It’s now time to get back to chemical probes and I’ll be taking a look at S2023 (Systematic literature review reveals suboptimal use of chemical probes in cell-based biomedical research) which has already been reviewed in blog posts from Practical Fragments, In The Pipeline and the Institute of Cancer Research. Readers of this blog are aware that PAINS filters usually crop up in posts on chemical probes but there are other things that I want to discuss and, in any case, references to PAINS in S2023 are minimal. Nevertheless, I’ll still stress that a substructural match of a chemical probe with a PAINS filter does not constitute a valid criticism of a chemical probe (it simply means that the chemical structure of the chemical probe shares structural features with compounds that have been claimed to exhibit frequent-hitter behaviour in a panel of six AlphaScreen assays) and one is more likely to encounter a bunyip than a compound that has actually been shown to exhibit pan-assay interference.
The authors of S2023 claim to have revealed “suboptimal use of chemical probes in cell-based biomedical research” and I’ll start by taking a look at the abstract (my annotations are italicised in red):
Chemical probes have reached a prominent role in biomedical research, but their impact is governed by experimental design. To gain insight into the use of chemical probes, we conducted a systematic review of 662 publications, understood here as primary research articles, employing eight different chemical probes in cell-based research. [A study such as S2023 that has been claimed by its authors to be systematic does need to say something about how the eight chemical probes were selected and why the literature for this particular selection of chemical probes should be regarded as representative of chemical probes literature in general.] We summarised (i) concentration(s) at which chemical probes were used in cell-based assays, (ii) inclusion of structurally matched target-inactive control compounds and (iii) orthogonal chemical probes. Here, we show that only 4% of analysed eligible publications used chemical probes within the recommended concentration range and included inactive compounds as well as orthogonal chemical probes. [I would argue that failure to use a chemical probe within a recommended concentration range is only a valid criticism if the basis for the recommendation is clearly articulated.] These findings indicate that the best practice with chemical probes is yet to be implemented in biomedical research. [My view is that the best practice with chemical probes is yet to be defined.] To achieve this, we propose ‘the rule of two’: At least two chemical probes (either orthogonal target-engaging probes, and/or a pair of a chemical probe and matched target-inactive compound) to be employed at recommended concentrations in every study. [The authors of S2023 do seem to moving the goalposts since the they’ve criticized studies for not using structurally matched target-inactive control compounds but are saying that using an additional orthogonal target-engaging probe makes it acceptable not to use a structurally matched target-inactive control compound. This suggestion does appear to contradict the Chemical Probes Portal criteria for 'classical' modulators which do require the use of a control compound defined as having a "similar structure with similar physicochemistry, non-binding against target".]
The following sentence does set off a few warning bells for me:
The term ‘chemical probe’ distinguishes compounds used in basic and preclinical research from ‘drugs’ used in the clinic, from the terms ‘inhibitor’, ‘ligand’, ‘agonist’ or ‘antagonist’ which are molecules targeting a given protein but are insufficiently characterised, and also from the term ‘probes’ which is often referring to laboratory reagents for biophysical and imaging studies.
First, the terms 'compound' and 'molecule' are not interchangeable and I would generally recommend using 'compound' when talking about biological activity or affinity. A more serious problem is that the authors of S2023 seem to be getting into homeopathic territory by suggesting that chemical probes are not ligands and this might have caused Paul Ehrlich (who died 26 years before Kaiser Wilhelm II) to spit a few feathers. Drugs and chemical probes are ligands for their targets by virtue of binding to their targets (the term 'ligand' is derived from the Latin 'ligare' which means 'to bind' and a compound can be a ligand for one target without necessarily being a ligand for another target) while the terms 'inhibitor', 'agonist' and 'antagonist' specify the consequences of ligand binding. I was also concerned by the use of the term 'in cell concentration' in S2023 given that uncertainty in intracellular concentration is an issue when working with chemical probes (as well as in PK-PD modelling). Although my comments above could be seen as nit-picking these are not the kind of errors that authors can afford to make if they’re going to claim that their “findings indicate that the best practice with chemical probes is yet to be implemented in biomedical research”.
