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Tenofovir (TFV) is the cornerstone nucleotide reverse transcriptase inhibitor (NtRTI) in many combination antiretroviral therapies prescribed to patients living with HIV/AIDS. Due to poor cell permeability and oral bioavailability, TFV is administered as one of two FDA-approved prodrugs, both of which metabolize prematurely in the liver and/or plasma. This premature prodrug processing depletes significant fractions of each oral dose and causes toxicity in kidney, bone, and liver with chronic administration. Although TFV exalidex (TXL), a phospholipid-derived prodrug of TFV, was designed to address this issue, clinical pharmacokinetic studies indicated substantial hepatic extraction, redirecting clinical development of TXL toward HBV. To circumvent this metabolic liability, we synthesized and evaluated ω-functionalized TXL analogues with dramatically improved hepatic stability. This effort led to the identification of compounds 21 and 23, which exhibited substantially longer t1/2 values than TXL in human liver microsomes, potent anti-HIV activity in vitro, and enhanced pharmacokinetic properties in vivo.

HIV protease is essential for processing the Gag polyprotein to produce infectious virions and is a major target in antiretroviral therapy. We have identified an unusual HIV-1 subtype C variant that contains insertions of leucine and asparagine (L38↑N↑L) in the hinge region of protease at position 38. This was isolated from a protease inhibitor naïve infant. Isothermal titration calorimetry showed that 10% less of L38↑N↑L protease was in the active conformation as compared with a reference strain. L38↑N↑L protease displayed a ±50% reduction in KM and kcat. The catalytic efficiency (kcat/KM) of L38↑N↑L protease was not significantly different from that of wild type although there was a 42% reduction in specific activity for the variant. An in vitro phenotypic assay showed the L38↑N↑L protease to be susceptible to lopinavir (LPV), atazanavir (ATV) and darunavir in the context of an unrelated Gag. However, in the presence of the related Gag, L38↑N↑L showed reduced susceptibility to darunavir while remaining susceptible to LPV and ATV. Furthermore, a reduction in viral replication capacity (RC) was observed in combination with the related Gag. The reduced susceptibility to darunavir and decrease in RC may be due to PTAPP duplication in the related Gag. The present study shows the importance of considering the Gag region when looking at drug susceptibility of HIV-1 protease variants.

Novel N-, O- and S-substituted pyridyl-benzamides, -benzylamines and -sulfonamides were prepared by means of palladium-catalysed cross-coupling reactions. The synthetic approach, using Pd2(dba)3 as palladium source and rac-BINAP as supporting ligand, proved to be successful for C N, C O and C S cross-coupling reactions. One of the substrates underwent an unexpected nucleophilic aromatic substitution of fluorine, rather than the expected C N cross-coupling reaction.

The wastewater treatment process, besides discharging pharmaceuticals into the environment, has been found to result in the formation of a variety of undescribed compounds. Here we investigate the laboratory scale chlorination of the commonly used anti-HIV drug Nevirapine, characterise its disinfection transformation products (DTPs), and using liquid chromatography with high resolution mass spectrometry, screen environmental surface water for these DTPs. Chlorination of Nevirapine was scaled up, fractioned by preparative chromatography and the fractions were tested in vitro for toxicity and anti-HIV activity. Nevirapine was found to be resistant to degradation at relevant chlorination levels, which may partially explain its ubiquitous presence in South African surface water. During simulated chlorination, a variety of DTPs with varying properties were formed, some of which were detected in the environment, close to wastewater treatment plants. Interestingly, some of these compounds, although not as toxic as Nevirapine, retained antiviral activity. Further purification and synthesis is required to fully characterise these novel molecules.

We have previously reported on HIV-1 infected patients who fail anti-retroviral therapy but manage to re-suppress without a regimen change despite harbouring major drug resistance mutations. Here we explore phenotypic drug resistance in such patients in order to better understand this phenomenon. Patients (n = 71) failing a non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimen, but who subsequently re-suppressed on the same regimen, were assessed for HIV-1 genotypic drug resistance through Sanger sequencing. A subset (n = 23) of these samples, as well as genotypically matched samples from patients who did not re-suppress (n = 19), were further assessed for phenotypic drug resistance in an in vitro single cycle assay. Half of the patients (n = 36/71, 51%) harboured genotypic drug resistance, with M184V (n = 18/36, 50%) and K103N (n = 16/36, 44%) being the most prevalent mutations. No significant difference in the median time to re-suppression (31–39 weeks) were observed for either group (p = 0.41). However, re-suppressors with mutant virus rebounded significantly earlier than those with wild-type virus (16 vs. 33 weeks; p = 0.014). Similar phenotypic drug resistance profiles were observed between patients who re-suppressed and patients who failed to re-suppress. While most remained susceptible to stavudine (d4T) and zidovudine (AZT), both groups showed a reduced susceptibility to 3TC and NNRTIs. HIV- 1 infected patients on an NNRTI-based regimen can achieve viral re-suppression on the same regimen despite harbouring viruses with genotypic and phenotypic drug resistance. However, re-suppression was less durable in those with resistance, reinforcing the importance of appropriate regimen choices, ongoing viral load monitoring and adherence counselling.

