Biological Evaluation of Arylpyrrole Series

New Stuff:
Online lab book hosting bioactivity data is here.
First set of compounds have been evaluated (Jan 2012) - here.
Initial phase of this project is to validate the biological activity of the two Tres Cantos leads. The promise of these compounds (and others) is discussed in a paper linked here.

Original activity data for the two compounds are here and here.

For this initial phase, the question is: What kind of biological (re)evaluation is needed? (not toxicology, just activity)

For experiments, Tres Cantos (Felix Calderon) kindly offered to re-evaluate these compounds. We also have links with other labs who have expressed an interest in this project (the Eskitis Institute in Queensland or Stuart Ralph's lab in Melbourne). Question is, what data are we looking for?

In our original proposal for this project, we assumed the following assays would be needed in general. Are all these needed for validation of the current two compounds, or only later during analog evaluation?

1) A primary whole cell parasite assay covering a sensitive and resistant falciparum strain (3D7, Dd2 and W2mef). (Screening for activity would use an image based anti-malarial HTS assay incorporating DAPI or SYBR-Green dyes to monitor parasite growth: asexual and, potentially, gametocytes.

2) Assay for information on the selectivity between drug resistant and sensitive falciparum strains, as well as possible cytotoxicity on mammalian cell lines (typically HepG2 or HEK293 cells), to check for a high therapeutic ratio.

3) For compounds that inhibit growth selectively, IC50s should be determined using serial dilutions of inhibitor in 48 and 96 h assays, which will allow us to screen for promising cell-permeable inhibitors and to discern immediate and delayed parasite death – suggesting whether inhibition is of cytosolic- or apicoplast-based targets.

In our correspondence with Felix, he said the following:

1) The antimalarial activity of these compounds is not affected by the presence or absence of folate in the culture medium, implying they are not inhibitors of the folate biosynthesis pathway. Is this of general significance since it steers clear of well-established resistance mechanisms? (review)

2) The compounds are neither bc1 nor DHODH inhibitors. Why is this important?

3) Felix would be happy to determine the IC50 for these compounds in the standard hypoxanthine incorporation assay (48 h). Determination in the original Tres Cantos dataset was measured at 72 h using the LDH assays. Is this difference in assay significant/desirable?

These questions are intentionally naive, because though there are many options, we need a consensus on what people will be looking for in validation of the existing compounds, and why.

Biological Results for First Set of Compounds

The screening data from three separate labs have been obtained for the first set of compounds on the project. Data were obtained from the Ralph Lab at the University of Melbourne, and a second data set was provided just before Christmas by the Avery Lab at Griffith University. Yesterday the third set was provided by GSK Tres Cantos in Spain, who originally discovered the hits we're starting with. The current list of available compounds in this open project is here, with those that have been evaluated by at least one lab indicated in the relevant column.

Having data on the same compounds from three labs using different screening methods is useful as it provides contrasting ways of assaying effectiveness. In any given screening experiment on this project it's going to be important to include known actives, so that we have benchmarks, and this was done in these cases. It's also very important to be 100% sure about the effectiveness of a compound before we become too attached to it...

The data (below, but all available through the relevant lab book) show that the original TCAMS compounds are certainly active, though perhaps not quite as active as suggested by the original screen. Paul Willis at MMV had suggested we also check out some "near neighbors" of these compounds that were in the original data set. We made a couple and one (a novel compound with the code PMY 14-1, shown below and synthesized here) has shown promising activity in all three screens, with Avery/GSK IC50s coming back as low nanomolar. (Note that this project will never involve patents or closed data, giving us the freedom to discuss the compounds freely.)

What's next? In the short term: We're waiting for confirmation of the Melbourne data via a re-run of some of the experiments. But what we need is an expert qualitative assessment of these bioactivity data by someone familiar with such screening assays. Either in comments below this post, or on G+, not by email. First item of business in the lab is to generate a few variants of PMY 14-1. We already have some new relevant compounds and are now planning others. What should we make - i.e. how ought we to change PMY 14-1? Sanjay Batra has students who are about to make steric variations in the aryl pyrrole, and these could then be employed in the synthesis of PMY 14-1 variants, for example, but shouldn't we also be interested in changes in the "upper half" of the molecule?

In the long term: It would be good to find other labs which already have analogous compounds to the actives. Paul and Zoe found a paper from the Roberts lab at Scripps describing a number of such compounds, and I will write to them to ask whether they are interested in having the compounds be screened for their antimalarial activity. If anyone knows of any other possible sources, that would be great, since using existing compounds saves a lot of time in the lab.


Biological Results for Second Set of Compounds

In January the first biological data for compounds from the open source drug discovery for malaria project came through. The compounds were based on two hits identified in the GSK Tres Cantos set (TCMDC-123812 and TCMDC-123794). The two originals performed well, and we also identified two other compounds that looked promising (PMY 14-1 and PMY 14-3-A). Biological data were obtained from three labs (the original GSK lab, Stuart Ralph's lab in Melbourne and Vicky Avery's lab in Brisbane) and compared to known antimalarials.

Since then Paul and Zoe have been making a second set of compounds, which we shipped last month. Details of those compounds are in this spreadsheet. They are intended to explore the most promising compounds from the first set.

