Carbonic Anhydrase - Relevance to Mechanism of Action

[mattodd]

I was speaking about the mechanism of action of Series 3 (OSM-S-106, MMV025100) at Monash University and David Shackleford was in the audience. He independently brought up the issue of carbonic anhydrase. We've discussed this before (@drc007 on twitter) and it remains of interest: OpenSourceMalaria/OSM_To_Do_List#561, OpenSourceMalaria/OSM_To_Do_List#573, OpenSourceMalaria/OSM_To_Do_List#503, #12

David just wrote:

"Being a primary aromatic sulfonamide, there's a substantial chance that it binds to carbonic anhydrase (CA).

While the plasmodial CA could possibly be a target (see paper from Krungkrai and Supuran), I'm intrigued by the possibility that the high expression of CA within erythrocytes could lead to a very high localised concentration where the (blood stage) plasmodium resides; another two papers (Maren and Matsumoto) attest to the erythrocytic accumulation of aromatic sulfonamides.

Evaluation of this would be a pretty simple in vitro whole blood-to-plasma partitioning ratio measurement."

This is a fascinating idea - that the intrinsically high concentration of erythrocytic CA could lead to a locally high concentration of the drug around the parasite. Further, if the drug is "sequestered" in the erythrocyte it might evade hepatic clearance. To be useful it'd obviously need to not be a tight binder of human CA.

David thought that Sandy Pang (http://www.pharmtox.utoronto.ca/faculty-k-sandy-pang-phd) had looked at this idea of "stealth" evasion of metabolic clearance with the drug harmol, but we couldn't find the relevant paper - I'll reach out to her to see if there's something already known about this.

[mattodd]

Sandy Pang wrote back by email. To paraphrase:

"Yes, we have worked on the concept of red cell carriage, something my colleague, Carl Goresky, is greatly interested in. We showed this for acetaminophen in multiple indicator dilution experiments. Trapping of drug by rbc reduced the clearance of drug. One can see it in experiments if rapid sampling could be conducted at the outflow of liver for a drug that is rapidly cleared – the concentration from the immediately fractionated plasma vs. the equilibrated plasma. Another paper is on digoxin: this or this.

[MFernflower]

I still feel a simple in vitro CA assay would be the first step in the whole process however @mattodd @drc007

[MFernflower]

@mattodd @drc007 Relevant paper I found https://www.sciencedirect.com/science/article/pii/S2211320716301269

[drc007]

Interesting paper, raises some questions. Is there only a single pf carbonic anhydrase? Do all compounds partition into parasite similarly?
Even if not the pf target, CA anhydrase inhibition in man would be a concern so still need in vitro data.

[MFernflower]

@mattodd @drc007 @mbhebhe perhaps we can send some compounds to the authors of this paper?

[mbhebhe]

Its an interesting paper with a simple-ish assay and they have assays for both Hs and Pf. We will try and contact them

[drc007]

@mbhebhe @mattodd More carbonic anhydrase inhibitors https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00437 I wonder if we could ask them to test a couple of compounds?

[mbhebhe]

We have been put into contact with Claudiu Trandafir Supuran and I will be sending the positive and negative controls soon

[mbhebhe]

Our positive control (OSM-S-106) and negative control (OSM-S-123) have been sent to Griffith University (Hs CA assay) and to the University of Florence (Pf CA assay).

[mbhebhe]

Hi all,
Further to the great suggestion by @mcoster on twitter and @drc007 on GH, many times!.. We now have results from the Uni of Florence done by Claudiu T. Supuran and his team.

Raw data can be found on here. The table below is the condensed version. Most importantly, OSM-S-106 seems to be inhibiting PfηCA a lot more than AAZ (standard). OSM-S-106 has a Ki of 2.2 nM and AAZ with a Ki of 170 nM. Details on the enzyme and its sulfonamide inhibition can be found in these two papers (doi.org/10.1080/14756366.2016.1217856 and http://dx.doi.org/10.1016/j.bmcl.2016.07.060)

	Ki (nM)			IC50 (uM)
	HsCAI	HsCAII	PfCA	HsCAI	HsCAII	PfCA
OSM-S-106	344.7	53.4	2.2	34.5	5.34	9.36
OSM-S-123	>10000	>10000	>10000	n/d	n/d	n/d
AAZ	250	12.1	170	38.3	8.02	735

I have done some reading on PfηCA and our lead compound seems to be the "most potent" so far. I compiled a list (at the end of this google doc ) of "known" inhibitors of PfηCA and our lead compound looks promising. PfηCA could be a potential target but we don't know how it could be linked to the asparagine-tRNA ligase #12 #16. Maybe PfηCA is the target and the asparagine-tRNA ligase is a compensatory mechanism?

