Synthesis and biological evaluation of truncated sarganaphthoquinoic acid derivatives as Hsp90 inhibitors

Hsp90 inhibition has been at the centre of attention in current research due to the possibility of “cracking down” on the entire process leading to the development of malignant cancers. Small underlying principles common in all types of cancers have been determined that govern the transformation of normal human cells into cancerous cells, with all relying on the ATPase activity of Hsp90 protein. Hsp90 protein is therefore an attractive drug target that if successfully inhibited can result in the remission of cancer tumours by one form of treatment. To date, no Hsp90 inhibitor has been sanctioned for cancer treatment as most are still in clinical development. Our research was therefore inspired by reports that indicated the potential of quinones / naphthoquinones to act as Hsp90 inhibitors. Preliminary results of a few selected marine natural product quinone systems i.e. sargaquinoic acid (SQA) (2.47) and lapachol (3.6) showed moderate cytotoxicity and weak interactions with the Hsp90 molecular chaperone, and evidence suggested C-terminal binding of these molecules. No correlation has been determined yet between cytotoxicity and Hsp90 inhibition, hence we aimed to develop natural product inspired molecules that exhibit both cytotoxic and Hsp90 inhibition properties. Due to limited amounts of the natural product that can be acquired from natural sources, synthetic analogues were opted for. Isolation of a few selected quinones was conducted to have material that could be used in biological assays. For structural modifications, a series of truncated naphthoquinone systems were prepared adopting the sarganaphthoquinoic acid (3.5) scaffold. The naphthoquinones were prepared via Diels-Alder reactions of relevant benzoquinones with myrcene, followed by aromatization reactions using MnO2. Various alkyl and aryl amines were then coupled to the C-2/3 position of the naphthoquinone using Michael’s addition reactions. Tricyclic naphthoquinones were also synthesized from reactions with hypotaurine and citral. Design of the analogues incorporated functionalities from known Hsp90 inhibitors e.g. geldanamycin (2.28) and its analogues. Preliminary results obtained showed that coupling of naphthoquinones with aryl-amines resulted in the most cytotoxic compounds (4.14-4.19) with IC50 values as low as 0.3 μM against Hs578T breast cancer carcinoma (triple negative). Most of the alkyl amines (4.20-4.25) had IC50 values greater than 50 μM except for 4.20 and 4.21 that showed IC50 values of 7.6 μM and 2.6 μM respectively. Tricyclic naphthoquinones (4.28-4.29) showed moderate cytotoxic activity of approximately 10 μM. Hsp90 inhibition was assessed by client protein degradation assays, of which SQA (2.47), showed the best Hsp90 inhibition properties, followed by compound 4.20. The most cytotoxic arylamino-naphthoquinone (4.16) and tricyclic naphthoquinones (4.28-4.29) showed only moderate inhibition. None of the compounds led to Hsp70 induction, suggesting possible binding to the C-terminus of Hsp90. Interactions at the binding site were assessed by molecular docking studies and saturation transfer difference (STD) NMR. Docking studies were conducted on the N-terminus of Hsp90 and better binding was observed for arylamino naphthoquinones (4.14-4.19) than for other series of compounds. Unfortunately, the co-crystal structure for the C-terminus of Hsp90 is unavailable, hence docking study comparisons on both domains could not be conducted. However, STD NMR offered a platform to assess binding interactions between the naphthoquinones and the N- or C-terminal domains of Hsp90. However no interactions were observed at both the N- and C- termini of Hsp90 due to either weak binding of ligands to the protein or poor water solubility of the ligands. From these preliminary results, naphthoquinones bind to Hsp90 protein but conclusive remarks to which terminal domain they bind to could not be made. The best candidate from amongst the series of naphthoquinones prepared that showed moderate cytotoxicity and promising Hsp90 inhibition was compound 4.20. We therefore succeeded in developing a new series of naphthoquinones that possess moderate cytotoxicity and show Hsp90 inhibition

