Gold compounds with anti-HIV and immunomodulatory activity

The human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS) that subsequently develops remain major health concerns even after three decades since the first cases were reported. Successful therapeutic measures to address HIV/AIDS consist mostly of combinations of drugs targeting viral enzymes including reverse transcriptase (RT), protease (PR) and integrase (IN) as well as entry steps of the viral life cycle. The remarkable benefits (e.g. improved quality of life) derived from the use of these agents are unfortunately limited by toxicity to the host and the development of drug resistant viral strains. Drug resistance limits the repertoire of drug combinations available. Unfortunately, because latent forms of the virus exists, therapy has to be life-long and with new infections occurring every day, resistant strains tend to spread. To circumvent these problems, new drugs that inhibit resistant strains or work against new viral targets have to be developed. The history of gold compounds as potential inhibitors of HIV prompted this study in which twenty seven compounds consisting of gold(I), gold(III) and precursors from five classes were tested for drug-likeness, anti-HIV and immunomodulatory effects using wet lab and in silico methodologies. Cytotoxicity determination was done using viability dyes and flow cytometry. Cell proliferation profiles were monitored using the carboxyflourescein succinimidyl ester dye dilution technology and a real time cell analyser for confirming viability dye findings. The compounds’ effects on viral enzymes was determined using direct enzyme assays and in silico molecular modelling techniques. H and P nuclear magnetic resonance spectroscopy studies for determining stability revealed that the backbone chemical shifts of the compounds were relatively unchanged after one week (-20 and 37 ºC) when dissolved in dimethylsulfoxide. Eight of the gold compounds had drug-like properties comparable to clinically available drugs when in silico predictions were performed. The 50% cytotoxic dose of the compounds in human cells was between 1 and 20 μM (clinically relevant concentrations for gold compounds). Three gold(I) compounds inhibited viral infectivity at non-toxic concentrations and two gold(III) compounds did so at cytostatic (anti-proliferative mechanism that is also antiviral) concentrations. In the immunomodulatory assay, cytokine levels were altered by five compounds with one gold(I) and a gold(III) compound significantly reducing the frequency of CD4+ cells (an anti-viral function) from HIV+ donors (p= 0.005 and 0.027 respectively) when multi-parametric flow cytometry was performed. Inhibition of RT activity was predicted in in silico studies to be through interactions with the ribonuclease (RNase) H site although with poor stereochemical orientation while favourable binding predictions with the IN cofactor binding site were observed for some gold(III) complexes. Compounds predicted to interact with the RNase H site of RT and the IN cofactor site require structural modification to improve drug-likeness and binding affinity. The drug-like compound(s) which inhibited viral infectivity and lowered CD4+ cell frequency have potential for incorporation into virostatic cocktails (combination of cytostatic and directly anti-viral agent). Cytostatic agents are known to be less prone to drug resistance and because they lower CD4+ cell frequency, such compounds can potentially limit HIV immune activation.Thesis (PhD)--University of Pretoria, 2011.Biochemistryunrestricte

Antioxidant and anti-inflammatory activity of Ocimum labiatum extract and isolated labdane diterpenoid

