Download Print version

In vitro and in vivo antimalarial activity of
novel harmine-analog heat shock protein 90
inhibitors: a possible partner for artemisinin.
Abebe Genetu Bayih, PhD
University of Gondar
Introduction
Malaria
• In 2015:
 3.2 billion people at risk
 214 million cases
 438,000 deaths
• 90% in sub-Saharan Africa
WHO
• Children most affected (WHO 2015 report)
• Significant reduction in mortality and morbidity
• Emergence of ART resistance – a challenge!
 Catastrophe if reaches Africa!
 NEW DRUGS NEEDED!
Source:
Introduction…
Heat shock proteins (Hsp):
• Molecular chaperone
• Function – protein folding, intracellular trafficking, gene
expression, cell cycle, and cell differentiation.
• Conserved – from bacterial to mammals
• Constitutive and induced
Hsp90 Structure (Bracher and Hartl, 2006)
• Temperature, nutrition, heavy metal exposure
 antimalarial and anti-cancer drug target
Introduction…
PfHsp90 inhibitors:
• Natural compounds (Geldanamycin and derivatives) (Kumar et al, 2003;
Banumathy et al, 2003)
• Harmine (Shahinas et al, 2010, 2012)
• Beta-carboline alkaloid
Harmine
• Preferentially binds to the ATP-binding domain of PfHsp90
• Inhibits P. falciparum in vitro
• Antimalarial effect in vivo in mice
Hypothesis:
Structural modification of harmine improves efficacy.
METHOD
45 novel harmine analogs screened for PfHsp90
binding
Two (17A and 21A) bind to PfHsp90
Cytotoxicity assay (HepG2 and
HeLa)
In vitro antimalarial assay
(P. falciparum)
Fixed dose in vivo antimalarial assay
(P. berghei ANKA in BALB/c mice)
21A - in vivo dose-ranging expt.
21A and DHA in vivo combination
expt.
RESULTS AND DISCUSSION
1. Competitive binding assay
• bis-ANS assay:
Mean EC50 ± SEM (µM) :
• 17A = 12.2 ± 2.3 ; 21A = 23.1 ± 8.8; Radicicol = 5.46 ± 2.42
2. In vitro antimalarial activity
• 72h in vitro susceptibility assay
• HRP-II ELISA
Mean IC50 ± SEM (µM) :
PF-W2:
17A = 4.2 ± 1.3
21A = 5.7 ± 1.7
CQ = 0.15 ± 0.04
PF-3D7: 21A = 13.5 ± 0.8
PF-MRA 1236:
21A = 9.2 ± 0.4
PF-MRA 1240:
21A = 9.6 ± 2.0
3. In vivo antimalarial activity
A. Fixed-dose harmine-derivatives against P. berghei in
BALB/c mice (100mg/kg)
17A
• At day-7 post-infection:
• Percent inhibition:
• 17A = 51.5%
• 21A = 56.1%
21A
Treatment
Percent Parasitemia
I. Parasitemia:
25
CQ
20
DMSO
15
10
*
5
0
0
2
4
6
8
Days post-infection
• 17A and 21A significantly lower parasitemia than vehicle
ctrl. (p<0.05).
II. Survival
• Drug treated mice showed better survival than the vehicle group
(DMSO).
• The Median Survival time: 17A = 11, 21A = 14d, DMSO = 8.5d.
• 21A resulted higher survival than 17A
• Significant difference b/n 21A and DMSO (Log-rank (Mantel-Cox)
test, p=0.0177)
B. Dose-ranging experiment
21A
Parasitemia:
Percent parasitemia inhibition
100
75
50
25
mg/kg mg/kg mg/kg mg/kg
D6
48.1
37.5
27.2
D7
45.9
33.4
18.9
18.4
• At D6, treatment with 100mg/kg, 75mg/kg and 50mg/kg
significantly reduced parasitemia.
• The Median Survival time: 100mg/kg = 11.5d, 75mg/kg = 10d,
50mg/kg = 10d, 25mg/kg = 8d, DMSO = 8d.
C. 21A-DHA combination experiment
• 21A (100mg/kg) showed additive effect with
DHA (10mg/kg)
• Parasite clearance:
• One day after two doses of drug:
21A = 0/5 (0%)
DHA = 2/5 (40%)
21A plus DHA = 5/5 (100%)
• One day after three doses of drug:
21A = 0/5 (0%)
DHA = 3/5 (60%)
21A plus DHA = 5/5 (100%)
• A combination of 21A and DHA improved
survival rate.
4. Cytotoxicity
• CCK-8 (Sigma):
• Based on water soluble WST-8
• Live/dead cell
Mean IC50 ± SEM (mM) :
HepG2:
17A = 0.31 ± 0.023
21A = 0.093 ± 0.002
Geldanamycin = 0.00038 ± 8.98E-05
Chloroquine = 0.24 ± 0.024
HeLa:
17A = 0.738 ± 0.14
21A = 0.436 ± 0.089
17A and 21A NOT toxic to HepG2 and HeLa cells.
SUMMARY
•
Out of 45 harmine analogs tested, 17A and 21A:
• Bind to ATP-binding domain of PfHsp90 effectively.
• Inhibit P. falciparum in vitro in micromolar concentration
• Significantly reduce parasitemia in vivo
• Prolong survival of infected mice (not seen in parent
molecule, Harmine)
• 21A showed a dose-dependent antimalarial effect.
• 21A showed additive effect with DHA
• Not toxic in HepG2 and HeLa cells in vitro
• 21A could be a potential standalone or a partner antimalarial
drug.
Acknowledgement:
Asongna Folefoc, PhD, University of Calgary, Canada
Abu Naser Mohon, MSc, University of Calgary, Canada
Scott Eagon, PhD, San Francisco State University, USA
Marc Anderson, PhD, San Francisco State University, USA
Dylan R. Pillai, MD, PhD, University of Calgary, Canada
Funding: Grandchallenges Canada to DRP
Towards a malaria-free happy world!