Antiviral Nucleoside Drug Delivery via Amino Acid Phosphoramidates

Abstract

Stable and water soluble amino acid phosphomonoester amidates of AZT were synthesized and shown to have potent anti-HIV-1 activity. Intracellular and cell extract metabolism studies revealed that these compounds are likely to be enzymatically converted to the corresponding monophosphates. In addition, we have shown that the half life and tissue distribution of a phosphoramidate of AZT is 5 and 10-fold greater, respectively, than AZT.     

Carbovir: A Carbocyclic Nucleoside with Potent and Selective Activity Against Human Immunodeficiency Virus (HIV) in Vitro

Abstract

Carbocyclic 2′, 3′-didehydro-2′,3′-dideoxyquanosine (carbovir), a novel nucleoside analog, emerged as a potent and selective anti-HIV agent from a primary screen of a large number of carbocyclic nucleosides.¹ Carbovir inhibited the infectivity and replication of HIV in T-cells at concentrations 200 to 400-fold below toxicity to host cells. Carbovir was also evaluated for its Inhibitory effects on the expression of viral antigen in HIV-infected CEM cells. Production of p 24 core antigen at optimal inhibitory concentrations of the antiviral agents indicated comparable results for AZT, ddA and carbovir.     

Design,Synthesis and Antiviral Activity of α-L-Arabinofuranosyl Derivatives of 2-Substituted-5,6-dichlorobenzimidazoles

Abstract

A number of 2-substituted-5,6-dichloro-l-(α-L-arabinofuranosyl)benzimidazoles have been prepared by condensation of 2-bromo-5,6-dichlorobenzimidazole or 2,5,6-trichlorobenzimidazole with tetra-O-acetyl-L-arabinofuranose. 2-Alkylamino derivatives were prepared by a substitution of the 2-chloro group with the appropriate amines. All target compounds were evaluated for activity against HCMV and HSV-1. The 2-chloro and 2-bromo derivatives showed moderate activity against HCMV at non-cytotoxic concentrations.     

Synthesis of Reagents for Fluorescence-Tagging of DNA

Abstract

A general route for the synthesis of alkynylamino nucleoside triphosphates is described. These nucleosides can be selectively coupled through the alkynylamino group to a variety of reporter groups and used to enzymatically label DNA.     

Synthesis and Biological Activity of Sugar-Fluorinated 2′,3′-dideoxy-4′-thioribofuranosyl Nucleosides

Abstract

The efficient DAST fluorination of deoxy-4′-thiopyrimidine nucleosides is reported. The cytidine analogue 3b was marginally effective against HIV.     

Inactivation of Nitric Oxide by Uric Acid

The 1980 identification of nitric oxide (NO) as an endothelial cell-derived relaxing factor resulted in an unprecedented biomedical research of NO and established NO as one of the most important cardiovascular, nervous and immune system regulatory molecule. A reduction in endothelial cell NO levels leading to “endothelial dysfunction” has been identified as a key pathogenic event preceding the development of hypertension, metabolic syndrome, and cardiovascular disease. The reduction in endothelial NO in cardiovascular disease has been attributed to the action of oxidants that either directly react with NO or uncouple its substrate enzyme. In this report, we demonstrate that uric acid (UA), the most abundant antioxidant in plasma, reacts directly with NO in a rapid irreversible reaction resulting in the formation of 6-aminouracil and depletion of NO. We further show that this reaction occurs preferentially with NO even in the presence of oxidants peroxynitrite and hydrogen peroxide and that the reaction is at least partially blocked by glutathione. This study shows a potential mechanism by which UA may deplete NO and cause endothelial dysfunction, particularly under conditions of oxidative stress in which UA is elevated and intracellular glutathione is depleted.     

