutic effect of Stampidine in CBA mice inoculated with intracerebral injections of the Josiah strain of Lassa virus. Mice were treated either with vehicle or nontoxic doses of stampidine administered intraperitoneally 24 hours prior to, 1 hour prior to, and 24 hours, 48 hours, 72 hours, and 96 hours after virus inoculation. RESULTS: The probability of survival following the Lassa challenge was significantly improved for stampidine treated mice (Kaplan Meier, Chi-squared = 11.7, df = 2, Log-Rank p-value = 0.003). CONCLUSION: Therefore, stampidine shows clinical potential as a new agent for treatment of viral hemorrhagic fevers caused by Lassa virus.

[Metalloproteinase inhibition: therapeutic application in rheumatic diseases]. / L'inibizione delle metalloproteasi: applicazioni terapeutiche nelle patologie reumatiche.

PURPOSE: Matrix metalloproteinases (MMPs) play an important role in the degradation of articular cartilage in several diseases, including osteorthritis and rheumatoid arthritis. Aiming at developing new drugs with selective inhibiting action against enzyme damaging the extracellular matrix, research is mainly directed towards the: 1) development of new drugs with specific inhibitory effect on MMPs; 2) better understanding of the pharmacologic profile of drugs already used in the treatment of rheumatic diseases, in order to identify those having an inhibiting action on degradative enzymes. MATERIALS AND METHODS: The interaction between rifamycins and collagenase type XI were studied using a fluorogenic substrate MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2. RESULTS: In our experimental conditions rifamycins showed a marked inhibition capacity with a IC50 ranging from 13 to 20.7 microM. This inhibition was reversible after extensive dialysis. CONCLUSIONS: Our results indicate that the effects of rifamycins in rheumatoid arthritis may correlate to the inhibitory activity of these molecules on collagenase activity.

Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency.

Autosomal recessive mutations in the thymidine kinase 2 gene (TK2) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 deficiency, we administered deoxycytidine and deoxythymidine monophosphates (dCMP+dTMP) to the Tk2 H126N (Tk2(-/-)) knock-in mouse model from postnatal day 4, when mutant mice are phenotypically normal, but biochemically affected. Assessment of 13-day-old Tk2(-/-) mice treated with dCMP+dTMP 200 mg/kg/day each (Tk2(-/-200dCMP/) (dTMP)) demonstrated that in mutant animals, the compounds raise dTTP concentrations, increase levels of mtDNA, ameliorate defects of mitochondrial respiratory chain enzymes, and significantly prolong their lifespan (34 days with treatment versus 13 days untreated). A second trial of dCMP+dTMP each at 400 mg/kg/day showed even greater phenotypic and biochemical improvements. In conclusion, dCMP/dTMP supplementation is the first effective pharmacologic treatment for Tk2 deficiency.

Stampidine as a promising antiretroviral drug candidate for pre-exposure prophylaxis against sexually transmitted HIV/AIDS.

INTRODUCTION: Pre-exposure prophylaxis (PrEP) is an evolving new approach to prevention of sexually transmitted HIV-1 that employs antiretroviral (ARV) agents prior to potential HIV-1 exposure in an attempt to reduce the likelihood of HIV-1 infection postexposure. The identification of new ARV agents with potent activity against multidrug-resistant HIV remains an unmet and urgent challenge in the field of PrEP. AREAS COVERED: This article reviews the preclinical and early clinical activity and safety profile of stampidine, a novel antiretroviral (ARV) drug candidate that exhibits remarkable subnanomolar to low nanomolar in vitro antiretroviral potency against genotypically and phenotypically nucleoside reverse transcriptase inhibitor (NRTI)-resistant primary clinical HIV isolates, non-nucleoside RT-resistant HIV-1 isolates. Stampidine has a favorable pharmacokinetic profile in mice, rats, dogs and cats with 25 or 50 mg/kg tolerable dose levels yielding micromolar plasma concentrations that are 1000-fold higher than its in vitro IC(50) value against HIV. Stampidine has a favorable, safety profile in mice, rats, dogs and cats and it showed significant in vivo ARV activity in HIV-infected Hu-PBL-SCID mice as well as FIV-infected domestic cats. Furthermore, it did not cause any maternal toxicity, developmental toxicity or teratogenicity in rabbits treated at 10 - 40 mg/kg/day dose levels. In a recently completed first-in-human Phase I clinical trial, stampidine did not cause dose-limiting toxicity at single dose levels ranging from 5 to 25 mg/kg. EXPERT OPINION: The favorable safety and activity profile of stampidine warrants its further development as a promising next-generation PrEP candidate to prevent the sexual transmission of HIV-1. The discovery of stampidine as a potent antiretroviral agent represents a significant step forward in the development of effective therapeutic as well as preventive strategies against HIV/AIDS.

