OpenSourceMalaria:Triazolopyrazine Series Mechanism of Action: Difference between revisions
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Revision as of 18:38, 9 June 2016
Open Source Malaria Series 4 and the ATP4 Ion Channel
About
Some Series Four compounds have been found to inhibit the PfATP4 ion channel in an assay performed by Kiaran Kirk's lab at Australian National University. This page will be regularly edited to update OSM's understanding of ion channel interactions, for both Series Four and antimalarials under investigation in other research groups.
A brief history of ATP4 and malaria
The Spiroindolones and KAE690 - 'Novartis Compounds'
Millions of compounds have been explored as a potential antimalarials as part of a concerted global effort to find new medicines to combat emerging resistance to existing drugs. Whole cell assays are an essential high throughput screening method for the identification of active compounds. In 2010, whole cell screening revealed a potent class of compounds, the spiroindolones, which were able to inhibit the growth of blood stage malaria parasites in vitro. [1]
Following a barrage of other in vitro assays, one of the spirondolones, KAE690, was progressed to Phase II human clinical trials, where it was shown to be effective against uncomplicated Plasmodium falciparum and P. vivax. Additionally, the drug was active in late stage gametocyte assays. The ability to inhibit gametocytes is highly prized in antimalarial candidates as gametocytes are the sexual form of the parasite transmitted back to the mosquito. Therefore drugs that terminate gametocytes play an important role in breaking the cycle of transmission.
KAE690 was next explored in drug pressure experiments. Microbial pathogens, such as plasmodium, are well known to develop resistance to drugs under prolonged exposure to medicines, both in laboratory experiments and in affected populations. Lab experiments can simulate the resistance developed in patients and also provide insight into the mechansims of resistance, which in turn can help to identify the mechanism of action of the therapeutic. Increasing concentrations of KAE609 were applied to particular parasite strain for a few months. The amount of drug required to inhibit parasetemia by 50% (IC50), was found to increase, indicating that the parasite had developed some resistance to the drug. Genetic analysis showed that the 'resistant' parasite had several mutations in a gene located within it's plasma membrane, PfATP4.
PfATP4
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Na+ regulation in the malaria parasite
Plasmodium falciparum is an intraerythrocytic (situated or occurring within the red blood cells) malaria parasite. On entering a red blood cell the environment experienced by the parasite changes from high Na+ concentration and low K+ concentration (blood plasma) to the low [Na+] and high [K+] media of the cytosol. Around 12 hours after invasion, the parasite establishes itself in the host and develops 'new permeability pathways' for the influx of Na+ and efflux of K+, which in turn provide the energy required transport nutrients across it's membrane. The new permeability routes lead to a change in erythroyte cytosol environment so that the ratio of Na+/K+ is again reversed to mirror that of blood plasma. Despite the change surrounding the parasite, it manages to maintain a low cytosolic [Na+] concentration. The mechanism of Na+ homeostasis within the plasmodium falciparum parasite has been investigated by Prof. Kiaran Kirk's group at Australian National University. Investigations by the Kirk lab have shown that the cation ATPase PfATP4 is responsible for maintaining low Na+ concentration in healthy parasites.
1. Proc Natl Acad Sci USA 105: 9059– 9064, Nature 465: 305–310, Nature 465: 311–315
Kirk Lab vs The Malaria Box
The Kirk lab have examined antimalarial compounds (known actives in whole cell assays) and looked at the way in which they perturb Na+ regulation within the parasite. OSM needs to profile the activity of more S4 compounds in the Kirk lab in order to gain better understanding of this possible MoA.
PfATP4 is the apparent target of a number of different chemotypes: Kirk Review
The Pyrazoleamides - 'Vaidya Compounds'
The Frontrunners
*note this section is linked to a GitHub Issue #400 (June 2016) and will be systematically edited to incorporate input from online discussion.
The following actives (MMV670246 is poorly active but included for comparison) have been identified as the "frontrunners" for further exploration by OSM. All but one of these compounds were found to be active in a whole cell assay, but to date (June 2016), only one has been tested in Kiaran Kirk's ion regulation assay.
A large range of data has been measured for the compounds and this is captured below in a condensed form of the master chemical list, which is colour coded to assist when glancing over the data (key: green = good, yellow = OK and red = poor).
In order to further probe the MoA of the series four compounds it is necessary to send these compounds for testing by the Kirk group at ANU. For example, three of the compounds have been tested in vivo and are able to kill the parasite in mice, but the MoA is still to be tested. We would also like to fill in the gaps in the data where possible as this will help when rationalising compound design and also for assembly of the S4 paper.
The active compounds are mainly ethers along with a few amides and one amine:
Ethers MMV663915 highly active, still to be tested in ion regulation assay. MMV670936 active with good cytotox and hERG profile, still to be tested in ion regulation assay. MMV639565 highly active in whole cell and in vivo, still to be tested in ion regulation assay.
Chiral ethers of particular interest owing to activity and the ability to explore/compare both enantiomers for each compound: MMV669844 highly active in whole cell and in vivo, still to be tested in ion regulation assay. Problematic hERG profile. MMV672687 good activity and cLogP but no further data yet. MMV688896 good activity and cLogP but no further data yet. MMV670652 highly activity, some clearance data but need to gather more. MMV670437 highly active, cLogP on target and basic amine centre is of interest for further exploration.
Amides MMV670246 was found to be a weakly hit in the ion regulation assay and to possess low activity in the whole cell assay. Hence, further data should be collected. MMV670767 is an amide with good whole cell activity but we should collect more data. MMV670944 is an active amide with lots of data - ion regulation data is required in order to produce a complete profile for the paper.
Amine MMV669848 active in the Kirk assay and has shown other promising properties, more exploration required.
Resynthesis is required in order to provide adequate quantities of the compounds for testing.
New Compounds We have shipped 12 compounds for potency evaluation and are awaiting data before folding the compounds into this issue. However, one existing compound MMV670947 has shown good activity and either this or the newly synthesised MMV693155 should be sent for testing.
We also need to look at some inactive/weakly active compounds and examine them in the Kirk assay and collect other data (hERG, Cytotox, clearance etc.). Some suggestions are:
Pyridine ethers MMV688895 - this compound should be re-tested in whole cell to confirm a lower potency than MMV670936 and also tested in the Kirk assay. pyridine ethers
Amides MMV675947 and MMV675946 are moderately active amides that would serve as good controls in Kirk assay. MMV675718 is an inactive amide that should be included as a negative control in Kirk assay.