BACKGROUND : Plants from the genus Ocimum are used as folk medicine for treating various diseases including inflammatory and immune-related diseases. Numerous reports have suggested plant extracts and their constituents as possible anti-inflammatory agents. Here, in vitro evidence of Ocimum labiatum’s immune-enhancing and antioxidant properties is presented for the first time. METHODS : The anti-inflammatory effect of O. labiatum ethanolic extract and an isolated diterpenoid was determined using a cytometric bead array (CBA) technique. The effect on phytohemagglutinin (PHA)-induced nitric oxide (NO) production in peripheral blood mononuclear cells (PBMCs) was also assessed. A battery of antioxidant assays were used for detecting antioxidant activity while the anti-inflammatory mechanism was evaluated using an ELISA-based activator protein (AP-1) (c-Jun) assay. Cytotoxicity was determined on TZM-bl and PBMCs using a tetrazolium dye and confirmed by a novel label-free real-time assay. RESULTS : A 25 μg/mL non-cytotoxic concentration of O. labiatum extract significantly (p < 0.05) inhibited the production of pro-inflammatory cytokines; IL-2, IL-4, IL-6 and IL-17A. Except for the dual acting pro- or anti-inflammatory cytokine, IL-6, which was upregulated, a non-cytotoxic 50 μM concentration of the isolated labdane diterpenoid compound significantly (p < 0.05) decreased the production of all the pro-inflammatory cytokines. In the anti-inflammatory pathway studies, the compound also inhibited AP-1 significantly (p < 0.05) at 50 μM. The extract demonstrated strong, dose dependent antioxidant activity with IC50 values ranging from 13 ± 0.8 to 54.86 ± 1.28 μg/mL while the terpene had no antioxidant property. The extract and diterpenoid decreased the production of the inflammatory mediator NO, at non-cytotoxic concentrations. The CC50 of the extract in TZM-bl and PBMCs was 62.6 ± 0.6 and 30.1 ± 0.4 μg/mL while that of the compound was 112.6 ± 0.2 and 70 ± 0.4 μM respectively. The real time studies confirmed tetrazolium dye assessed viability and also detected a unique growth pattern for the plant materials compared to untreated cells. CONCLUSIONS : O. labiatum extract demonstrated promising anti-inflammatory and antioxidant properties while the terpenoid showed anti-inflammatory but no antioxidant activity. The anti-inflammatory mechanism of the terpene was a result of inhibition of AP-1. These data represents promising first steps towards the development of naturally derived anti-inflammation drugs.Southern African Biochemistry and Informatics for Natural Products (SABINA), the Technology Innovation Agency (TIA, South Africa), Margaret McNamara Memorial Fund (MMMF), the Namibian Ministry of Education and the University of Pretoria.http://www.journal-inflammation.comhb2016Biochemistr

New bis(thiosemicarbazonate) gold(III) complexes inhibit HIV replication at cytostatic concentrations : potential for incorporation into virostatic cocktails

Four bis(thiosemicarbazonate)gold(III) complexes (1–4) with a general formula [Au(L)]Cl {L=L1, glyoxal-bis (N4-methylthiosemicarbazone); L2, glyoxal-bis(N4-ethylthiosemicarbazone); L3, diacetyl-bis(N4- methylthiosemicarbazone); L4, diacetyl-bis(N4-ethylthiosemicarbazone)} were synthesised and screened for activity against the human immunodeficiency virus (HIV). Complexes 1–4 were characterised using 1H-NMR and IR spectroscopy; and their purity established by micronanalysis. Complex 3 inhibited viral infection of TZM-bl cells by 98% (IC50=6.8±0.6 μM) at a non toxic concentration of 12.5 μM while complex 4 inhibited infection of these cells by 72 and 98% (IC50=5.3±0.4 μM) at concentrations of 6.25 and 12.5 μMrespectively. Themechanism of inhibition of infection in TZM-bl cells is presumably as a result of the cytostatic or anti-proliferative activity that was observed for complex 4 in real time cell electronic sensing (RT-CES) and carboxyflourescein succinimidyl ester (CFSE) analysis. Treatment of T lymphocytes from HIV infected individuals with 4 decreased CD4+ T cell expression (p=0.0049) as demonstrated by multi-parametric flow cytometry without suppressing cytokine production. None of the ligands (L1–L4) demonstrated anti-viral activity, supporting the importance of metal (gold) complexation in these potential drugs. Complexes 3 and 4were shown to have ideal lipophilicity values thatwere similarwhenshake flask (0.97±0.5 and 2.42±0.6) and in silico prediction (0.8 and 1.5) methods were compared. The activity and drug-like properties of complexes 3 and 4 suggests that these novel metal-based compounds could be combined with virus inhibitory drugs to work as cytostatic agents in the emerging class of anti-HIV drugs known as virostatics.The South African National Research Foundationhttp://www.elsevier.com/locate/jinorgbionf201