Natural Infection of Burkholderia pseudomallei in an Imported Pigtail Macaque (Macaca nemestrina) and Management of the Exposed Colony

Identification of the select agent Burkholderia pseudomallei in macaques imported into the United States is rare. A purpose-bred, 4.5-y-old pigtail macaque (Macaca nemestrina) imported from Southeast Asia was received from a commercial vendor at our facility in March 2012. After the initial acclimation period of 5 to 7 d, physical examination of the macaque revealed a subcutaneous abscess that surrounded the right stifle joint. The wound was treated and resolved over 3 mo. In August 2012, 2 mo after the stifle joint wound resolved, the macaque exhibited neurologic clinical signs. Postmortem microbiologic analysis revealed that the macaque was infected with B. pseudomallei. This case report describes the clinical evaluation of a B. pseudomallei-infected macaque, management and care of the potentially exposed colony of animals, and protocols established for the animal care staff that worked with the infected macaque and potentially exposed colony. This article also provides relevant information on addressing matters related to regulatory issues and risk management of potentially exposed animals and animal care staff.Abbreviations: CDC, Centers for Disease Control and Prevention; IHA, indirect hemagglutination assay; PEP, postexposure prophylacticBurkholderia pseudomallei, formerly known as Pseudomonas pseudomallei, is a gram-negative, aerobic, bipolar, motile, rod-shaped bacterium. B. pseudomallei infections (melioidosis) can be severe and even fatal in both humans and animals. This environmental saprophyte is endemic to Southeast Asia and northern Australia, but it has also been found in other tropical and subtropical areas of the world.^7,22,32,42 The bacterium is usually found in soil and water in endemic areas and is transmitted to humans and animals primarily through percutaneous inoculation, ingestion, or inhalation of a contaminated source.^{8, 22,28,32,42} Human-to-human, animal-to-animal, and animal-to-human spread are rare.^8,32 In December 2012, the National Select Agent Registry designated B. pseudomallei as a Tier 1 overlap select agent.³⁹ Organisms classified as Tier 1 agents present the highest risk of deliberate misuse, with the most significant potential for mass casualties or devastating effects to the economy, critical infrastructure, or public confidence. Select agents with this status have the potential to pose a severe threat to human and animal health or safety or the ability to be used as a biologic weapon.³⁹Melioidosis in humans can be challenging to diagnose and treat because the organism can remain latent for years and is resistant to many antibiotics.^12,37,41 B. pseudomallei can survive in phagocytic cells, a phenomenon that may be associated with latent infections.^19,38 The incubation period in naturally infected animals ranges from 1 d to many years, but symptoms typically appear 2 to 4 wk after exposure.^13,17,35,38 Disease generally presents in 1 of 2 forms: localized infection or septicemia.²² Multiple methods are used to diagnose melioidosis, including immunofluorescence, serology, and PCR analysis, but isolation of the bacteria from blood, urine, sputum, throat swabs, abscesses, skin, or tissue lesions remains the ‘gold standard.’^9,22,40,42 The prognosis varies based on presentation, time to diagnosis, initiation of appropriate antimicrobial treatment, and underlying comorbidities.^7,28,42 Currently, there is no licensed vaccine to prevent melioidosis.There are several published reports of naturally occurring melioidosis in a variety of nonhuman primates (NHP; 2,10,13,17,25,30,31,35 The first reported case of melioidosis in monkeys was recorded in 1932, and the first published case in a macaque species was in 1966.³⁰ In the United States, there have only been 7 documented cases of NHP with B. pseudomallei infection.^2,13,17 All of these cases occurred prior to the classification of B. pseudomallei as a select agent. Clinical signs in NHP range from subclinical or subacute illness to acute septicemia, localized infection, and chronic infection. NHP with melioidosis can be asymptomatic or exhibit clinical signs such as anorexia, wasting, purulent drainage, subcutaneous abscesses, and other soft tissue lesions. Lymphadenitis, lameness, osteomyelitis, paralysis and other CNS signs have also been reported.^{2,7,10,22,28,32} In comparison, human''s clinical signs range from abscesses, skin ulceration, fever, headache, joint pain, and muscle tenderness to abdominal pain, anorexia, respiratory distress, seizures, and septicemia.^7,9,21,22

Table 1.

