title: In vitro and in vivo anti-malarial activity of novel harmine-analog heat shock protein 90 inhibitors: a possible partner for artemisinin.
abstract: BACKGROUND
  The emergence of artemisinin-resistant Plasmodium falciparum strains poses a serious challenge to the control of malaria. This necessitates the development of new anti-malarial drugs. Previous studies have shown that the natural beta-carboline alkaloid harmine is a promising anti-malarial agent targeting the P. falciparum heat-shock protein 90 (PfHsp90). The aim of this study was to test the anti-malarial activity of harmine analogues.
  
  
  METHODS
  Forty-two harmine analogues were synthesized and the binding of these analogues to P. falciparum heat shock protein 90 was investigated. The in vitro anti-malarial activity of two of the analogues, 17A and 21A, was evaluated using a 72-h growth inhibition assay. The in vivo anti-malarial activity was tested in Plasmodium berghei infection of BALB/c mice. The potential of 21A for a combination treatment with artemisinin was evaluated using in vivo combination study with dihydro-artemisinin in BALB/c mice. Cytotoxicity of the harmine analogues was tested in vitro using HepG2 and HeLa cell lines.
  
  
  RESULTS
  17A and 21A bound to PfHsp90 with average IC50values of 12.2 ± 2.3 and 23.1 ± 8.8 µM, respectively. They also inhibited the P. falciparum W2 strain with average IC50values of 4.2 ± 1.3 and 5.7 ± 1.7 µM, respectively. In vivo, three daily injections of P. berghei-infected BALB/c mice with 100 mg/kg of either 17A or 21A showed significant reduction in parasitaemia with a 51.5 and 56.1% reduction, respectively. Mice treated with 17A and 21A showed a median survival time of 11 and 14 days, respectively, while the vehicle control mice survived a median of only 8.5 days. A dose-ranging experiment with 21A showed that the compound has a dose-dependent anti-malarial effect. Furthermore, treatment of infected mice with a combination of 21A and dihydroartemisinin (DHA) showed a dramatic reduction in parasitaemia compared to treatment with DHA alone.
  
  
  CONCLUSIONS
  A novel and non-toxic harmine analogue has been synthesized which binds to PfHsp90 protein, inhibits P. falciparum in vitro at micromolar concentration, reduces parasitaemia and prolongs survival of P. berghei-infected mice with an additive anti-malarial effect when combined with DHA.
authors.affiliation: Department of Pathology and Laboratory Medicine, MIID and Medicine, University of Calgary, Calgary, AB, Canada. abebegenetu@gmail.com.|Department of Medical Parasitology, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia. abebegenetu@gmail.com.
authors.forename: Abebe Genetu
authors.initials: AG
authors.lastname: Bayih
authors.affiliation: Department of Pathology and Laboratory Medicine, MIID and Medicine, University of Calgary, Calgary, AB, Canada.
authors.forename: Asongna
authors.initials: A
authors.lastname: Folefoc
authors.affiliation: Department of Pathology and Laboratory Medicine, MIID and Medicine, University of Calgary, Calgary, AB, Canada.
authors.forename: Abu Naser
authors.initials: AN
authors.lastname: Mohon
authors.affiliation: Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, CA, USA.
authors.forename: Scott
authors.initials: S
authors.lastname: Eagon
authors.affiliation: Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Anderson
authors.affiliation: Department of Pathology and Laboratory Medicine, MIID and Medicine, University of Calgary, Calgary, AB, Canada.
authors.forename: Dylan R
authors.initials: DR
authors.lastname: Pillai
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000008:administration & dosage / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D037621:Artemisinins / Q000008:administration & dosage / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004195:Disease Models, Animal; D004357:Drug Synergism; D005260:Female; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors*; D006247:Harmine / Q000008:administration & dosage / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D020128:Inhibitory Concentration 50; D008288:Malaria / Q000188:drug therapy; D008807:Mice, Inbred BALB C; D010962:Plasmodium berghei / Q000187:drug effects*; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000254:growth & development; D011485:Protein Binding; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors; D016896:Treatment Outcome
chemical_list: D000962:Antimalarials; D037621:Artemisinins; D018841:HSP90 Heat-Shock Proteins; D015800:Protozoan Proteins; D006247:Harmine; C031327:artemisinin
keywords: Anti-malarial drugs; Heat-shock protein 90; Malaria; Plasmodium falciparum