Regina Appiah-Opong

HIV-1 Proteases from Drug-Naive West African Patients Are Differentially Less Susceptible to Protease Inhibitors

BACKGROUND Now that highly active antiretroviral therapy (HAART) is being initiated on a large scale in West Africa, it remains controversial whether protease inhibitors (PIs), originally designed and tested against human immunodeficiency virus type 1 (HIV-1) subtype B, are equally effective against the non-B subtypes that are prevalent in West African countries. In this study, we investigated whether Ghanaian HIV-1 isolates, as representatives of West African isolates, are susceptible to PIs. METHODS We first generated an HIV-1 protease cassette vector proviral DNA carrying a luciferase gene, which allows patient-derived HIV-1 proteases to be inserted and to be subjected to both genotypic and phenotypic assays. HIV-1 protease genes derived from 39 treatment-naive Ghanaian patients were used in this experiment as representatives of West African strains. The cloned patient-derived HIV-1 protease genes were first sequenced and then genetically compared. Phenotypic analysis was performed with Ghanaian HIV-1 protease-chimeric viruses in the presence of 6 different PIs. Structural models of HIV-1 protease homodimers were constructed by the molecular modeling software. RESULTS Genetic analysis of cloned patient-derived HIV-1 protease genes indicated that most of the Ghanaian HIV-1 proteases are placed as subtype CRF02_AG strains, which are phylogenetically distant from subtype B strains, and that Ghanaian HIV-1 proteases do not harbor known major mutations influencing drug resistance but commonly carry 2-3 minor mutations. Phenotypic analysis performed with HIV-1 protease-recombinant viruses in the presence of 6 different PIs revealed that Ghanaian HIV-1 proteases are differentially less susceptible to the PIs. In support of this finding of differential susceptibility, structural analysis showed a significant distortion of nelfinavir, but not of amprenavir, in the Ghanaian protease pocket, suggesting nelfinavir might be less insertable into the Ghanaian protease than into the protease of subtype B. CONCLUSIONS These findings provide implications for the combination of PIs during the introduction of HAART into West Africa.

Toxicity of low levels of methylglyoxal: depletion of blood glutathione and adverse effect on glucose tolerance in mice.

Methylglyoxal, a metabolic by-product of glycolysis is also formed during food processing and has serious toxicological effects when in excess. In this study, ddY mice were exposed to low levels of methylglyoxal (1% v/v) via drinking water while in utero continuing until 2 months of age when investigations on blood GSH status and selected GSH dependent functions in the blood, and glucose tolerance were carried out. The results showed that GSH content was significantly decreased in the blood of methylglyoxal exposed mice when compared with controls (mean, 0.756 mmol/l vs. 1.090 mmol/l, p < 0.001). The data showed significant (p < 0.001) decreases in blood GSH-S-transferase activity and red blood cell (rbc) capacity to refract oxidative stress. Impaired glucose tolerance was 5.3 times more prevalent in the methylglyoxal exposed mice when compared with the controls. The results indicate that chronic intake of methylglyoxal, at levels that could be attained in food, is toxic by depletion of blood GSH and could have adverse effect on some GSH dependent functions in vivo.

Current perspectives in drug discovery against tuberculosis from natural products

Currently, one third of the worlds population is latently infected with Mycobacterium tuberculosis (MTB), while 8.9-9.9 million new and relapse cases of tuberculosis (TB) are reported yearly. The renewed research interests in natural products in the hope of discovering new and novel antitubercular leads have been driven partly by the increased incidence of multidrug-resistant strains of MTB and the adverse effects associated with the first- and second-line antitubercular drugs. Natural products have been, and will continue to be a rich source of new drugs against many diseases. The depth and breadth of therapeutic agents that have their origins in the secondary metabolites produced by living organisms cannot be compared with any other source of therapeutic agents. Discovery of new chemical molecules against active and latent TB from natural products requires an interdisciplinary approach, which is a major challenge facing scientists in this field. In order to overcome this challenge, cutting edge techniques in mycobacteriology and innovative natural product chemistry tools need to be developed and used in tandem. The present review provides a cross-linkage to the most recent literature in both fields and their potential to impact the early phase of drug discovery against TB if seamlessly combined.

Medicinal plants used to treat TB in Ghana.