Triterpenoids from ocimum labiatum activates latent HIV-1 expression in vitro : potential for use in adjuvant therapy

Abstract: Latent HIV reservoirs in infected individuals prevent current treatment from eradicating infection. Treatment strategies against latency involve adjuvants for viral reactivation which exposes viral particles to antiretroviral drugs. In this study, the effect of novel triterpenoids isolated from Ocimum labiatum on HIV-1 expression was measured through HIV-1 p24 antigen capture in the U1 latency model of HIV-1 infection and in peripheral blood mononuclear cells (PBMCs) of infected patients on combination antiretroviral therapy (cART). The mechanism of viral reactivation was determined through the compound’s effect on cytokine production, histone deacetylase (HDAC) inhibition, and protein kinase C (PKC) activation. Cytotoxicity of the triterpenoids was determined using a tetrazolium dye and flow cytometry. The isolated triterpene isomers, 3-hydroxy-4,6a,6b,11,12,14b-hexamethyl-1,2,3,4,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-octadecahydrop icene-4,8a-dicarboxylic acid (HHODC), significantly (p < 0.05) induced HIV-1 expression in a dose-dependent manner in U1 cells at non-cytotoxic concentrations. HHODC also induced viral expression in PBMCs of HIV-1 infected patients on cART. In addition, the compound up-regulated the production of interleukin (IL)-2, IL-6, tumour necrosis factor (TNF)-_, and interferon (IFN)- but had no effect on HDAC and PKC activity, suggesting cytokine upregulation as being involved in latency activation. The observed in vitro reactivation of HIV-1 introduces the adjuvant potential of HHODC for the first time here

Impedance technology reveals correlations between cytotoxicity and lipophilicity of mono and bimetallic phosphine complexes

Label free impedance technology enables the monitoring of cell response patterns post treatment with drugs or other chemicals. Using this technology, a correlation between the lipophilicity of metal complexes and the degree of cytotoxicity was observed. Au(L1)Cl (1), AuPd(L1)(SC4H8)Cl3 (1a) and Au(L2)Cl (2) [L1 = diphenylphosphino-2-pyridine; L2 = 2-(2-(diphenylphosphino)ethyl)-pyridine] were synthesised, in silico drug-likeness and structure– activity relationship monitored using impedance technology. Dose dependent changes in cytotoxicity were observed for the metal complexes resulting in IC50s of 12.5 ± 2.5, 18.3 ± 8.3 and 16.9 ± 0.5 lM for 1, 1a and 2 respectively in an endpoint assay. When a real time impedance assay was used, dose-dependent responses depicting patterns that suggested slower uptake (at a toxic 20 lM) and faster recovery of the cells (at the less toxic 10 lM) of the bimetallic complex (1a) compared to the monometallic complexes (1 and 2) was observed. These data agreed with the ADMET findings of lower aqueous solubility of 1a and non-ideal lipophilicity (AlogP98 of 6.55) over more water soluble 1 and 2 with ideal lipophilicity (4.91 and 5.03 respectively) values. The additional coordination of a Pd atom to the nitrogen atom of a pyridine ring, the sulfur atom of a tetrahydrothiophene moiety and two chlorine atoms in 1a could be contributing to the observed differences when compared to the monometallic complexes. This report presents impedance technology as a means of correlating drug-likeness of lipophilic phosphine complexes containing similar backbone structures and could prove valuable in filtering drug-like compounds in a drug discovery project.Technology Innovation Agency (TIA), the University of Pretoria.Organization for Women in Science for the Developing World (OWSD) formerly Third World Organization for Women in Science (TWOWS) and University of Johannesburg.http://link.springer.com/journal/105342016-08-31hb201

In vitro Antimycobacterial, Apoptosis-Inducing Potential, and Immunomodulatory Activity of Some Rubiaceae Species

