Re-emerging Aspartic Protease Targets: Examining Cryptococcus neoformans Major Aspartyl Peptidase 1 as a Target for Antifungal Drug Discovery.

Cryptococcosis is an invasive infection that accounts for 15% of AIDS-related fatalities. Still, treating cryptococcosis remains a significant challenge due to the poor availability of effective antifungal therapies and emergence of drug resistance. Interestingly, protease inhibitor components of antiretroviral therapy regimens have shown some clinical benefits in these opportunistic infections. We investigated Major aspartyl peptidase 1 (May1), a secreted Cryptococcus neoformans protease, as a possible target for the development of drugs that act against both fungal and retroviral aspartyl proteases. Here, we describe the biochemical characterization of May1, present its high-resolution X-ray structure, and provide its substrate specificity analysis. Through combinatorial screening of 11,520 compounds, we identified a potent inhibitor of May1 and HIV protease. This dual-specificity inhibitor exhibits antifungal activity in yeast culture, low cytotoxicity, and low off-target activity against host proteases and could thus serve as a lead compound for further development of May1 and HIV protease inhibitors.

Per-residue energy decomposition pharmacophore model to enhance virtual screening in drug discovery: a study for identification of reverse transcriptase inhibitors as potential anti-HIV agents.

A novel virtual screening approach is implemented herein, which is a further improvement of our previously published "target-bound pharmacophore modeling approach". The generated pharmacophore library is based only on highly contributing amino acid residues, instead of arbitrary pharmacophores, which are most commonly used in the conventional approaches in literature. Highly contributing amino acid residues were distinguished based on free binding energy contributions obtained from calculation from molecular dynamic (MD) simulations. To the best of our knowledge; this is the first attempt in the literature using such an approach; previous approaches have relied on the docking score to generate energy-based pharmacophore models. However, docking scores are reportedly unreliable. Thus, we present a model for a per-residue energy decomposition, constructed from MD simulation ensembles generating a more trustworthy pharmacophore model, which can be applied in drug discovery workflow. This work is aimed at introducing a more rational approach to the field of drug design, rather than comparing the validity of this approach against those previously reported. We recommend additional computational and experimental work to further validate this approach. This approach was used to screen for potential reverse transcriptase inhibitors using the pharmacophoric features of compound GSK952. The complex was subjected to docking, thereafter, MD simulation confirmed the stability of the system. Experimentally determined inhibitors with known HIV-reverse transcriptase inhibitory activity were used to validate the protocol. Two potential hits (ZINC46849657 and ZINC54359621) showed a significant potential with regard to free binding energy. Reported results obtained from this work confirm that this new approach is favorable in the future of the drug design industry.

Towards novel therapeutics for HIV through fragment-based screening and drug design.

Fragment-based drug discovery has been applied with varying levels of success to a number of proteins involved in the HIV (Human Immunodeficiency Virus) life cycle. Fragment-based approaches have led to the discovery of novel binding sites within protease, reverse transcriptase, integrase, and gp41. Novel compounds that bind to known pockets within CCR5 have also been identified via fragment screening, and a fragment-based approach to target the TAR-Tat interaction was explored. In the context of HIV-1 reverse transcriptase (RT), fragment-based approaches have yielded fragment hits with mid-µM activity in an in vitro activity assay, as well as fragment hits that are active against drug-resistant variants of RT. Fragment-based drug discovery is a powerful method to elucidate novel binding sites within proteins, and the method has had significant success in the context of HIV proteins.

Medicinal attributes of Solanum xanthocarpum fruit consumed by several tribal communities as food: an in vitro antioxidant, anticancer and anti HIV perspective.

BACKGROUND: Solanum xanthocarpum (Solanaceae) has been used for treatment of many infectious and degenerative diseases in traditional medicine. Present study reports the medicinal efficacy of S. xanthocarpum fruit as antioxidant, anticancer and anti HIV agents. METHODS: Extracts were prepared using Soxhlet apparatus and partially characterized by thin layer chromatography (TLC). Total flavonoid content was determined spectrophotometrically. Reducing power, DPPH radical scavenging activity and lipid peroxidation inhibition assays were used for measurement of antioxidant potential. Cytotoxic (SRB assay) and anti-HIV RT inhibition (RT assay kit, Roche) activities were determined using ELISA. RESULTS: TLC revealed the diversity of phytoconstituents in various sequential extracts of S. xanthocarpum fruit. Total flavonoid contents in extracts ranged between 10.22-162.49 µg quercetin equivalent/mg. Spectroscopic scanning of water soluble phenolics showed maximum absorbance at 250 and 280 nm. Polar extracts displayed potent radical scavenging activity (>80%). Several sub-fractions (spots) of extracts separated on TLC plates also exhibited powerful radical scavenging activity. Considerable reducing power was observed in extracts. Hexane fraction provided 55% lipoprotection in rat kidney homogenate. Non-polar extracts exhibited appreciable cytotoxic activity (70-91%) against leukemia (THP-1) and lung cancer (HOP-62) cell lines. Lower inhibitory activity was observed in extracts against HIV Reverse Transcriptase enzyme. CONCLUSION: The study demonstrated considerable antioxidant and anticancer activities in S. xanthocarpum fruit.