AIMS The current study was designed to document medicinal plant species that are traditionally used to treat tuberculosis (TB) by Ghanaian communities. METHODS The medicinal plants used against TB or its signs and symptoms were selected using library and online published data searches. A guided questionnaire interview was also conducted with a botanist involved in plant collection at the Centre for Scientific Research into Plant Medicine (CSRPM) at Mampong. Data obtained were entered in Excel and summarized into means and frequencies using SPSS 12.0.1 for windows, and expressed as tables and bar graphs. RESULTS A total of 15 medicinal plant species distributed between 13 genera and 13 families were documented. The following medicinal plant species were found to be used against TB in Greater Accra and Eastern parts of Ghana: Azadirachta indica A. Juss. Stem bark (Meliaceae), Hygrophila auriculata Heine, whole plant (Acanthaceae), Chenopodium ambrosioides L. leaves (Amaranthaceae), Coix lacryma-jobi L. glumes (Poaceae), Solanum torvum Sw. unripe fruits (Solanaceae), Solanum torvum Sw. leaves (Solanaceae), Bidens pilosa L. whole plant (Asteraceae), Phyllanthus fraternus G.L. Webster leaves (Phyllanthaceae), Dissotis rotundifolia (Sm.) Triana, leaves (Melastomataceae), Cymbopogon giganteus Chiov. Leaves (Poaceae), Cyperus articulatus L. roots (Cyperaceae), Allium sativum L. bulb (Amaryllidaceae), Zingiber officinale Roscoe, rhizomes (Zingiberaceae), Allium cepa L. bulbs (Amaryllidaceae), Allium cepa L. leaves (Amaryllidaceae), Aloe vera var. barbadensis aqueous extract from leaves (Xanthorrhoeaceae), Aloe vera var. barbadensis organic extract from leaves (Xanthorrhoeaceae), Cocos nucifera Linn, water (Arecaceae) and Cocos nucifera Linn. Husk (Arecaceae). CONCLUSIONS The collected plant species could be a source of a new class of drugs against TB. Bioactivity guided fractionation is recommended to identify lead compounds for antimycobacterial activity. The current paper documents for the first time medicinal plant species used by Ghanaian communities to treat TB. These results are a basis for selection of plants for further pharmacological, toxicological and phytochemical studies in developing new plant-based antimycobacterial drugs.

Antimycobacterial and cytotoxic activity of selected medicinal plant extracts

Ethnopharmacological relevance Tuberculosis (TB) caused by Mycobacterium tuberculosis remains an ongoing threat to human health. Several medicinal plants are used traditionally to treat tuberculosis in Ghana. The current study was designed to investigate the antimycobacterial activity and cytotoxicity of crude extracts from five selected medicinal plants. Material and methods The microplate alamar blue assay (MABA) was used for antimycobacterial studies while the CellTiter 96® AQueous Assay, which is composed of solutions of a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine methosulfate) PMS, was used for cytotoxic studies. Correlation coefficients were used to compare the activity of crude extracts against nonpathogenic strains and the pathogenic Mycobacterium tuberculosis subsp.tuberculosis. Results Results of the MIC determinations indicated that all the crude extracts were active on all the three tested mycobacterial strains. Minimum inhibitory concentration values as low as 156.3 µg/mL against M. tuberculosis; Strain H37Ra (ATCC® 25,177™) were recorded from the leaves of Solanum torvum Sw. (Solanaceae). Cytotoxicity of the extracts varied, and the leaves from S. torvum had the most promising selectivity index. Activity against M. tuberculosis; Strain H37Ra was the best predictor of activity against pathogenic Mycobacterium tuberculosis subsp.tuberculosis (correlation coefficient=0.8). Conclusion The overall results of the present study provide supportive data on the use of some medicinal plants for tuberculosis treatment. The leaves of Solanum torvum are a potential source of anti-TB natural products and deserve further investigations to develop novel anti-TB agents against sensitive and drug resistant strains of M. tuberculosis.

Antiproliferative and Pro-Apoptotic Activity of Diarylheptanoids Isolated from the Bark of Alnus japonica in Human Leukemia Cell Lines.

Alnus japonica Steud is a tree that grows in damp areas of mountain valleys and has been used as a traditional medicine in Asia. We investigated the antiproliferative activity of hirsutanone (Hir) and oregonin (Ore) in human cancer cell lines and elucidated their mechanisms of action. A cytotoxicity study using a panel of 12 human cancer and 4 normal cell lines indicated that Hir exhibited potent antiproliferative activity against 4 leukemia (Jurkat, U937, THP-1, and HL-60) and 2 colon cancer cell lines (HCT-15 and Colo205). Although Ore suppressed the cell growth of Jurkat and THP-1, its inhibitory potency was weaker than that of Hir. The IC50 values of Hir and Ore in Jurkat were 11.37 μM and 22.16 μM, respectively. Further analysis on Jurkat cells demonstrated that Hir caused a sequence of events involved in apoptosis, including nuclear morphological changes and accumulation of cells with sub-G1 DNA content. Hir led to the cleavage of poly(ADP-ribose) polymerase (PARP) and activation of caspase-3, -8, and -9. In addition, Hir-induced PARP cleavage was completely abolished by specific inhibitors to these caspases. Our data suggested that Hir is a potent antiproliferative compound against the 4 leukemia cell lines and the 2 colon cancer cell lines tested. Furthermore, Hir exerts antiproliferative actions via caspase-dependent apoptotic cell death.