Stampidine as a novel nucleoside reverse transcriptase inhibit with potent anti-HIV activity.

Stavudine (STV, d4T, 2',3'-didehydro-3'-deoxythymidine, CAS 3056-17-5) is a standard anti-HIV drug. Stampidine (STAMP, DDE-113, HI-113, N-[p-(4-bromophenyl)-2',3'-didehydro-3'-deoxy-5'-thymidylyl]-L-alanine methyl ester, CAS 217178-62-6) is a novel aryl phosphate derivative of stavudine with more potent anti-HIV activity and more favorable pharmacodynamic features. The remarkable potency of stampidine against clinical HIV-1 isolates with NRTI- or NNRTI-resistance (NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor) warrants the further development of this new anti-HIV agent.

In vitro anti-HIV potency of stampidine alone and in combination with standard anti-HIV drugs.

The purpose of the present study was compare the in vitro anti-HIV potency stampidine (CAS 217178-62-6), a novel aryl phosphate derivative of stavudine (CAS 3056-17-5), and drug combinations containing stampidine to the anti-HIV tency of the standard drugs zidovudine (CAS 30516-87-1), stavudine, lamivudine (CAS 134678-17-4), nelfinavir (CAS 159989-65-8), and nevirapine (CAS 129618-40-2) as well as their combinations. Stampidine inhibited the laboratory HIV-1 strain HTLV(IIIB) (B-envelope subtype) as well as the primary clinical HIV-1 isolates BR/92/025 (C-envelope subtype) and BR/93/20 (F-envelope sub-type) with subnanomolar IC50 values. Stampidine was as effective as zidovudine against HTLV(IIIB) and BR/92/025 and 3-logs more effective than zidovudine against BR/93/20. Stampidine was more effective than stavudine, lamivudine, nelfinavir, and nevirapine against all three HIV-1 isolates. The combination of stampidine with zidovudine + lamivudine was more effective than the combination of nelfinavir or nevirapine with zidovudine lamivudine against all three HIV-1 isolates. The combination of stampidine with nelfinavir was more effective than zidovudine + lamivudine as well as the combination of zidovudine + lamivudine with nelfinavir. The combination of stampidine with lamivudine + nelfinavir was more effective than the combination of zidovudine with lamivudine + nelfinavir. The combination of stampidine with lamivudine + nevirapine was more effective than the combination of stavudine with lamivudine + nevirapine. These findings demonstrate that (a) stampidine, as well as its combinations with the standard anti-HIV drugs zidovudine, lamivudine, nelfinavir or nevirapine, are potent inhibitors of HIV-1 replication in human peripheral blood mononuclear cells, and (b) replacement of either zidcovudine, zidovudine+lamivudine or stavudine in 3-drug cocktails with stampidine resulted in greater anti-HIV potency in vitro.

Prophylaxis of Pneumocystis carinii pneumonia in patients infected with the human immunodeficiency virus type 1.