Summary of reported cases of naturally occurring Burkholderia pseudomalleiinfections in nonhuman primates

Quantitative Proteomic Analysis in Metastatic Renal Cell Carcinoma Reveals a Unique Set of Proteins with Potential Prognostic Significance

Metastatic renal cell carcinoma (RCC) is one of the most treatment-resistant malignancies, and patients have a dismal prognosis, with a <10% five-year survival rate. The identification of markers that can predict the potential for metastases will have a great effect in improving patient outcomes. In this study, we used differential proteomics with isobaric tags for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS analysis to identify proteins that are differentially expressed in metastatic and primary RCC. We identified 1256 non-redundant proteins, and 456 of these were quantified. Further analysis identified 29 proteins that were differentially expressed (12 overexpressed and 17 underexpressed) in metastatic and primary RCC. Dysregulated protein expressions of profilin-1 (Pfn1), 14–3-3 zeta/delta (14–3-3ζ), and galectin-1 (Gal-1) were verified on two independent sets of tissues by means of Western blot and immunohistochemical analysis. Hierarchical clustering analysis showed that the protein expression profile specific for metastatic RCC can distinguish between aggressive and non-aggressive RCC. Pathway analysis showed that dysregulated proteins are involved in cellular processes related to tumor progression and metastasis. Furthermore, preliminary analysis using a small set of tumors showed that increased expression of Pfn1 is associated with poor outcome and is a potential prognostic marker in RCC. In addition, 14–3-3ζ and Gal-1 also showed higher expression in tumors with poor prognosis than in those with good prognosis. Dysregulated proteins in metastatic RCC represent potential prognostic markers for kidney cancer patients, and a greater understanding of their involved biological pathways can serve as the foundation of the development of novel targeted therapies for metastatic RCC.Renal cell carcinoma (RCC)¹ is the most common neoplasm of the adult kidney. Worldwide incidence and mortality rates of RCC are rising each decade (1). Seventy-five percent of kidney tumors are of the clear cell (ccRCC) subtype (2). Although modern imaging techniques for abdominal screening have led to increased incidental detection of renal tumors (3), unfortunately ∼25% to 30% of patients still have metastases at presentation.The prognosis with RCC is quite variable. The greatest risk of recurrence following nephrectomy is within the first 3 to 5 years (4). The ability to predict which tumors will metastasize would have a significant effect on patient outcomes, because the likelihood of a favorable response to treatment is greater when the metastatic burden is limited, and surgical resection of a single or limited number of metastases can result in longer survival (5). Furthermore, ∼3% of patients will develop a second primary renal tumor, either synchronous or metachronous. Currently, patient prognosis is assessed based on histological parameters and a multivariate analysis developed at Memorial Sloan Kettering (6), but neither is sufficiently accurate. A more accurate assessment of prognosis is urgently needed to better guide patient management.Although surgery can be curative for localized disease, many patients eventually relapse. Metastatic RCC is one of the most treatment-resistant malignancies, with chemotherapy and radiotherapy having limited effect. The five-year survival rate for metastatic RCC is ≤10% (7). Although there has been much progress in RCC treatment with the new era of antiangiogenic therapy, the majority of patients ultimately suffer a relapse and die from progression of the cancer. A more in-depth understanding of the pathogenesis of metastasis will be a cornerstone in the development of new targeted therapies. A number of prognostic markers have previously been identified based on comparative analysis of primary and metastatic tumors, including C-reactive protein, tetraspanin 7, hypoxia-inducible factor 1 α, phos-S6, U3 small nucleolar ribonucleoprotein protein, carbonic anhydrase IX, and microvascular density (8–14). However, no biomarker has yet had an established clinical role independent of stage (15). Differential protein expression between primary RCC and normal tissues was previously studied (16–18). Also, differential expression between primary and metastatic kidney disease has been investigated at the microRNA level (19, 20). Molecular analyses hold the promise of providing a better understanding of the pathogenesis of kidney cancer (21).In this study, we aimed to elucidate the pathogenesis of RCC metastasis through proteomic analysis and to identify potential prognostic markers for kidney cancer. We performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS to identify proteins that were dysregulated in metastatic RCC relative to primary RCC. Differential expressions of selected biologically interesting proteins—profilin-1 (Pfn1), 14–3-3 zeta/delta (14–3-3ζ), and galectin-1 (Gal-1)—were validated on two independent sets of tumors by means of western blot (WB) analysis and immunohistochemistry (IHC). Hierarchical clustering analysis showed that differential protein expression can distinguish between aggressive and non-aggressive tumors. In order to explore the role of these dysregulated proteins in tumor progression, we performed Gene Ontology (GO) and pathway analyses. In addition, we carried out a preliminary analysis to assess the potential of Pfn1, 14–3-3ζ, and Gal-1 as prognostic markers in RCC.     