[Research development of HIV drug resistance].

Highly active antiretroviral combination therapy significantly reduced the mortality, but in the high-speed copying, high genetic variation and drug selection pressure under the effect of the increasingly serious problem of drug resistance greatly weakened the role of HAART inhibit viral replication and reduce antiviral treatment. This paper reports the latest trends in HIV drug-resistance in order to develop anti-HIV drugs in clinical programs, research and development of new guidance anti-HIV-1 strategy to bring guidance.

A drug discovery platform: a simplified immunoassay for analyzing HIV protease activity.

Although numerous methods for the determination of HIV protease (HIV-PR) activity have been described, new high-throughput assays are required for clinical and pharmaceutical applications due to the occurrence of resistant strains. In this study, a simple enzymatic immunoassay to identify HIV-PR activity was developed based on a Ni(2+)-immobilized His(6)-Matrix-Capsid substrate (H(6)MA-CA) is cleaved by HIV protease-His(6) (HIV-PRH(6)) which removes the CA domain and exposes the free C terminus of MA. Following this cleavage, two monoclonal antibodies specific for either the free C-terminal MA or CA epitope are used to quantify the proteolytic activity using a standard ELISA-based system. Specificity for detection of the HIV-PRH(6) activity was confirmed with addition of protease inhibitor (PI), lopinavir. In addition, the assay was able to detect an HIV-PR variant activity indicating that this assay is capable of assessing viral mutation affect HIV-PR activity. The efficacy of commercially available PIs and their 50% inhibitory concentration (IC(50)) were determined. This assay provides a high-throughput method for both validating the efficiency of new drugs in vitro and facilitating the discovery of new PIs. In addition, it could serve as a method for examining the influence of various mutations in HIV-PRs isolated from drug-resistant strains.

A novel non-radioactive assay for HIV-RT (RdDp) based on pyrosequencing for high-throughput drug screening.

Current in vitro assays for the activity of HIV-RT (reverse transcriptase) require radio-labeled or chemically modified nucleotides to detect reaction products. However, these assays are inherently end-point measurements and labor intensive. Here we describe a novel non-radioactive assay based on the principle of pyrosequencing coupled-enzyme system to monitor the activity of HIV-RT by indirectly measuring the release of pyrophosphate (PP(i)), which is generated during nascent strand synthesis. The results show that our assay could monitor HIV-RT activity with high sensitivity and is suitable for rapid high-throughput drug screening targeting anti-HIV therapies due to its high speed and convenience. Moreover, this assay can be used to measure primase activity in an easy and sensitive manner, which suggests that this novel approach could be wildly used to analyze the activity of PP(i)-generated and ATP-free enzyme reactions.

Preclinical evaluation of 1H-benzylindole derivatives as novel human immunodeficiency virus integrase strand transfer inhibitors.