Procyanidin trimer C1 derived from Theobroma cacao reactivates latent human immunodeficiency virus type 1 provirus

Despite remarkable advances in combination antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) infection remains incurable due to the incomplete elimination of the replication-competent virus, which persists in latent reservoirs. Strategies for targeting HIV reservoirs for eradication that involves reactivation of latent proviruses while protecting uninfected cells by cART are urgently needed for cure of HIV infection. We screened medicinal plant extracts for compounds that could reactivate the latent HIV-1 provirus and identified a procyanidin trimer C1 derived from Theobroma cacao as a potent activator of the provirus in human T cells latently infected with HIV-1. This reactivation largely depends on the NF-κB and MAPK signaling pathways because either overexpression of a super-repressor form of IκBα or pretreatment with a MEK inhibitor U0126 diminished provirus reactivation by C1. A pan-PKC inhibitor significantly blocked the phorbol ester-induced but not the C1-induced HIV-1 reactivation. Although C1-induced viral gene expression persisted for as long as 48 h post-stimulation, NF-κB-dependent transcription peaked at 12 h post-stimulation and then quickly declined, suggesting Tat-mediated self-sustainment of HIV-1 expression. These results suggest that procyanidin C1 trimer is a potential compound for reactivation of latent HIV-1 reservoirs.

Antitrypanosomal Activities and Mechanisms of Action of Novel Tetracyclic Iridoids from Morinda lucida Benth.

ABSTRACT Trypanosoma brucei parasites are kinetoplastid protozoa that devastate the health and economic well-being of millions of people in Africa through the disease human African trypanosomiasis (HAT). New chemotherapy has been eagerly awaited due to severe side effects and the drug resistance issues plaguing current drugs. Recently, there has been an emphasis on the use of medicinal plants worldwide. Morinda lucida Benth. is a popular medicinal plant widely distributed in Africa, and several research groups have reported on the antiprotozoal activities of this plant. In this study, we identified three novel tetracyclic iridoids, molucidin, ML-2-3, and ML-F52, from the CHCl3 fraction of M. lucida leaves, which possess activity against the GUTat 3.1 strain of T. brucei brucei. The 50% inhibitory concentrations (IC50) of molucidin, ML-2-3, and ML-F52 were 1.27 μM, 3.75 μM, and 0.43 μM, respectively. ML-2-3 and ML-F52 suppressed the expression of paraflagellum rod protein subunit 2, PFR-2, and caused cell cycle alteration, which preceded apoptosis induction in the bloodstream form of Trypanosoma parasites. Novel tetracyclic iridoids may be promising lead compounds for the development of new chemotherapies for African trypanosomal infections in humans and animals.

Application of multi-target phytotherapeutic concept in malaria drug discovery: a systems biology approach in biomarker identification

There is an urgent need for new anti-malaria drugs with broad therapeutic potential and novel mode of action, for effective treatment and to overcome emerging drug resistance. Plant-derived anti-malarials remain a significant source of bioactive molecules in this regard.The multicomponent formulation forms the basis of phytotherapy. Mechanistic reasons for the poly-pharmacological effects of plants constitute increased bioavailability, interference with cellular transport processes, activation of pro-drugs/deactivation of active compounds to inactive metabolites and action of synergistic partners at different points of the same signaling cascade. These effects are known as the multi-target concept. However, due to the intrinsic complexity of natural products-based drug discovery, there is need to rethink the approaches toward understanding their therapeutic effect.This review discusses the multi-target phytotherapeutic concept and its application in biomarker identification using the modified reverse pharmacology - systems biology approach. Considerations include the generation of a product library, high throughput screening (HTS) techniques for efficacy and interaction assessment, High Performance Liquid Chromatography (HPLC)-based anti-malarial profiling and animal pharmacology. This approach is an integrated interdisciplinary implementation of tailored technology platforms coupled to miniaturized biological assays, to track and characterize the multi-target bioactive components of botanicals as well as identify potential biomarkers. While preserving biodiversity, this will serve as a primary step towards the development of standardized phytomedicines, as well as facilitate lead discovery for chemical prioritization and downstream clinical development.

Update on Medicinal Plants with Potency on Mycobacterium ulcerans.

Mycobacterium ulcerans disease has been a serious threat for people living in rural remote areas. Due to poverty or availability of traditional medicine these populations rely on herbal remedies. Currently, data on the anti-Mycobacterium ulcerans activity of plants, so far considered community-based knowledge, have been scientifically confirmed, concomitantly with some medicinal plants used to treat infectious diseases in general. Products derived from plants usually responsible for the biological properties may potentially control Mycobacterium ulcerans disease; numerous studies have aimed to describe the chemical composition of these plant antimicrobials. Thus, the present work provides the first compilation of medicinal plants that demonstrated inhibitory potential on Mycobacterium ulcerans. This work shows that the natural products represent potential alternatives to standard therapies for use as curative medicine for Mycobacterium ulcerans disease.