Impacts of 3 years of elevated atmospheric CO2 on rhizosphere carbon flow and microbial community dynamics

Carbon (C) uptake by terrestrial ecosystems represents an important option for partially mitigating anthropogenic CO₂ emissions. Short‐term atmospheric elevated CO₂ exposure has been shown to create major shifts in C flow routes and diversity of the active soil‐borne microbial community. Long‐term increases in CO₂ have been hypothesized to have subtle effects due to the potential adaptation of soil microorganism to the increased flow of organic C. Here, we studied the effects of prolonged elevated atmospheric CO₂ exposure on microbial C flow and microbial communities in the rhizosphere. Carex arenaria (a nonmycorrhizal plant species) and Festuca rubra (a mycorrhizal plant species) were grown at defined atmospheric conditions differing in CO₂ concentration (350 and 700 ppm) for 3 years. During this period, C flow was assessed repeatedly (after 6 months, 1, 2, and 3 years) by ¹³C pulse‐chase experiments, and label was tracked through the rhizosphere bacterial, general fungal, and arbuscular mycorrhizal fungal (AMF) communities. Fatty acid biomarker analyses and RNA‐stable isotope probing (RNA‐SIP), in combination with real‐time PCR and PCR‐DGGE, were used to examine microbial community dynamics and abundance. Throughout the experiment the influence of elevated CO₂ was highly plant dependent, with the mycorrhizal plant exerting a greater influence on both bacterial and fungal communities. Biomarker data confirmed that rhizodeposited C was first processed by AMF and subsequently transferred to bacterial and fungal communities in the rhizosphere soil. Over the course of 3 years, elevated CO₂ caused a continuous increase in the ¹³C enrichment retained in AMF and an increasing delay in the transfer of C to the bacterial community. These results show that, not only do elevated atmospheric CO₂ conditions induce changes in rhizosphere C flow and dynamics but also continue to develop over multiple seasons, thereby affecting terrestrial ecosystems C utilization processes.     

Phosphatidate phosphatase,a key regulator of lipid homeostasis

Yeast Pah1p phosphatidate phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol. PAP plays a crucial role in lipid homeostasis by controlling the relative proportions of its substrate phosphatidate and its product diacylglycerol. The cellular amounts of these lipid intermediates influence the synthesis of triacylglycerol and the pathways by which membrane phospholipids are synthesized. Physiological functions affected by PAP activity include phospholipid synthesis gene expression, nuclear/endoplasmic reticulum membrane growth, lipid droplet formation, and vacuole homeostasis and fusion. Yeast lacking Pah1p PAP activity are acutely sensitive to fatty acid-induced toxicity and exhibit respiratory deficiency. PAP is distinguished in its cellular location, catalytic mechanism, and physiological functions from Dpp1p and Lpp1p lipid phosphate phosphatases that utilize a variety of substrates that include phosphatidate. Phosphorylation/dephosphorylation is a major mechanism by which Pah1p PAP activity is regulated. Pah1p is phosphorylated by cytosolic-associated Pho85p–Pho80p, Cdc28p-cyclin B, and protein kinase A and is dephosphorylated by the endoplasmic reticulum-associated Nem1p–Spo7p phosphatase. The dephosphorylation of Pah1p stimulates PAP activity and facilitates the association with the membrane/phosphatidate allowing for its reaction and triacylglycerol synthesis. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.