We have identified 1H-benzylindole analogues as a novel series of human immunodeficiency virus (HIV) integrase inhibitors with antiretroviral activities against different strains of HIV type 1 (HIV-1), HIV-2, and simian immunodeficiency virus strain MAC(251) [SIV(MAC(251))]. Molecular modeling and structure-activity relationship-based optimization resulted in the identification of CHI/1043 as the most potent congener. CHI/1043 inhibited the replication of HIV-1(III(B)) in MT-4 cells at a 50% effective concentration (EC(50)) of 0.60 microM, 70-fold below its cytotoxic concentration. Equal activities against HIV-1(NL4.3), HIV-2(ROD), HIV-2(EHO), and SIV(MAC(251)) were observed. CHI/1043 was equally active against virus strains resistant against inhibitors of reverse transcriptase or protease. Replication of both X4 and R5 strains in peripheral blood mononuclear cells was sensitive to the inhibitory effect of CHI/1043 (EC(50), 0.30 to 0.38 microM). CHI/1043 inhibited integrase strand transfer activity in oligonucleotide-based enzymatic assays at low micromolar concentrations. Time-of-addition experiments confirmed CHI/1043 to interfere with the viral replication cycle at the time of retroviral integration. Quantitative Alu PCR corroborated that the anti-HIV activity is based upon the inhibition of proviral DNA integration. An HIV-1 strain selected for 70 passages in the presence of CHI/1043 was evaluated genotypically and phenotypically. The mutations T66I and Q146K were present in integrase. Cross-resistance to other integrase strand transfer inhibitors, such as L-708,906, the naphthyridine analogue L-870,810, and the clinical drugs GS/9137 and MK-0518, was observed. In adsorption, distribution, metabolism, excretion, and toxicity studies, antiviral activity was strongly reduced by protein binding, and metabolization in human liver microsomes was observed. Transport studies with Caco cells suggest a low oral bioavailability.

[Efficacy of raltegravir: from healthy volunteers to phase III trials]. / Eficacia del raltegravir: desde los voluntarios sanos a la fase III.

Raltegravir is the first in a new class of antiretroviral treatments called integrase inhibitors, which work by preventing HIV from inserting its genetic material into the DNA of the human chromosome. Phase I-III studies have shown this drug to have potent antiretroviral action, which is more rapid than that of protease inhibitors. The dose selected in efficacy studies is 400 mg every 12 h. However, due to the favorable pharmacokinetic profile of raltegravir, the possibility of administration of 600 to 800 mg once daily is under study. Given that this drug is not metabolized by the cytochrome P450 system, the potential for pharmacological interactions is low. Moreover, because humans lack a cellular homologue for HIV integrase, raltegravir has a low potential for toxicity. Raltegravir has an intermediate genetic barrier and consequently there may be cross-resistance across the integrase inhibitor class. For all these reasons, raltegravir is an attractive option in treatment-naive and pretreated patients and in those receiving simplification regimens.

Anti-HIV activities of organic and aqueous extracts of Sutherlandia frutescens and Lobostemon trigonus.

A screening process was applied to extracts made from Sutherlandia frutescens (L.) R. Br (Fabaceae) and Lobostemon trigonus (Boraginaceae) as identified by the Botany Department, University of Port Elizabeth to detect if any of the extracts inhibited the human immunodeficiency virus (HIV). For purposes of dereplication, sulphated polysaccharides were removed and bovine serum albumin (BSA) was included in the assays to adsorb non-specific tannins potentially present. In the reverse transcriptase (RT) assay, an aqueous extract of the Lobostemon leaves inhibited HIV-1 RT with an IC50 value of 49 microg/ml, while in the protease assay no inhibition was seen. In the alpha- and beta-glucosidase assays, no significant inhibition was seen with the inclusion of BSA, indicating tannin-based inhibitory effects on these two enzymes. The beta-glucuronidase inhibitory activity, however, was retained in the presence of BSA. The study shows that Sutherlandia extracts contain inhibitory compounds active against HIV target enzymes, while aqueous Lobostemon leaf extracts contain a potent HIV-1 RT inhibitor, thus showing a potential mechanistic action of these plants in aiding HIV-positive patients.

The challenge of antiretroviral-drug-resistant HIV: is there any possible clinical advantage?

Resistance to antiretroviral drugs is associated with reduced treatment options and, therefore, increased risk of disease progression or death. Despite the main goal of antiretroviral therapy should be achievement of complete suppression of HIV replication, the accumulation of resistance mutations in patients with multiple treatment failure makes this objective often difficult, or even impossible to obtain. Thus, clinicians should be aware about the complex relationship between drug pressure and viral replication capacity and about some potential advantages related to antiretroviral drug resistance. The two main biological mechanisms that can be at the origin of these clinical benefits are: reduction of viral fitness and viral hypersusceptibility. The term "fitness" indicates the ability of HIV to maintain high rate of replication capacity in presence of antiretroviral drugs. Consequently, replication capacity, high in presence of wild type virus, tends to decrease when HIV must adapt its enzymes to work in presence of drugs. A reduction of viral fitness is observed in patients harbouring mutations conferring resistance to all the three classes of antiretroviral drugs currently in use. Particularly, the effects on reduction of replication capacity related to M184V mutation in reverse transcriptase are analyzed. Viral isolates with reduced susceptibility or resistance to some antiretroviral drugs may exhibit significant increased susceptibility to other drugs acting on the same enzyme. This phenomenon is known as hypersusceptibility and can be demonstrated in vitro by phenotypic assays. Phenotypic hypersusceptibility has been demonstrated for all three drug classes. Particularly, NNRTI hypersusceptibility, associated with NRTI mutations, is analyzed and discussed.