Procalcitonin serum levels in tertian malaria

BACKGROUND: Procalcitonin (PCT) is closely correlated with parasite burden and clinical outcome in falciparum malaria. The role of PCT in tertian malaria has not previously been investigated. PATIENTS AND METHODS: PCT serum levels in 37 patients with tertian malaria were analysed. Clinical and laboratory parameters were assessed and statistically correlated both to the initial PCT levels and during the course of the disease. RESULTS: PCT levels rose for one day after commencing treatment and declined thereafter. However, there was no significant correlation with parasite burden, clinical parameters, laboratory values, or the presence of semi-immunity. Before treatment, the majority of patients showed normal or slightly elevated PCT levels (< 2.5 ng/ml), but PCT was markedly elevated (4.8 – 47 ng/ml) in one third of the population. The two groups did not differ by any other of the assessed parameters. Thus, while the post-treatment course of PCT resembles falciparum malaria, the lack of correlation between disease severity and even high PCT levels in a large proportion of patients is intriguing. CONCLUSIONS: There is a fundamental difference in the relationship of PCT with tertian malaria not seen in other infectious diseases in which elevated PCT levels have been observed. This suggests distinct pathophysiological pathways in malaria

The synthesis and breast cancer inhibitory activity of cinnamic acid analogues based on the halogenated monoterpene pharmacophore

Breast cancer is one of the leading causes of death, with mortality rate estimates of 465 000 deaths per annum. It is estimated that 1.3 million women are diagnosed with the disease each year especially in the developing countries. Current chemotherapy relies on the use of high doses of non-specific toxic agents that possess adverse side effects and compromise patient’s compliance and adherence to treatment. Paclitaxel, one of the common drugs used in breast cancer chemotherapy results in sensory and motor neuropathy, whilst hormonal therapy e.g. Herceptin causes severe cardiovascular, gastrointestinal and cutaneous side effects. There has been a demand in developing newer cancer agents that demonstrate selective cytoxicity with minimal effect on normal body tissue. Numerous studies have shown that marine organisms produce a wide range of halogenated compounds that possess cytotoxic properties, and hence can be a source of new drug hits or leads for cancer therapy. Halomon, a polyhalogenated monoterpene from Portieria hornemannii, displayed interesting activity against brain, renal and lung cancer tumours with selective/differential cytotoxicity. This inspired us to focus our project on halogenated monoterpenes isolated from the same Rhodophyta class as P. hornemannii but with particular attention to Plocamium species. Several metabolites have been isolated from P. cornutum, P. corallorhiza and P. suhrii that possess interesting cytotoxicities against a breast cancer cell line (MCF7) and an oesophageal cancer line (WHCO1). The aim of the project was therefore centred at isolating target compounds for preliminary structure-activity studies against a breast cancer cell line, and use this information to synthesize a series of analogues that are more stable than the natural products and yet as active using a fragment-based type approach to map out pharmacophoric elements. Five metabolites were isolated from P. cornutum and five from P. corallorhiza. Cell-based assays were conducted using an MTT assay kit against MCF7 and MDA-MB-231 breast cancer cell lines and (1E,3E,5S,6R)-1,5,6-trichloro-2-(dichloromethyl)-6-methylocta-1,3,7-triene, isolated from P. cornutum was the most active with IC50 values of 3.0 μM and 6.15 μM respectively. Introduction of a terminal aromatic ring to enhance stability, together with varying substituents (H, CH3, CF3, Br, CN, CHO, CHCl2) on position 7 of the molecule, gave rise to a series of cinnamate ester derivatives inspired by (1E,3E,5S,6R)-1,5,6-trichloro-2-(dichloromethyl)-6-methylocta-1,3,7-triene. The analogues were synthesized from their benzaldehyde precursors via Aldol condensation, esterification and Wittig reactions. Their carboxylic acid counterparts were synthesized by hydrolysis of the parent esters in an attempt to promote water solubilities of the analogues. Biological activity assays were then conducted with the cinnamate analogues against the MDA-MB-231 breast cancer cell line using an MTT assay kit. Ester derivatives with -CHO and -CHCl2 functionalities had IC50 values of 43.45 μM and 100.01 μM respectively whilst the other ester derivatives were inactive. It was concluded that either an aldehyde (-CHO) or gem-dichlorides (-CHCl2) is specifically required for cytotoxic activity to be observed. None of the carboxylic acids were active which could have been due to failure of the compounds to enter the breast cancer cells and reach the target site because of their polar nature. Compounds with -CHO and -CHCl2 functionalities were therefore selected for future SARs studies

Assessment of potential anti-cancer stem cell activity of marine algal compounds using an in vitro mammosphere assay

