PPIs, protein pocket predictions and some endogenous allosteric modulators

A simplified format for this and likely subsequent blogs from my literature surveys. which I hope you will find useful.  This blog covers a number of topics relating to protein protein interactions

Discovery of Tricyclic Indoles That Potently Inhibit Mcl-1 Using Fragment-Based Methods and Structure-Based Design Jason P. Burke, et al J. Med. Chem., Article ASAP, DOI: 10.1021/jm501984f, Publication Date (Web): April 17, 2015. Copyright © 2015, American Chemical Society

A nice story describing the elaboration of a micromolar tricyclic indole MCL-1 inhibitor to a low nanomolar compound as a follow up to earlier work from the same group highlighting another tractable PPI target

Solvent Accessible Surface Area-Based Hot-Spot Detection Methods for Protein–Protein and Protein–Nucleic Acid Interfaces Cristian R. Munteanu, António et al J. Chem. Inf. Model., Article ASAP , DOI: 10.1021/ci500760m, Publication Date (Web): April 17, 2015, Copyright © 2015, American Chemical Society http://pubs.acs.org/doi/abs/10.1021/ci500760m

Keeping with protein-protein interactions is another method for identifying hot-spots for protein-protein and protein-nucleic acid interactions. A couple of web based tools are provided for respectively predicting protein nucleic acid and protein-protein interactions

PockDrug: A Model for Predicting Pocket Druggability That Overcomes Pocket Estimation Uncertainties Alexandre Borrel, Leslie et al Camproux J. Chem. Inf. Model., Article ASAP DOI: 10.1021/ci5006004 Publication Date (Web): April 16, 2015, Copyright © 2015, American Chemical Society,

Predicting druggability of a pocket of a protein is a tricky business as highlighted in this report outlining a new method which appears to be more accurate than existing methods. The tool PockDrug which is reported to be better able to cope with uncertainty in pocket boundaries and highlights the importance of getting the estimate of a pocket to give improved druggability prediction.

Endogenous Allosteric Modulators of G Protein–Coupled Receptors Emma T. van der Westhuizen, Celine Valant, Patrick M. Sexton, and Arthur Christopoulos J Pharmacol Exp Ther May 2015 353:246-260; published ahead of print February 3, 2015, doi:10.1124/jpet.114.221606 http://jpet.aspetjournals.org/content/353/2/246.abstract

Finally a report looking at endogenous allosteric modulators of GPCR’s a topic that perhaps doesn’t get the recognition it deserves and arguably relates to the PPI topics already discussed. After all many PPI’s are regulating the activity of other proteins via what is effectively an allosteric interaction which then begs the question how many small molecule allosteric modulators are in fact acting at PPI sites


More screening PAIN

Another valuable contribution form the Monash group on PAINS 1 – frequent hitters in screening assays. This time aminothiazoles are in the spot light – those favourites of early parallel synthesis aficionados with the mix of alpha-bromomethylketone and substituted thiourea. The authors observed issues with this motif in fragment libraries and in conventional screening decks – not necessarily that they were really bad just that they did hit frequently and were challenging to attempt to optimise – best steer clear.

In another paper 2 where the authors were screening for HDAC inhibitors using a thiol trap fluorescence read-out several series of compounds were highlighted showing activity due to thiol trapping which had not been detected with their cheminformatics filters prior to running the assay.  Compounds tended to show non-specific thiol trapping, furthermore these same compounds were shown to have activity across a range for targets (Pubchem) and unfortunately had been reported as tools for other targets. This paper is sumamrised nicely by Erlanson 3 with additional commentary on PAINS compounds

  1. S. M. Devine et al J. Med. Chem., Article ASAP DOI: 10.1021/jm501402x Publication Date (Web): January 16, 2015 Copyright © 2015, American Chemical Society
  2. J. L. Dahlin, et al, J. Med. Chem., Article ASAP DOI: 10.1021/jm5019093 Publication Date (Web): February 21, 2015 Copyright © 2015, American Chemical Society
  3. D. A. Erlanson J. Med. Chem., Article ASAP, DOI: 10.1021/acs.jmedchem.5b00294, Publication Date (Web): February 24, 2015 Copyright © 2015, American Chemical Society

A different take on kinase inhibitors and selective inhibition of substrate phosphorylation

An article 1 that I really found fascinating and potentially with substantial consequences in the way we think about the “activity” of compounds and the way we set up biological screens is the work from AZ discussing selective inhibitors of kinase substrate activation. Specifically the AZ team have identified ATP competitive inhibitors of p38 kinase, which as with many kinases has multiple substrates, that selectivity inhibit the phosphorylation of one substrate over another. Compounds from two chemical series were identified and the authors showed that the substrate selectivity was due to a specific polar interaction. The key to identifying these compounds was the use of the endogenous target and off-target substrates in the primary screen rather than an artificial substrate that virtually everyone else in the field has been using.  I am not aware of this approach being used previously but will be happy to be proved wrong.