Tuberculosis (TB), a disease caused by microorganisms of the Mycobacterium tuberculosis complex, infects almost one-third of the world’s population. The TB epidemic has been further exacerbated by the emergence of multi, extensively, and totally-drug-resistant (MDR, XDR, and TDRTB) strains. An effective immune response plays a crucial role in determining the establishment of TB infection. Therefore, the modulation of the immune system has been considered as a vital approach for the treatment or control of various immune-related diseases such as TB. In this study, the antimycobacterial, immunomodulatory, and apoptosis-inducing effects of six Rubiaceae species were evaluated. A twofold serial dilution method was used to determine the minimum inhibitory concentration values of the plant extracts. The effect of the extracts on the activity of 15-lipoxygenase was investigated. The levels of six different cytokines, IL-2, IL-4, IL-5, IL-10, IFN-γ, and TNF-α, were measured in LPS-activated U937 cell line while the apoptosis-inducing effect of the extracts was evaluated using an annexin V/PI assay using a flow cytometer. The results obtained revealed that all the six extracts tested had antimycobacterial activity against M. tuberculosis H37Rv, M. tuberculosis ATCC 25177, and Mycobacterium bovis ATCC 27299 strains, with MIC values ranging from 39 to 312 μg/mL. The extracts of Cremaspora triflora and Cephalanthus natalensis were the most active against M. tuberculosis (MIC = 39 μg/mL), followed by Pavetta lanceolata and Psychotria zombamontana against M. bovis (MIC = 78 μg/mL). The extracts of P. zombamontana and Psychotria capensis had remarkable IC50 values of 4.32 and 5.8 μg/mL, respectively, better than that of quercetin. The selected extracts promoted Th1/Th2 balances in an in vitro model at the tested concentration which may suggest the therapeutic value of the plant in diseases where inflammation is a significant factor such as TB. The addition of the crude extracts of C. triflora, P. capensis, and P. zombamontana at the tested concentrations to the cell culture medium induced apoptosis in a time- and dose-dependent manner. This interesting preliminary result generated from this study encourages further investigations of these extracts owing to the LOX-inhibitory effect, immunomodulatory, and apoptotic-inducing properties in addition to their antimycobacterial properties

In vitro reactivation of latent HIV-1 by cytostatic bis (thiosemicarbazonate) gold(III) complexes

BACKGROUND : A number of cytostatic agents have been investigated for the ability to reactivate latent viral reservoirs, which is a major prerequisite for the eradication of HIV-1 infection. Two cytostatic bis(thiosemicarbazonate) gold(III) complexes (designated 1 and 2) were tested for this potential in the U1 latency model of HIV-1 infection. METHODS : Cell viability in the presence or absence of 1 and 2 was determined using a tetrazolium dye and evidence of reactivation was assessed by p24 antigen capture following exposure to a latency stimulant, phorbol myristate acetate (PMA) and or test compounds. The latency reactivation mechanism was explored by determining the effect of the complexes on protein kinase C (PKC), histone deacetylases (HDAC) and proinflammatory cytokine production. RESULTS : The CC50 of the complexes in U1 cells were 0.53 ± 0.12 μM for 1 and 1.0 ± 0.4 μM for 2. In the absence of PMA and at non toxic concentrations of 0.2 and 0.5 μM, 1 and 2 significantly (p ≤ 0.02) reactivated virus in U1 cells by 2.7 and 2.3 fold respectively. In comparison, a 2.6 fold increase (p = 0.03) in viral reactivation was observed for hydroxyurea (HU), which was used as a cytostatic and latent HIV reactivation control. Viral reactivation was absent for the complexes during co-stimulation with PMA indicating the lack of an additive effect between the chemicals as well as an absence of inhibition of PMA induced HIV reactivation but for HU inhibition of the stimulant’s activity was observed (p = 0.01). Complex 1 and 2 activated PKC activity by up to 32% (p < 0.05) but no significant inhibition of HDAC was observed. Increases in TNF-α levels suggested that the reactivation of virus by the complexes may have been due to contributions from the latter and the activation of PKC. CONCLUSION : The ethyl group structural difference between 1 and 2 seems to influence bioactivity with lower active concentrations of 1, suggesting that further structural modifications should improve specificity. The cytostatic effect of 1 and 2 and now HIV reactivation from a U1 latency model is consistent with that of the cytostatic agent, HU. These findings suggest that the complexes have a potential dual (cytostatic and reactivation) role in viral “activation/elimination”.AuTEK Biomed (Mintek and Harmony Gold),Technology Innovation Agency (TIA) and the University of Pretoria.http://www.biomedcentral.com/bmcinfectdis/hb201

