Can we predict compound precipitation in DMSO stocks?

Collections of compounds used within drug discovery screening projects have to be tested in a variety of different assay types and therefore are stored in a liquid form. The most widely used solvent for this purpose is DMSO (Dimethyl sulfoxide).

However some compounds do not remain in solution and fall out forming a precipitate. A team at GlaxoSmithKline investigated this issue, in this publication – (Ioana Popa-Burke and John Russell, “Compound Precipitation in High-Concentration DMSO Solutions.,” Journal of biomolecular screening, 19 (2014), 1302–8 .

In the article the team noted that from one library of compounds – the “Tox Set” stored at 100mM they measured a 15.17% precipitation rate (by means of visual inspection). This was compared to a collection of fragment based compounds (at 100mM concentration) which had a 4.76% precipitation observed. It should be noted the there was a difference in the total number of theses set of compounds 422 versus 1995 respectively, which may explain some differences. The team also investigated other fragment and diversity compound collections with a higher number of members but at a lower concentration (a 40mM Fragment set with 7137 members and 10mM diversity set of 38,360 members). These both gave similar observed precipitates of 3.45% and 3.11% respectively.
The water content of all the DMSO samples was measured using Echo 555 acoustic dispenser, and this was similar across the samples ranging from 90.9% to 92.0% in all collections apart from the fragments at 100mM which had 85.8% DMSO. It was therefore assumed that % water content was not to a cause for precipitation. As all these compounds had been prepared and solubilised in the same manner, it was concluded that a chemical property must be the driver of precipitation of these compounds.

To determine if a physicochemical property could be identified as a correlating factor for increased precipitation rate in the Tox set compared to the fragment set , the MW, clogP, fsp3 (fraction of sp3 carbons) and TPSA (total polar surface area) were analysed with three different data mining techniques however no relationship was uncovered.
The identified precipitating compounds soluble concentration and purity was determined using LC-UV-MS-ELSD system. This revealed that concentration was, as to be expected lower in the free solution of precipitated samples, however the level of purity was similar for precipitating compounds as fully soluble compounds, suggesting that impurities are not the causative factor in the production of precipitates.

To determine if number of freeze thaw cycles would increase the number of precipitates observed, the team took the identified 110 precipitating compounds from the 100mM tox and 100mM Fragment set and made fresh solution in DMSO at different concentrations. These samples were then exposed to 1, 2, 5, and 10 freeze thaw cycles, and the number of precipitates was observed.
Number of Freeze thaw cycles did not have a significant increase in the number of compounds that precipitated however there was a correlation with compound concentration.
A key result was that from this set of precipitating compounds was 86% were unable to go into a solution at 100mM initially before any freeze thaw cycle.

So to answer the question – no, from this latest publication the authors cannot predict which compounds will precipitate from a DMSO solution. Compound concentration does have an effect on the number of precipitates observed, which is important to remember when thinking about compound library composition and stored concentration.

Fragment Screening: False Positives – (2,5-Dimethyl-1H-pyrrol-1-yl)benzoic acid

After a recent screening campaign in the Translational Drug Discovery Group (TDDG), using a well-known fragment library, we were looking into confirming our hits by repurchasing them and purifying these samples. When searching for 4-(2,5-dimethyl-1H-pyrrol-1-yl)benzoic acid (3) we came across this paper by Rolf Hartmann et al( Chem. Eur. J. 2013, 19, 8397 – 8400). in which they describe their elucidation of a screening artefact contained within their sample of 3.
Hartmann was screening fragments in the search for novel bacterial RNA polymerase (RNAP) inhibitors and came across pyrrol 1 as a positive hit from their screening library. Upon further reading of the literature they found that structurally related compounds 2-4 had also previously been described as RNAP inhibitors. However, once compounds 1-4 had been resynthesized and tested no activity was observed. Hartmann did notice, as had we, that after purification these compounds are colourless but quickly develop a red tone when a solution of them is left open to the air on the bench.

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Hartmann postulated that the observed activity that he and others had seen from these compounds was coming from a decomposition impurity rather than the parent pyrrol. To accelerate their decomposition the purified inactive parent pyrrol 1 was heated to 50°C in DMSO for 10 days and the resulting HPLC trace and NMR spectra can be seen in figure 2.

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As the C-H protons from the pyrrol ring (5.7 ppm) of 1 had disappeared and a broad new signal was observed in the aromatic region (7.0-8.5 ppm) it was thought that the decomposition product was a polymer. The decomposition sample was subjected to ultrafiltration (cut-off 3.5kDa) and it was shown that the active component was of a high molecular weight which they named P1. Figure S3 shows a broad molecular weight distribution of P1 by gel permeation chromatography (GPC) with a weight-average molecular weight of 40kDa

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The UV/Vis spectroscopy of P1 shows an absorption peak at 498 nm (2.49 eV) which is associated with a π-π^* transition indicating a well conjugated π electron system in the backbone (figure S4). This absorption at 498 nm was also seen in the commercial starting material but not in the purified pyrrol 1.

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The decomposition of compounds 2-5 were carried out and the corresponding polymers P2-P5 were isolated. A carboxylic acid signal was observed by IR for P1-P4 which was supported that they could be easily dissolved in water at basic pH in contrast to P5. The polyanionic structure of P1 was confirmed by gel electrophoresis. At pH 3.6 P1 and P5 remained at the starting point but at pH 7.8 P1 migrated to the anode and P5 remained at the starting point.
Using all of the above analysis Hartmann has postulated that the structure of P1 (figure 3).

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Compounds P1-P5 were tested against E. coli RNAP in an in vitro transcription assay (table 1). None of the monomers affected transcription but all of the carboxylic acid containing polymers displayed a concentration dependant inhibition.

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Figure 4 shows the positively charged DNA binding channel which was used to explain why P5 is inactive against RNAP but the other polyanionic P1-P4 show inhibitory activity. Further experiments were conducted that all supported this hypothesised mechanism of P1 RNAP inhibition.

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When P1 was tested against 8 other RNA polymerases and only 1 (bacteriophage T7) was strongly inhibited. Unfortunately P1 showed no inhibition in growth of E. coli and Pseudomonas aeruginosa. This lack of in vivo activity has been put down to P1 being to hydrophilic for passive diffusion and too large to permeate the porins. Hartmann was hopeful that if he could reduce the average size of the P1 polymers then he would able to achieve in vivo activity by permeating these porins.
Hartmann has taken the time in this paper to thoroughly investigation a false positive from their own work and also confirmed that the other close analogues that had previously been described as RNAP inhibitors were also false positives. Not only is this an interesting paper as Hartmann has been able to identify the previously unknown inhibitor but it also helps the wider community potentially avoid wasting resources pursuing these Pan Assay Interference Compounds (PAINS).
Key words
Fragment screening, false positives, PAINS