[Image: see text] Since the discovery of HIV as the etiological agent of AIDS, the virus has infected millions of people each year. Fortunately, with the use of HAART, viremia can be suppressed to below detectable levels in the infected individuals, which significantly improves their quality of life and prevents the onset of AIDS. However, HAART is not curative and issues relating to adherence and drug resistance may lead to the re-emergence of viremia, the development of AIDS, and ultimately death. To address a pressing need for the development of new and efficacious antiretroviral agents with activity against viruses bearing prevalent resistant mutations, we have designed two generations of benzimidazolone derivatives as HIV non-nucleoside reverse transcriptase inhibitors. The first generation benzimidazolone inhibitors were found to be potent inhibitors of wild-type HIV reverse transcriptase but were ineffective in the presence of common resistance mutations such as K103N and Y181C. A second generation benzimidazolone inhibitor (compound 42) not only showed inhibitory activity against wild-type HIV but also remained active against HIV containing the K103N, Y181C, and K103N/Y181C drug resistance mutations.

Studies have shown a low frequency of HIV-1 protease drug resistance mutations in patients failing protease inhibitor (PI)-based therapy. Recent studies have identified mutations in Gag as an alternate pathway for PI drug resistance in subtype B viruses. We therefore genotyped the Gag and protease genes from 20 HIV-1 subtype C-infected pediatric patients failing a PI-based regimen. Major protease resistance mutations (M46I, I54V, and V82A) were identified in eight (40%) patients, as well as Gag cleavage site (CS) mutations (at codons 373, 374, 378, 428, 431, 449, 451, and 453) in nine (45%) patients. Four of these Gag CS mutations occurred in the absence of major protease mutations at PI failure. In addition, amino acid changes were noted at Gag non-CS with some predicted to be under HLA/KIR immune-mediated pressure and/or drug selection pressure. Changes in Gag during PI failure therefore warrant further investigation of the Gag gene and its role in PI failure in HIV-1 subtype C infection.

The objective of this study was to assess the phenotypic susceptibility of HIV-1 subtype C isolates, with nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance-associated amino acid changes, to newer NNRTIs. A panel of 52 site-directed mutants and 38 clinically derived HIV-1 subtype C clones was created, and the isolates were assessed for phenotypic susceptibility to etravirine (ETR), rilpivirine (RPV), efavirenz (EFV), and nevirapine (NVP) in anin vitrosingle-cycle phenotypic assay. The amino acid substitutions E138Q/R, Y181I/V, and M230L conferred high-level resistance to ETR, while K101P and Y181I/V conferred high-level resistance to RPV. Y181C, a major NNRTI resistance-associated amino acid substitution, caused decreased susceptibility to ETR and, to a lesser extent, RPV when combined with other mutations. These included N348I and T369I, amino acid changes in the connection domain that are not generally assessed during resistance testing. However, the prevalence of these genotypes among subtype C sequences was, in most cases

The human immunodeficiency virus (HIV) pandemic remains a significant problem, especially in developing nations where the social and economic impacts are severe. Until a cure or vaccine for the disease is found, a constant supply of new compounds to fill the drug development pipeline is a requirement, given the tendency for the virus to rapidly develop resistance to current therapies. Here we disclose our efforts to improve upon the efficacy of cyclopropyl-indole derivatives developed as NNRTIs in our laboratories. To this end, modifications to the functionality occupying the small Val179 pocket have resulted in nearly two orders of magnitude increase in potency.

The development of novel anti-HIV agents remains an important medicinal chemistry challenge given that no cure for the disease is imminent, and the continued use of current NNRTIs inevitably leads to problems associated with resistance. Inspired by the pyrazole-containing NNRTI lersivirine (LSV), we embarked upon a study to establish whether 1,2,3-triazole heterocycles could be used as a new scaffold for the creation of novel NNRTIs. An especially attractive feature of triazoles used for this purpose is the versatility in accessing variously functionalised systems using either the thermally regulated Huisgen cycloaddition, or the related 'click' reaction. Employing three alternative forms of these reactions, we were able to synthesise a range of triazole compounds and evaluate their efficacy in a phenotypic HIV assay. To our astonishment, even compounds closely mimicking LSV were only moderately effective against HIV.