The first biological data are now back - from Vicky Avery's lab. We have some super-potent compounds, which is very exciting. One is picomolar (the data below are the average of two runs on 3D7). The data are posted raw here, and are summarized below (direct link to picture file).

A few obvious points:
1) We're eagerly awaiting the data from the other two labs, to see if the activity is confirmed.
2) The QSAR isn't flat - i.e. changes to the structure of the molecules make a difference to the bioactivity.
3) The aryl pyrrole appears to be needed in all sets.
4) Replacement of the ester with an amide in the original GSK compounds is seriously deleterious.

What's needed:
1) The most potent compounds have high logP. We're going to need to make them more aqueous soluble.
2) The best four from the first round and four from this second round are going to be shipped for basic metabolism assays to Sue Charman at Monash.
3) We're hoping to send 2-3 compounds for in vivo evaluation. Possibly the two originals, plus one of the super-potent compounds. Awaiting confirmation that we can do that.
4) The work that Sanjay Batra at CDRI is doing on installing sterically demanding groups on the aryl ring in place of F will be an important addition here.

1) What do we do to decrease logP?
2) There have already been some good suggestions on how to change these compounds by modification of/introduction of other heterocycles. We think this is still the way to go for round three. Everyone agree?
3) Does the lack of activity for compound ZYH 23-1, and its laughable lack of reactivity towards hydride reduction, suggest we need not worry about these compounds being PAINS?

If you've any other gut feelings about these compounds, or if you'd like to play with them in your lab, or if you spot some chemistry you'd like to do to make a related scaffold, please say.

To re-state the obvious: this is open source, meaning you can join the project, or take what we've done and use it in your own research, with attribution (CC-BY-3.0).

Biological Results for Third Set of Compounds



A summary of the biological activities obtained for the third set of compounds in 2012 - those arising from the consultation for which synthetic and commercial compounds that were most wanted. See also some links for analysis of trends in the data.

First set (Oct 19th) Data, and these were discussed briefly in an online meeting.

Second set (Nov 8th) Data, and discussion

Third set (Dec 10) Data (essentially inactive, aside from mild activity for OSM-S-103


Previous discussion of these data, highlighting trends.

Importance of primary amide side chain.

Impact of replacing ester with amides and amines. And impact on the two original GSK compounds.

Dramatic impact of methylation of the hit compound.

Low efficacy of pyrazoles.

Prodrug hypothesis II and III

Reminder of the efficacy of the near neighbour thiazolidinones.

Suggestion of next compounds, including hybrids, and the WANTED! compounds.



Late Stage Gametocyte Assay for Arylpyrroles



Four of the arylpyrroles/near neighbors have been tested in a late stage anti-gametocyte imaging assay, with interesting results.

This assay is less usual than other malarial assays (because it is technically more challenging). See this paper for a clear description of the importance of the gametocyte stage of malaria. The upshot is that drugs targeting this form of the parasite (the late stage gametocyte) are particularly valuable because they could help prevent the transmission of malaria.

In fact in the above paper many antimalarial compounds did not display activity against LSG, with only methylene blue reaching an IC50 of 12 nM. Indeed more generally it seems that there are few compounds that have been identified with this activity.

Interestingly the novel compounds screened from the arylpyrrole set (but not the original GSK compound OSM-S-5) were highly potent (nanomolar) in this assay. The data are posted in the lab notebook. That's pretty interesting.


Metabolic Studies on a Set of Arylpyrroles



Eight compounds - two GSK originals and 6 promising-looking compounds made during this project were examined by Sue Charman's lab at Monash for metabolic degradation in vivo. In human terms that means they were tested (on a simplistic level) to see whether the compounds would last long in the blood or whether they would likely be metabolised. As part of these studies the solubilities of the compounds were evaluated.

Compounds were as follows. Note the use of the new "OSM-S" notation which we're introducing to give a compound a unique ID tag, independent of source, though there will probably always be trailing synthetic-prep tags.

The results are posted here.

As is often the case low degradation rates (good) come at the cost of low solubility. We kind of expected this based on the high logP values for some of these compounds. Presumably analogous compounds could be re-examined when more soluble.

hERG Assay for Two Potent OSM-S Compounds



One of the most potent compounds identified to date in the OSDD malaria project, the near-neighbour analog OSM-S-35 (ZYH3) was subjected to the hERG assay along with one of the original TCAMS GSK compounds which in this project has the tag OSM-S-5.

Raw data are here, plus spreadsheet here.

These results suggest that these compounds are "misses" in this assay, implying that they, and perhaps the series as a whole, would not have cardiac side effects as drugs.

Courtesy of Paul Willis at MMV: The human ether-a-go-go related gene (hERG) encodes a potassium channel in the heart (IKr) which is involved in cardiac repolarisation. Inhibition of the hERG channel can cause ‘QT interval prolongation’ resulting in a potentially fatal ventricular tachyarrhythmia called Torsade de Pointes. A number of drugs have been withdrawn from either the market or from late stage clinical trials due to these cardiotoxic effects, therefore it is important to identify hERG inhibitors early in drug discovery. Therefore the fact that these compounds are inactive at hERG is good news (Much is understood about the pharmacophores that hit the hERG channel so I was not expecting an issue for these compounds but it is always good to confirm).  A hERG inhibition at an early stage is not a show stopper but a clear issue that has to be addressed in the optimization process.