  1. J. G. Cumming et al J. Med. Chem., 2015, 58 (1), 278–293

Aryl halides

A couple of papers looking at different aspects of halogens in drug discovery. Firstly 1 the frequency of para-substituted chlorophenyl compounds both in commercial reagents and in marketed drugs is disproportionately high relative to ortho- or meta- substitution. The authors speculate that the high frequency of para-chlorophenyl in drugs may reflect both reagent availability and the use of Topliss decision tree strategies for substituent selection. Interestingly meta-substitution is quite rare while ortho is comparatively ore common at about 25% the frequency of a para-chlorosubstituent certainly conformational effects will be playing a big role in this. Of course introducing an ortho-substituent is often the reaction that fails in array chemistry requiring bespoke synthesis.

Halogen bonding is getting an increasing profile thus scoring functions to recognise opportunities for halogen bonding are valuable 2. Perhaps an ortho chloro substituent has greater difficulty in achieving a good alignment for a robust interaction although that doesn’t explain the infrequent occurrence of meta-substituted arylchlorides.

  1. Understanding Our Love Affair with p-Chlorophenyl: Present Day Implications from Historical Biases of Reagent Selection Dean G. Brown, Moriah M. Gagnon and Jonas Boström J. Med. Chem., Article ASAP DOI: 10.1021/jm501894t Publication Date (Web): February 19, 2015 Copyright © 2015, American Chemical Society http://pubs.acs.org/doi/abs/10.1021/jm501894t
  2. Evaluating the Potential of Halogen Bonding in Molecular Design: Automated Scaffold Decoration Using the New Scoring Function XBScore Markus O. Zimmermann, Andreas Lange and Frank M. Boeckler J. Chem. Inf. Model., Article ASAP DOI: 10.1021/ci5007118 Publication Date (Web): February 19, 2015 Copyright © 2015, American Chemical Society, http://pubs.acs.org/doi/abs/10.1021/ci5007118


Redox potentials and drug discovery

A paper just out, that set me thinking a bit about are we missing a trick or two, discusses redox potentials of hydroxycinnamic acids such as caffeic and ferulic acid this are of particular current interest in health because of their anti-oxidant activity. It’s estimated that this class may constitute one third of the phenolic compounds in our diet. The paper determines redox potentials for a range of these acids alongside their antioxidant activity showing that they correlate with each other and allows development of structure property activity relationships.

All this is well and good assuming stuffing ourselves with anti-oxidants really is a good thing, though I will keep drinking the red wine just in case, but what about use of redox potentials more generally in drug design – are we missing a trick. My past experience and from what I see in the literature I generally read is very little use of the technique to help understand how our molecules will behave – when I tried to get some redox potentials measured while working in big pharma I was told the kit was no longer available – note the no longer. I guess where I am coming from is can we use measured redox potentials as part of our understanding of improving stability of molecules in aggressive redox environments particularly within metabolic enzymes. A quick look on google did pick up this thesis sponsored by Roche looking at developing high throughput redox measurement techniques. The starting point they come from is that redox potentials can be considered equivalents of ionisation potentials. So could we be looking at some sort of cascade of calculated ionisation potentials during in silico design followed by redox potentials after synthesis and who knows perhaps exploit electrochemistry during synthesis another under used area perhaps? It would be good to hear people’s thoughts on this.


Dimorphism matters

While I suspect most people working in drug discovery are aware of gender differences in the way drugs can behave in the clinic, dimorphism in cells is perhaps less well recognised. A Nature comment from Elizabeth Pollitzer highlights the importance of recording the gender of cells in experiments citing examples of differences in response of muscle derived stem cells from male and female subjects and responses of cells to stress. Dr Pollitzer also notes that ten prescription drugs were withdrawn between 1997 and 2001 due to gender differences in adverse events a figure which did surprise me a little. While hormonal differences can explain some differences in both exposure and efficacy/safety others have been attributed to differences in metabolic pathways.

Thinking within the context of a drug screening cascade at what stage should one be checking for dimorphism –Dr Pollitzer recommends ideally running separate cell based studies from both males and females which seems very reasonable. Perhaps (and admittedly much more demanding) does this need extending to the in vivo situation running male/female PK/PD, on late leads, at least where other evidence suggests this may be appropriate. From a metabolic perspective, one thing I am not clear about with microsomal studies – are pooled microsomes or hepatocytes pooled from both male and female or from single gender. Will we see drugs being developed which only achieve exposure/efficacy in one gender – clearly I am not considering treatments for more obviously gender specific diseases here?  Perhaps also we may see an improvement of reproducibility of experiments from the literature if (again as Dr Pollitzer urges) journal editors and referees insist on the reporting of the source gender of cells

Finally in similar vein reinforcing the importance of recognising and understanding gender differences two EU reports. The first on gendered innovations that considers where there may be opportunities for innovation based on gender biases in population analysis – eg in cardiovascular disease historically it has been mainly males that have been studied – is this information as relevant to the female as the male? The second I guess reinforces the first by emphasising that the absence of thought about dimorphism in running and reporting of experiments is a bigger concern than any funding or recruitment gender inequities.