In a previous communication we described a series of indole based NNRTIs which were potent inhibitors of HIV replication, both for the wild type and K103N strains of the virus. However, the methyl ether functionality on these compounds, which was crucial for potency, was susceptible to acid promoted indole assisted SN1 substitution. This particular problem did not bode well for an orally bioavailable drug. Here we describe bioisosteric replacement of this problematic functional group, leading to a series of compounds which are potent inhibitors of HIV replication, and are acid stable.

Surveillance of human immunodeficiency virus type 1 transmitted drug resistance (TDR) was conducted among pregnant women in South Africa over a 5-year period after the initiation of a large national antiretroviral treatment program. Analysis of TDR data from 9 surveys conducted between 2005 and 2009 in 2 provinces of South Africa suggests that while TDR remains low (

Objectives: Single-dose nevirapine (sdNVP) used to prevent mother-to-child transmission of HIV-1 results in the selection of genotypic drug resistance mutations. To assess the levels of phenotypic resistance conferred by these mutations, we examined the ability of sample-derived HIV-1 reverse transcriptase to function in the presence of nevirapine (NVP). Methods: Plasma samples from HIV-1 pregnant women before and after exposure to sdNVP were used to extract viral reverse transcriptase for the Cavidi ExaVir Drug susceptibility assay. The fold increases in phenotypic resistance for each sample were compared with the genotypic profiles determined by population-based sequencing. Results: None of the women sampled before sdNVP exposure had phenotypic resistance (median fold increase 0.7). Seven weeks after sdNVP, there was a 16-fold increase in phenotypic resistance among women who had NVP resistance mutations compared with only a 1.9-fold increase among NVP-exposed women with wild-type virus. Phenotypic resistance decayed with time coincident with the fading of genotypic mutations, and by 18 months, all samples were phenotypically susceptible. Conclusions: Exposure of pregnant women to sdNVP was associated with the transient appearance of viral populations that displayed phenotypic resistance to NVP. Overall, there was good concordance between phenotypic resistance to NVP, as measured with this enzymatic assay, and the presence of NVP genotypic mutations.

Protease inhibitors (PIs) are used as a first-line regimen in HIV-1-infected children. Here we investigated the phenotypic consequences of amino acid changes in Gag and protease on lopinavir (LPV) and ritonavir (RTV) susceptibility among pediatric patients failing PI therapy. The Gag-protease from isolates from 20 HIV-1 subtype C-infected pediatric patients failing an LPV and/or RTV-based regimen was phenotyped using a nonreplicativein vitroassay. Changes in sensitivity to LPV and RTV relative to that of the matched baseline (pretherapy) sample were calculated. Gag and protease amino acid substitutions associated with PI failure were created in a reference clone by site-directed mutagenesis and assessed. Predicted phenotypes were determined using the Stanford drug resistance algorithm. Phenotypic resistance or reduced susceptibility to RTV and/or LPV was observed in isolates from 10 (50%) patients, all of whom had been treated with RTV. In most cases, this was associated with protease resistance mutations, but substitutions at Gag cleavage and noncleavage sites were also detected. Gag amino acid substitutions were also found in isolates from three patients with reduced drug susceptibilities who had wild-type protease. Site-directed mutagenesis confirmed that some amino acid changes in Gag contributed to PI resistance but only in the presence of major protease resistance-associated substitutions. The isolates from all patients who received LPV exclusively were phenotypically susceptible. Baseline isolates from the 20 patients showed a large (47-fold) range in the 50% effective concentration of LPV, which accounted for most of the discordance seen between the experimentally determined and the predicted phenotypes. Overall, the inclusion of thegaggene and the use of matched baseline samples provided a more comprehensive assessment of the effect of PI-induced amino acid changes on PI resistance. The lack of phenotypic resistance to LPV supports the continued use of this drug in pediatric patients.

The HIV pandemic represents one of the most serious diseases to face mankind in both a social and economic context, with many developing nations being the worst afflicted. Due to ongoing resistance issues associated with the disease, the design and synthesis of anti-HIV agents presents a constant challenge for medicinal chemists. Utilizing molecular modeling, we have designed a series of novel cyclopropyl indole derivatives as HIV non-nucleoside reverse transcriptase inhibitors and carried out their preparation. These compounds facilitate a double hydrogen bonding interaction to Lys101 and efficiently occupy the hydrophobic pockets in the regions of Tyr181/188 and Val179. Several of these compounds inhibited HIV replication as effectively as nevirapine when tested in a phenotypic assay.