Background: The cancer stem cell (CSC) theory proposes that tumours arise from and are sustained by a subpopulation of cells with both cancer and stem cell properties. One of the key hallmarks of CSCs is the ability to grow anchorage-independently under serum-free culture conditions resulting in the formation of tumourspheres. It has further been reported that these cells are resistant to traditional chemotherapeutic agents. Methods: In this study, the tumoursphere assay was validated in MCF-7 cells and used to screen novel marine algal compounds for potential anti-cancer stem cell (CSC) activity in vitro. Results: MCF-7 breast cancer cells were observed to generate tumourspheres or mammospheres after 3-5 days growth in anchorage-independent conditions and an apparent enrichment in potential CSCs was observed by an increase in the proportion of CD44high/CD24low marker-bearing cells and Oct4 expression compared to those in the bulk population grown in regular adherent conditions. Using this assay, a set of algal metabolites was screened for the ability to inhibit mammosphere development as a measure of potential anti-CSC activity. We report that the polyhalogenated monoterpene stereoisomers RU017 and RU018 isolated from the red alga Plocamium cornutum, both of which displayed no cytotoxicity against either adherent MCF-7 breast cancer or MCF-12A non-transformed breast epithelial cells, were able to prevent MCF-7 mammosphere formation in vitro. On the other hand, neither the brown algal carotenoid fucoxanthin nor the chemotherapeutic paclitaxel, both of which were toxic to adherent MCF-7 and MCF-12A cells, were able to inhibit mammosphere formation. In fact, pre-treatment with paclitaxel appeared to enhance mammosphere formation and development, a finding which is consistent with the reported resistance of CSCs to traditional chemotherapeutic agents. Conclusion: Due to the proposed clinical significance of CSC in terms of tumour initiation and metastasis, the identification of agents able to inhibit this subpopulation has clinical significance

Assessment of potential anti-cancer stem cell activity of marine algal compounds using an in vitro mammosphere assay:

The cancer stem cell (CSC) theory proposes that tumours arise from and are sustained by a subpopulation of cells with both cancer and stem cell properties. One of the key hallmarks of CSCs is the ability to grow anchorage-independently under serum-free culture conditions resulting in the formation of tumourspheres. It has further been reported that these cells are resistant to traditional chemotherapeutic agents

Plasmodium vivax: paroxysm-associated lipids mediate leukocyte aggregation

<p>Abstract</p> <p>Background</p> <p>Paroxysms are recurrent febrile episodes, characteristic of <it>Plasmodium vivax </it>infections, which coincide with the rupture of schizont-infected erythrocytes in the patients' circulation. The present study describes the formation of prominent aggregates of leukocytes <it>in vitro </it>in the presence of parasite and host factors released during paroxysms.</p> <p>Methods</p> <p>Whole blood cells from uninfected malaria-naïve donors were incubated with plasma taken during a paroxysm or normal human plasma as a control and cell smears were observed under the microscope for the presence of leukocyte aggregates. Plasma factors involved in mediating the leukocyte aggregation were identified using immune depletion and reconstitution experiments. Furthermore, biochemical characterization was carried out to determine the chemical nature of the active moieties in plasma present during paroxysms.</p> <p>Results</p> <p>Leukocyte aggregates were seen exclusively when cells were incubated in plasma collected during a paroxysm. Immune depletion and reconstitution experiments revealed that the host cytokines TNF-alpha, GM-CSF, IL-6 and IL-10 and two lipid fractions of paroxysm plasma comprise the necessary and sufficient mediators of this phenomenon. The two lipid components of the paroxysm plasmas speculated to be of putative parasite origin, were a phospholipid-containing fraction and another containing cholesterol and triglycerides. The phospholipid fraction was dependent upon the presence of cytokines for its activity unlike the cholesterol/triglyceride-containing fraction which in the absence of added cytokines was much more active than the phospholipids fraction. The biological activity of the paroxysm plasmas from non-immune patients who presented with acute <it>P. vivax </it>infections was neutralized by immune sera raised against schizont extracts of either <it>P. vivax </it>or <it>Plasmodium falciparum</it>. However, immune sera against <it>P. vivax </it>were more effective than that against <it>P. falciparum </it>indicating that the parasite activity involved may be antigenically at least partially parasite species-specific.</p> <p>Conclusion</p> <p>Leukocyte aggregation was identified as associated with paroxysms in <it>P. vivax </it>infections. This phenomenon is mediated by plasma factors including host-derived cytokines and lipids of putative parasite origin. The characteristics of the phospholipid fraction in paroxysm plasma are congruent with those of the parasite-derived, TNF-inducing GPI moieties described by others. The more active cholesterol/triglyceride(s), however, represent a novel malarial toxin, which is a new class of biologically active lipid associated with the paroxysm of <it>P. vivax </it>malaria.</p