Immunosuppression due to human immunodeficiency virus type 1 (HIV) infection has led to a marked increase in Pneumocystis carinii pneumonia (PCP). Prophylaxis against PCP is standard practice in pediatric cancer patients but is associated with unique problems in HIV-infected patients, including the need for lifelong therapy, adverse reactions, and drug interactions. HIV-infected patients at highest risk for PCP are those with a prior episode of PCP and/or a CD4 lymphocyte count of less than 200 cells/microL. A combination of trimethoprim and sulfamethoxazole is effective prophylactically, although a significant rate of adverse reactions makes long-term prophylaxis difficult. Other oral medications such as dapsone and a combination of pyrimethamine and sulfadoxine are promising but not yet adequately tested. Inhalation of aerosolized pentamidine is an effective and safe means of prophylaxis if the proper dose and nebulizer are used. The only common adverse effects with the latter are airway irritation manifested by cough or wheezing. Zidovudine appears to have a synergistic benefit in further reducing the attack rate of PCP when used with aerosolized pentamidine.

In vivo antiretroviral activity of stampidine in chronically feline immunodeficiency virus-infected cats.

Here we report the antiretroviral activity of the experimental nucleoside reverse transcriptase inhibitor (NRTI) compound stampidine in cats chronically infected with feline immunodeficiency virus (FIV). Notably, a single oral bolus dose of 50 or 100 mg of stampidine per kg resulted in a transient >/=1-log decrease in the FIV load of circulating peripheral blood mononuclear cells in five of six FIV-infected cats and no side effects. A 4-week stampidine treatment course with twice-daily administration of hard gelatin capsules containing 25 to 100 mg of stampidine per kg was also very well tolerated by cats at cumulative dose levels as high as 8.4 g/kg and exhibited a dose-dependent antiretroviral effect. One of three cats treated at the 25-mg/kg dose level, three of three cats treated at the 50-mg/kg dose level, and three of three cats treated at the 100-mg/kg dose level (but none of three control cats treated with placebo pills) showed a therapeutic response, as evidenced by a >/=1-log reduction in the FIV load in peripheral blood mononuclear cells within 2 weeks. The previously documented in vitro and in vivo antiretroviral activity of stampidine against primary clinical human immunodeficiency virus type 1 isolates with genotypic and/or phenotypic NRTI resistance, together with its favorable animal toxicity profile, pharmacokinetics, and in vivo antiretroviral activity in FIV-infected cats, warrants further development of this promising new NRTI compound.

Antiretroviral spermicide WHI-07 prevents vaginal and rectal transmission of feline immunodeficiency virus in domestic cats.

WHI-07 [5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-bromophenyl)-methoxy alaninyl phosphate] is a novel dual-function aryl phosphate derivative of zidovudine with potent anti-human immunodeficiency virus (HIV) and spermicidal activities. WHI-07 was active against the feline immunodeficiency virus (FIV). This study evaluated whether topical application of WHI-07 as a single agent and in combination with an organometallic vanadium complex, vanadocene dithiocarbamate (VDDTC), via a nontoxic gel microemulsion can block vaginal as well as rectal transmission of feline AIDS (FAIDS) by chronically FIV-infected feline T cells in the natural host model. Genital transmission of FIV was monitored in recipient cats by the appearance of viral antibodies to FIV Gag proteins and by virus isolation of blood leukocytes as measured by FIV reverse transcriptase activity and FIV-specific PCR. Microbicidal activity was considered effective when the treated cats did not show evidence of FIV infection for up to 18 weeks postchallenge. An aggregate analysis of 46 specific-pathogen-free cats revealed that a single dose of the infected cell inoculum efficiently transmitted FIV infection when delivered into the vagina (100%) or rectum (66%). Pretreatment of the vagina or rectum with 2% WHI-07 alone or in combination with 0.25% VDDTC significantly (P = 0.004) protected cats from genital transmission by the highly infectious inoculum (7 million FIV(Bangston)-infected feline T cells). Collectively, using the vaginal and rectal transmucosal model for FAIDS, our studies demonstrated that WHI-07 either alone or in combination with a vanadocene has clinical potential for the development of a dual-function anti-HIV microbicide for sexually active women.