In the mean time, Sally-Ann Poulsen at Griffith Uni are working on a protein X-ray crystal structure determination of OSM-S-106 with human CA II. We will let you know when we get the data.
It would also be great if we could do the same with PfηCA. Does anyone know anyone who could help us with that?

In this issue Anyachan suggested that CA was a potential target.

[drc007]

@mbhebhe @mattodd This is fabulous news, and an excellent example of collaboration moving the science forward.

I had a quick look at the sequences of the human and Plasmodium falciparum carbonic anhydrase and they appear very different. A crystal structure of the ligand bound to the human enzyme would be interesting with respect to off target activity. Getting a crystal structure of PfηCA would be great, however I suspect at the moment the main challenge will be to find a lab that is open.

Would be interesting to get pharmacokinetics etc.

It looks like a great starting point, if only we had chemistry labs open......

[mbhebhe]

@drc007 thank you for pushing us to do this. Also OSM and collaborating for the win!
We will have to wait until this all blows over. Hopefully it will be worth the wait

[holeung]

We could do the crystallography of Pf CA with OSM-S-106. But our labs are shut down right now.

[drc007]

@mattodd @MFernflower @mbhebhe Looking at the data summary for OSM-S-106 here https://github.com/OpenSourceMalaria/Series3/wiki/The-Data-on-the-Original-Hit the presentation of the HERG data is a bit confusing, I suspect the in vitro potency quoted is not activity at the HERG ion channel?

[drc007]

With many labs closed around the world it might be worth compiling a list of what experiments we might want to do with OSM-S-106 so that when labs reopen they can perhaps pick up items on the list immediately.

Resynthesis of OSM-S-106 and retest of fresh batch in both PfηCA and human Carbonic Anhydrase. Would be useful to have a larger batch for further work. Has it been crystallised? What is the mpt?

Identify lab(s) that can run the PfηCA assay routinely. Get together a few positive controls we might want to include (Dorzolamide, Acetazolamide, Brinzolamide, Dichlorphenamide)?

Since the sulphonamide probably binds to the Zinc in the active site of PfηCA, are there other Zinc metalloenzymes we might want to counter-screen against?

Obtain a crystal structure of PfηCA with ligand bound in addition to crystal structure of human enzyme.

Preliminary in vitro data suggests low turnover, however there are other clearance routes in vivo. Would be useful to get pharmacokinetic data, perhaps rat PK to start with.

Look at CYP interactions in vitro, plasma protein binding

Would also be interesting to look at erythrocyte accumulation of aromatic sulfonamides as suggested by @mattodd here #14 (comment)

Should we look to test a compound in vivo?

Start designing novel ligands, given that lab capacity is likely to be constrained we should use the time to really focus on novel ligands that address particular questions.

[mbhebhe]

The hERG data is the in vitro potency from a dose response curve.

I can try and re-synthesize OSM-S-106, if I have time. The batch sent for the CA assays was synthesized by a MSc student about 2 months before shipping it. He made a lot of the lead compound. We haven't tried recrystallizing it but it decomposes at 279 degrees Celsius.

I have synthesized a positive control for the PfCA (Ki: 80nM). I was also in the middle of synthesizing the only other known inhibitor of Asn Ligase before the labs shut down. We are planning on sending these two positive controls and OSM-S-106 to the Creek Lab for their metabolics studies so that we could compare the controls to our lead to try and understands it's MoA.

[drc007]

@mbhebhe But in vitro potency in which assay? I presume not HERG since the HERG IC50 is quoted as >10 uM?

[cdsouthan]

Good stuff but have the PubChem neighbours been tested in parasite and enzyme assays?

https://www.ncbi.nlm.nih.gov/pccompound?cmd=Link&LinkName=pccompound_pccompound&from_uid=44528665

[mattodd]

@drc007 @mbhebhe hERG data: I assume the potency is asexual blood stage assay of that sample, no? The link back to the ELN (once it loads, jeez a bit slow, right?) just gives the sheet from Adam Hill at the Victor Chang in Sydney, and they would not have done that potency assay, so yes, it's a bit confusing. Tha, can you please check whether you have anything more on this and I'll do the same. Can you please in any case amend the page so that it's clear who created the data?

[mbhebhe]

@cdsouthan out of the 18 only 4 compounds, including OSM-S-106 (https://pubchem.ncbi.nlm.nih.gov/compound/31981178, https://pubchem.ncbi.nlm.nih.gov/compound/47015523#section=2D-Structure, https://pubchem.ncbi.nlm.nih.gov/compound/11499583#section=2D-Structure) have been tested in the parasite. Data can be found here

@drc007 @mattodd I have updated the ELN, with the assay info.