Bioactive kaurane diterpenoids from Annona glabra.

Phytochemical analysis of the fruits of Annona glabra yielded two new kaurane diterpenoids, annoglabasin A (methyl-16 beta-acetoxy-19-al-ent-kauran-17-oate)(1) and annoglabasin B (16 alpha-hydro-19-acetoxy-ent-kauran-17-oic acid)(2), along with 11 known kaurane derivatives (3-13). The structures of the new compounds were established by spectral and chemical evidence. Among these, methyl-16 alpha-hydro-19-al-ent-kauran-17-oate (11) exhibited mild activity against HIV replication in H9 lymphocyte cells, and 16 alpha-17-dihydroxy-ent-kauran-19-oic acid (4) showed significant inhibition of HIV-reverse transcriptase.

Bioactive acylphloroglucinol derivatives from Eucalyptus species.

The acyphloroglucinol derivatives produced by Eucalyptus species are reviewed. Aspects of their chemistry, stereochemistry, biological activity and biogenesis are discussed.

A screen in Escherichia coli for nucleoside analogs that target human immunodeficiency virus (HIV) reverse transcriptase: coexpression of HIV reverse transcriptase and herpes simplex virus thymidine kinase.

Human immunodeficiency virus (HIV) reverse transcriptase substitutes for temperature-sensitive DNA polymerase I (Pol Its) in Escherichia coli, providing a screen for anti-HIV reverse transcriptase nucleoside analogs in bacteria. Since phosphorylation of nucleosides in E. coli is limited to thymidine and its derivatives, we coexpressed herpes simplex virus thymidine kinase, an enzyme that phosphorylates a wide variety of nucleoside analogs, together with HIV reverse transcriptase. Coexpression of herpes simplex virus thymidine kinase and HIV reverse transcriptase rendered Pol Its cells sensitive to dideoxycytidine. Studies with different nucleoside analogs indicate that this bacterial screening system is able to select and identify nucleoside analogs that specifically target HIV reverse transcriptase.

Human immunodeficiency virus reverse transcriptase substitutes for DNA polymerase I in Escherichia coli.

We present evidence that human immunodeficiency virus (HIV) reverse transcriptase (RT) can substitute for DNA polymerase I in bacteria. Expression of HIV RT enables an Escherichia coli mutant, polA12 recA718, containing a temperature-sensitive mutation in DNA polymerase I, to grow at a nonpermissive temperature. The plasmid pBR322 contains a DNA polymerase I-dependent origin of replication. Expression of HIV RT enables the same E. coli mutant to maintain this plasmid at a nonpermissive temperature. Furthermore, expression of HIV RT in this mutant renders it sensitive to 3'-azido-3'-deoxythymidine, a commonly used anti-AIDS drug that targets HIV RT. These combined findings on the genetic complementation of DNA polymerase I by HIV RT provide a bacterial assay to screen for drugs directed against HIV RT. Genetic complementation provides a method for positive selection of large numbers of functional HIV RT mutants for studies on structure-function relationships.

Thalifaberidine, a cytotoxic aporphine-benzylisoquinoline alkaloid from Thalictrum faberi.

From Thalictrum faberi, thalifaberidine [1], a new aporphine-benzylisoquinoline alkaloid, together with four known alkaloids, thalifaramine [2], thalifaricine [3], thalifarazine [4], and thalifaronine [5], were isolated. Thalifaberidine [1] was identified as 6',8-desmethylthalifaberine, and its 1H- and 13C-nmr data were completely assigned through the use of one- and two-dimensional nmr techniques. Thalifaberidine [1], thalifaberine [6], and thalifasine [7] showed cytotoxic activity against several human cancer cell lines, as well as antimalarial activity.

Screening anti-HIV Chinese materia medica with HIV and equine infectious anemic virus reverse transcriptase.