Combinations of Host Biomarkers Predict Mortality among Ugandan Children with Severe Malaria: A Retrospective Case-Control Study

Background: Severe malaria is a leading cause of childhood mortality in Africa. However, at presentation, it is difficult to predict which children with severe malaria are at greatest risk of death. Dysregulated host inflammatory responses and endothelial activation play central roles in severe malaria pathogenesis. We hypothesized that biomarkers of these processes would accurately predict outcome among children with severe malaria. Methodology/Findings: Plasma was obtained from children with uncomplicated malaria (n = 53), cerebral malaria (n = 44) and severe malarial anemia (n = 59) at time of presentation to hospital in Kampala, Uganda. Levels of angiopoietin-2, von Willebrand Factor (vWF), vWF propeptide, soluble P-selectin, soluble intercellular adhesion molecule-1 (ICAM-1), soluble endoglin, soluble FMS-like tyrosine kinase-1 (Flt-1), soluble Tie-2, C-reactive protein, procalcitonin, 10 kDa interferon gamma-induced protein (IP-10), and soluble triggering receptor expressed on myeloid cells-1 (TREM-1) were determined by ELISA. Receiver operating characteristic (ROC) curve analysis was used to assess predictive accuracy of individual biomarkers. Six biomarkers (angiopoietin-2, soluble ICAM-1, soluble Flt-1, procalcitonin, IP-10, soluble TREM-1) discriminated well between children who survived severe malaria infection and those who subsequently died (area under ROC curve&gt;0.7). Combinational approaches were applied in an attempt to improve accuracy. A biomarker score was developed based on dichotomization and summation of the six biomarkers, resulting in 95.7% (95% CI: 78.1-99.9) sensitivity and 88.8% (79.7-94.7) specificity for predicting death. Similar predictive accuracy was achieved with models comprised of 3 biomarkers. Classification tree analysis generated a 3-marker model with 100% sensitivity and 92.5% specificity (cross-validated misclassification rate: 15.4%, standard error 4.9%). Conclusions: We identified novel host biomarkers of pediatric severe and fatal malaria (soluble TREM-1 and soluble Flt-1) and generated simple biomarker combinations that accurately predicted death in an African pediatric population. While requiring validation in further studies, these results suggest the utility of combinatorial biomarker strategies as prognostic tests for severe malaria

iNOS polymorphism modulates iNOS/NO expression via impaired antioxidant and ROS content in P. vivax and P. falciparum infection.

Nitric oxide (NO) has dicotomic influence on modulating host-parasite interplay, synchronizing physiological orchestrations and diagnostic potential; instigated us to investigate the plausible association and genetic regulation among NO level, components of oxidative stress, iNOS polymorphisms and risk of malaria. Here, we experimentally elucidate that iNOS promoter polymorphisms are associated with risk of malaria; employing mutation specific genotyping, functional interplay using western blot and RT-PCR, quantitative estimation of NO, total antioxidant content (TAC) and reactive oxygen species (ROS). Genotyping revealed significantly associated risk of P. vivax (adjusted OR = 1.92 and 1.72) and P. falciparum (adjusted OR = 1.68 and 1.75) infection with SNP at iNOS-954G/C and iNOS-1173C/T positions, respectively; though vivax showed higher risk of infection. Intriguingly, mutation and infection specific differential upregulation of iNOS expression/NO level was observed and found to be significantly associated with mutant genotypes. Moreover, P. vivax showed pronounced iNOS protein (2.4 fold) and mRNA (2.5 fold) expression relative to healthy subjects. Furthermore, TAC and ROS were significantly decreased in infection; and differentially decreased in mutant genotypes. Our findings endorse polymorphic regulation of iNOS expression, altered oxidant-antioxidant components and evidences of risk association as the hallmark of malaria pathogenesis. iNOS/NO may serve as potential diagnostic marker in assessing clinical malaria