Let’s take a look at the criteria by which the authors of S2023 have assessed the use of chemical probes. They assert that “Even the most selective chemical probe will become non-selective if used at a high concentration” although I think it’d be more correct to state that the functional selectivity of a probe depends on binding affinity of the probe for target and anti-targets as well as the concentration of the probe (at its site of action). Selectivity also depends on the concentration of anything that binds competitively with the probe and, when assessing kinase selectivity, it can be argued that assays for ATP-competitive kinase inhibitors should be run at a typical intracellular ATP concentration (here’s a recent open access review on intracellular ATP concentration). The presence of serum in cell-based assays should also be considered when setting upper concentration limits since chemical probes may bind to serum proteins such as albumin which means that the concentration of a compound that is ‘seen’ by the cells is lower than the total concentration of the compound in the assay. In my experience binding to albumin tends to increase with lipophilicity and is also favored by the presence of an acidic group such as carboxylate in a molecular structure.
I’m certainly not suggesting that chemical probes be used at excessive concentrations but if you’re going to criticise other scientists for exceeding concentration thresholds then, at very least, you do need to show that the threshold values have been derived in an objective and transparent manner. My view that it would not be valid to criticise studies publicly (or in peer review of submitted manuscripts) simply because the studies do not comply with recommendations made by the Chemical Probes Portal. It is significant that the recommendations from different groups of chemical probe experts with respect to the maximum concentration at which UNC1999 should be used differ by almost an order of magnitude:
As the recommended maximal in-cell concentration for UNC1999 varies between the Chemical Probes Portal and the Structural Genomics Consortium sites (400 nM and 3 μM, respectively), we analysed compliance with both concentrations.
One of the eight chemical probes featured in S2023 is THZ1 which is reported to bind covalently to CDK7 and the electrophilic warhead is acrylamide-based, suggesting that binding is irreversible. Chemical probes that form covalent bonds with their targets irreversibly need to be considered differently to chemical probes that engage their targets reversibly (see this article). Specifically, the degree of target engagement by a chemical probe that binds irreversibly depends on time as well as concentration (if you wait long enough then you’ll achieve 100% inhibition). This means that it’s not generally possible to quantify selectivity or to set concentration thresholds objectively for chemical probes that bind to their targets irreversibly. It’s not clear (at least to me) why an irreversible covalent inhibitor such as THZ1 was included as one of the eight chemical probes covered by the S2023 study so I checked to see what the Chemical Probes Portal had to say about THZ1 and something doesn’t look quite right. The on-target potency is given as a Kd (dissociation constant which is a measure of affinity) value of 3.2 nM and the potency assay is described as “time-dependent binding established supporting covalent mechanism”. However, Kd is a measure of affinity (and therefore not a time-dependent) and my understanding is that it is generally difficult to measure Kd for irreversible covalent inhibitors which are typically characterized by kinact (inactivation rate constant) and Ki (inhibition constant) values obtained from analysis of enzyme inhibition data. The off-target potency of THZ1 is summarized as “KiNativ profiling against 246 kinases in Loucy cells was performed showing >75% inhibition at 1 uM of: MLK3, PIP4K2C, JNK1, JNK2, JNK3, MER, TBK1, IGF1R, NEK9, PCTAIRE2, and TBK1, but in vitro binding to off-target kinases was not time dependent indicating that inhibition was not via a covalent mechanism”. The results from the assays used to measure on-target and off-target potency for THZ1 do not appear to be directly comparable.
It’s now time to wrap up and I suggest that it would not be valid to criticise (either publicly or in peer review) a study simply on the grounds that it reported results of experiments in which a chemical probe was used at a concentration exceeding a recommended maximum value. The S2023 authors assert that an additional orthogonal target-engaging probe can be substituted for a matched target-inactive control compound but this appears to contradict criteria for classical modulators given by the Chemical Probes Portal.