Chronobiology, chronotoxicity, chronoefficacy!

Just picked up this article on Wee1 expression in the circadian rhythm dependent intestinal damage induced by docetaxel from Fujimura’s group which identifies increased expression of Wee1 (controlled by the clock gene complex clock/bmal), phosphorylated CDK1, and cleaved Caspase-3 and lower levels of survivin in animals dosed with docetaxel 14 hours after lights on (HALO) compared with those dosed 2 HALO. The 14 HALO group showed increased intestinal damage compared with the 2 HALO group although the mechanistic relationship of the changes in protein expression and intestinal damage is unclear. Drug exposures were the same from the two dosing protocols. It has previously been suggested that docetaxel is more efficacious when dosed during the light period time point while another recent report highlighted the circadian nature of toxicity and efficacy of oxaliplatin. The idea of chronotherapy is, of course, not new with various reviews written on the field . Finally chronotoxicity as determined by MTD and efficacy for celecoxib is discussed with respect to efficacy in a breast cancer mouse xenograft model I found the results quite impressive.

However, this did set me thinking a few thoughts. Do we as research scientists consider sufficiently the timing of drug dosing either for efficacy or for minimising side effects either preclinically or clinically? Is this a variable that needs tighter control in the preclinical setting? What are the consequences on drug efficacy for those who suffer from disrupted sleep patterns (be it due to ill health, life style or required work patterns)? Could co-administration (or with a 12hr off-set) of an appropriate hypnotic (and its not entirely clear what would be an appropriate hypnotic) or improved sleep hygiene improve efficacy of some therapeutics?

Selective versus Specific

Am I the only one who worries about what might be a trivial point except for the ability to mislead when the word specific is used to describe how selective a compound is.  Specific to me implies that said compound interacts with one target and one target only – this we all know is nonsense.  At best a compound shows good selectivity (greater than 100 fold, greater than 1000 fold – whatever you might choose to define selective) against the targets it has been tested against and that of course assumes the assays can really be compared in such a simple way – is radioligand binding  a good measure of selectivity – what about the kinetics at target, functional efficacy etc.  Anyway back to selective v. specific the danger as I see it is when a compound gets described as “specific” any pharmacology seen with that compound is then ascribed to interaction with that target – which may be true but there again may not thus we can end up with a lot of fairly misleading literature.  Of course the worst case is when a compound is shown to have weak interaction against one target, tested against no another target and promptly described as specific – you will have all seen examples.

Perhaps journal editors and referee’s should ban the word “specific” from any article discussing the pharmacological profile of any compound – or am I being just being an unfair nit-picker!

Non-covalent hydrophobic tags and protein degradation

An article that recently caught my eye set me thinking about the liabilities of hydrophobicity – where a ligand binds to a protein, leaving a lipophilic motif exposed to cytosol. Long et al in Chemistry and Biology 2012, 19, 629 (see also Neklesa and Crews, Nature 2012, 487, 308  discuss the role of lipophilic motifs binding non-covalently to proteins, which directs the protein to the proteasome for degradation. Specifically, the authors investigated the binding of (Boc)3Arg conjugated Trimethoprim to DHFR and the resultant reduction of DHFR levels in mammalian cells. The effect was blocked by Trimethoprim or by proteasome inhibitors. Ethacrynic acid, a covalent inhibitor of GST, also induced degradation of GST when tagged with Boc3Arg.

The main thrust of the articles is in the context of a therapeutic strategy, but arguably, in the case of off-target proteins, could unanticipated degradation due to “tagging” by a drug result in toxic events possibly only manifested over the long term, if the lipophilicity enhanced degradation is modest?

While the authors discuss ligands with a Boc3Arg motif, it is not a huge leap to think of, for example, drugs with lipophilic biaryl groups that become exposed, on binding of a polar head group, to an off-target protein, with consequent demolition by the proteasome.

If this is truly physiologically relevant, can the (unwanted) degradation phenomenon be reduced with a more uniform distribution of polarity in a drug molecule? Or, put another way, is it better to have a molecule of lipophilicity X with a reasonably uniform surface distribution of polarity, or a compound with the same lipophilicity but a non-uniform surface distribution of polarity? Intuitively I would prefer the former, but if anyone has seen any analysis of the distribution of polarity and developability, I would love to see it. Given that lipophilicity seems to be discussed as a global phenomenon for a molecule, perhaps considering local lipophilicity may be useful as well.

A couple of additional relevant articles related to covalent binding of a lipophilic tag and proteasome degradation are Neklesa et al Nature Chem. Biol., 2011, 7, 538 and H. S. Tae et al ChemBioChem 2012, 13, 538