Chrysotherapy: evaluating gold compounds for anti-HIV activity

M.Sc.Background: The continuous emergence of drug resistant strains of HIV as a result of errors made by reverse transcriptase coupled with undesirable side effects of available drugs, latency problems, cost etc, warrants the continuous search for new drug candidates. Chrysotherapy which is the use of gold compounds for the treatment of various ailments has been practiced since 2500 BC. The use of gold compounds such as auranofin for the treatment of rheumatoid arthritis has lead to remission of this disease. Gold compounds such as auranofin not only prevented the progression of arthritis but also increased the CD4+ count of an HIV positive patient who was not on antiretrovirals. These compounds have been implicated in the treatment of cancers, autoimmune diseases and microorganism infections. Objectives: In this work, novel gold compounds were evaluated with the aim of identifying lead compound(s) that can eventually serve as anti-HIV agents. Materials and Methods: Eleven gold (I) phosphine complexes, four of their corresponding ligands (compound without gold atom), and a gold (III) complex were tested for the ability to inhibit reverse transcriptase (RT) and protease (PR) in direct enzyme assays. Uptake of the compounds by host cells was evaluated with inductively coupled plasma atomic emission spectrometry (ICP-AES). Potential toxicity of the gold compounds was screened for by viability dyes and flow cytometry assays. To determine inhibition of whole virus by other mechanisms in addition to RT or PR, p24 production by infected cells was evaluated. Prior to all these analysis, stability of compounds in solution was determined by 31P nuclear magnetic resonance (NMR) and UV-visible spectroscopy. Results: The compounds were shown to be stable in solution over a one week period and were taken up by both continuous cell lines and primary cells. Eight of the gold compounds significantly inhibited HIV-1 reverse transcriptase at concentrations of 25 and 250 μM while four compounds and the four ligands did not. In a fluorogenic assay against HIV-1 PR, four of the gold compounds demonstrated inhibitory activity. The gold compounds were toxic to cells lines but not to primary cells. One of the complexes (EK231) significantly reduced p24 (p=0.0042) production at a concentration of 25 μM. Conclusion: Data provided here suggests that the therapeutic benefits of these gold containing compounds as potential HIV-1 reverse transcriptase and protease inhibitors should be considered

Novel gold(I) phosphine compounds inhibit HIV-1 enzymes

The increasing incidence of human immunodeficiency virus (HIV) infection and the associated acquired immune deficiency syndrome (AIDS) mortality rates as well as the sometimes severe side effects of highly active anti retroviral therapy (HAART) warrants the continuous search for new, less toxic drug candidates. The anti-HIV activity (inhibition of reverse transcriptase-RT and protease-PR in direct enzyme assays) of eleven gold(I) phosphine compounds are reported here. Uptake of the compounds by peripheral blood mononuclear cells (PBMCs) was demonstrated by inductively coupled plasma atomic emission spectroscopy (ICP-AES) while the effect of the compounds on cell viability was assessed using flow cytometry with annexin V and propidium iodide (PI). Of the 11 gold compounds tested, 7 significantly (p o 0.05) inhibited RT activity at concentrations of 25 and 250 mM while 3 compounds significantly inhibited its activity at 6.25 mM. In the anti-protease assay, 4 of the compounds significantly inhibited the enzyme (p o 0.05) at 100 mM. All of the compounds were taken up by PBMCs (demonstrated by ICP-AES) and were non toxic to these cells at clinically tolerable concentrations. The potential of these novel gold(I) phosphine compounds as anti-HIV agents is therefore promising and worthy of further investigation