The inhibitory activities of human immunodeficiency virus reverse transcriptase (HIV-RT) inhibitors PFA and Suramin against HIV-RT and equine infectious anemic virus reverse transcriptase (EIAV-RT) were studied in this paper. The 50% inhibitory concentration (IC50) of HIV-RT and EIAV-RT treated by PFA and Suramin were 0.2 mumol, 9.8 mumol and 17 mumol, 19.9 mumol, respectively. More than thirty Chinese medicines, including recipes, herbs, extracts of traditional materia medica and isolated compounds were tested using HIV-RT and EIAV-RT as target enzymes. It was found that 5 crude extracts such as Rhizoma Polygoni Cuspidati, and Radix Notoginseng, 7 isolated compounds like Flavone of Ramulus Visci, Flavone of Ajuga Decumbens Thumb, and Aristolochic acid, as well as the extract of the complex prescription Xiao Chai Hu Tang have shown various inhibitory actions on these two enzymes. The activities of the two enzymes were comparable, but HIV-RT was more sensitive, suggesting that HIV-RT can be used as index for the screening of anti-aids drugs.

The mechanism of human immunodeficiency virus reverse transcriptase-catalyzed strand transfer from internal regions of heteropolymeric RNA templates.

The mechanism of human immunodeficiency virus reverse transcriptase-catalyzed strand transfer synthesis (i.e. switching of the primer to a new template) from internal regions of natural sequence RNA was investigated. The system consisted of a 142-nucleotide RNA template (donor) primed with a specific 20-nucleotide DNA oligonucleotide used to initiate synthesis. DNA oligonucleotides with homology to internal regions of the donor were used as acceptor templates. In reactions performed in the absence of acceptor template, a prominent DNA synthesis product 75 nucleotides in length resulting from pausing DNA synthesis within the homology zone was observed. Prominent donor RNA degradation products of 47 or 54 nucleotides were also observed, in reactions with 80 or 150 mM KCl, respectively. The lengths indicated a potential 13- or 20-nucleotide long, respectively, complementary region between the DNA and RNAs. The 54-, but not the 47-, nucleotide RNA was susceptible to Escherichia coli RNase H, indicating that the DNA was annealed only to the 54-mer. When acceptor was added, a portion of the 75-nucleotide DNA was chased into transfer product at both salt concentrations, and a portion of the 54-mer RNA became resistant to E. coli RNase H. Evidently, this donor RNA was annealed to the 75-nucleotide long DNA but could be actively displaced by the acceptor. Overall, these observations support two mechanisms for transfer. In one, the pause site-specific DNA dissociates from the donor template before transferring. In the other, the acceptor actively displaces the DNA from the donor.

Parameters that influence the binding of human immunodeficiency virus reverse transcriptase to nucleic acid structures.

We have investigated the binding of human immunodeficiency virus reverse transcriptase (HIV-RT) to various hybrid RNA-DNA or DNA-DNA nucleic acid structures. Binding was measured by preequilibrating the RT with the nucleic acid substrate in the presence or absence of Mg2+ and then initiating synthesis or RNase H degradation reactions in the presence of excess "trap" polymer [poly(rA)-oligo(dT)]. The trap polymer sequestered RT molecules as soon as they dissociated from the substrate, such that the amount of synthesis or degradation on the substrate was proportional to the amount of bound RT. On hybrid substrates that had the 3' terminus of a complementary DNA oligomer recessed on a longer DNA or RNA template, binding to the RNA-DNA hybrid was more stable. Both the dissociation rate constant (k(off)) and equilibrium constant (Kd) values were larger for the DNA-DNA substrates by 5-10-fold. The difference was clearly in dissociation, since the association rate constant (k(on)) for both types of substrates was similar. On hybrid structures that had the 3' termini of a complementary RNA or DNA oligomer recessed on a longer DNA template, k(off) values are approximately the same on either structure. Although binding of the RT to DNA-DNA hybrid structures did not require Mg2+, its presence during the preequilibration period greatly stabilized binding. An approximate 20-60-fold decrease in the k(off), depending on the substrate structure, was observed with Mg2+. Measurements on one particular DNA-DNA hybrid indicated that the k(on) decreased by approximately 2 orders of magnitude with Mg2+. The relevance of these results to HIV replication is discussed.

Non-nucleoside inhibitors of HIV reverse transcriptase: screening successes--clinical failures.

A little less than two years ago, the first report describing non-nucleoside inhibitors of HIV reverse transcriptase (RT) led to the high anticipation that a range of new drugs could soon be available for the treatment of AIDS. The intervening period has given rise to several such agents but recent clinical trial data has indicated this optimism to be premature. This short review seeks to trace the brief history of the drug discovery process and to assess whether there are lessons to be learnt from the episode.