To investigate the involvement of cytochrome P450s in the metabolism of plants treated with xenobiotic agrochemicals, bean leaves were treated with 3,5-dichlorosalicylic acid (DC-SA), a priming agent of plant defense and 2,6-dichloroisonicotinic acid (DC-INA), a chemical inducer of systemic acquired resistance. Through the use of directed differential display reverse transcription polymerase chain reactions, a differentially expressed cDNA amplicon, found to be up-regulated by both DC-SA and DC-INA treatment, was identified as a cytochrome P450 cDNA, CYP98A5. The nucleotide sequence indicates extensive homology to 3'-hydroxylases of p-coumaroyl esters. Dot blot analysis of leaves treated with various SA and isonicotinic acid derivatives showed enhanced expression of CYP98A5 due to DC-SA and DC-INA. Northern blot analysis of a time-dependent induction study of CYP98A5 in treated bean leaves indicated that DC-SA induces CYP98A5 mRNA transcripts earlier than DC-INA. Both inducers resulted in high transcript levels 24-48 h after treatment. The up-regulation of CYP98A5 is supportive of the conditioning and sensitizing effects of DC-SA and DC-INA to elicit a more rapid and effective defense response.

Following the rapid scale-up of the programme for universal access to antiretroviral therapy (ART) in southern Africa, resistance to antiretroviral medications will occur. A detectable viral load must be treated as an emergency and should trigger intensive patient tracking and adherence counselling. In contrast to the developed world, the incidence of transmitted resistance is still low in most areas in the region. Therefore, in this consensus statement we do not recommend resistance testing in HIV-infected adults upon diagnosis or ART initiation. However, baseline resistance testing is recommended for children who have been exposed to ART for prevention of mother-to-child-transmission therapy and subsequently become HIV-infected. Resistance testing is also recommended after virological failure of first- and second-line ART regimens.

Indigofera daleoides Benth. ex Harv. & Sond. (Fabaceae) is used by traditional healers in Limpopo Province for treatment of diarrhoea. The bioassay-guided fractionation of the ethylacetate extract from I. daleoides led to the isolation of two pure compounds which were identified as compound 1 (6,2-O-[3-nitropropanoyl-β-D-glucopyranose]) and compound 2 with a novel skeleton (6,3′,4′-trihydroxyflavan 5′-O-glucopyranoside). These compounds were isolated for the first time from I. daleoides. Isolated compounds were tested for antibacterial and cytotoxic activities in vitro using micro dilution method and sodium-2,3-bis-[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) assay, respectively. The results showed that compound 2 (flavonoid), isolated from I. daleoides, exhibited the highest antibacterial activity against S. aureus, S. typhy, V. cholera, E. coli and S. dysentery with MIC values ranging between 25–50 μg/ml. Compound 1 (nitrocompound) was found to be less active than compound 2 (flavonoid) against most of the bacteria tested. Staphylococcus aureus had higher susceptibility to compound 2 (flavonoid) as compared to the other bacteria tested. Compound 1 (nitrocompound) and 2 (flavonoid) isolated from I. daleoides had relatively high fifty per cent inhibitory concentration (IC50 values) (349.4 and 364.5 μg/ml, respectively) as compared to the crude extract (IC50 254 μg/ml), thus suggesting these compounds are less cytotoxic to Vero cells than the crude ethanol extract. The results of this study suggest possible antidiarrhoeal potential of these compounds, especially compound 2 (flavonoid) from I. daleoides which need to be further investigated. This is the first report on the antibacterial activity and cytotoxicity of purified compounds from I. daleoides.

Pigmentation has become an important phenotypical characteristic, in the pharmaceutical, medicinal as well as in the cosmetic field. Plants and their extracts are inexpensive and rich resources of active compounds that can be utilized to inhibit tyrosinase activity as well as melanin production. Natural and synthetic chemical agents can frequently modulate the metabolism of pigmentation produced. Methanol extracts of seven plants were evaluated for their inhibitory effect on the monophenolase and diphenolase activated forms of tyrosinase in vitro . Active extracts were also investigated for their inhibitory effect on melanogenesis in B16 melanoma cells. Extracts of Hyaenanche globosa and Myrsine africana showed 92% and 83% inhibition of tyrosinase activity and 42% and 62% inhibition of DOPA auto-oxidation at 500 μg/ml respectively. Rooibos tea which is known for its antioxidant activity did not show any inhibition of tyrosinase or DOPA oxidation activity. M. africana demonstrated 16% and 18% inhibition of melanin production at 6.25 and 12.50 μg/ml respectively.