I’ll start this post by with reference to a disease that some of you many never have heard of. Chagas disease is caused by the very nasty T. cruzi parasite (not to be confused with the even nastier American politician) and is of particular interest in Latin America where the disease is endemic. T. cruzi parasites have an essential requirement for ergosterol and, as discussed in C2010, are potentially vulnerable to inhibition of sterol 14α-demethylase (CYP51), which catalyzes the conversion of lanosterol to ergosterol. However, the CYP51 inhibitor posaconazole (an antifungal medication) showed poor efficacy in a clinical trials for chronic Chagas disease. Does this mean that CYP51 is a bad target? The quick answer is “maybe but maybe not” because we can’t really tell whether the lack of efficacy is due to irrelevance of the target or inadequate exposure.
We commonly invoke the free drug hypothesis (FDH) in drug design which means that we assume that the free concentration at the site of action is the same as the free plasma concentration (the term ‘free drug theory’ is also commonly used although I prefer FDH). The FDH is covered in the S2010 (see Box 1 and 2) and B2013 articles and, given that the targets of small molecule drugs tend to be intracellular, I’ll direct you to the excellent Smith & Rowland perspective on intracellular and intraorgan concentrations of drugs. When we invoke the FDH we’re implicitly assuming that the drug can easily pass through barriers, such as the lipid bilayers that enclose cells, to get to the site of action. In the absence of active transport, the free concentration at the site of action of a drug will tend to lag behind the free plasma concentration with the magnitude of the lag generally decreasing with permeability. Active transport (which typically manifests itself as efflux) is a more serious problem from the design perspective because it leads to even greater uncertainty in the free drug concentration at the site of action and it’s also worth remembering that transporter expression may vary with cell type. It’s worth mentioning that uncertainty in the free concentration at the site of action is even greater when targeting intracellular pathogens, as is the case for Chagas disease, malaria and tuberculosis.
Some may see chemical probes as consolation prizes in the drug discovery game and, while this may sometimes be the case, we really need to be thinking of chemical probes as things that need to be designed. As is well put in “A conversation on using chemical probes to study protein function in cells and organisms” that was recently published in Nature Communications:
“But drugs are different from chemical probes. Drugs don’t necessarily need to be as selective as high-quality chemical probes. They just need to get the job done on the disease and be safe to use. In fact, many drugs act on multiple targets as part of their therapeutic mechanism.”
High selectivity and affinity are clear design objectives and, to some extent, optimization of affinity will tend to lead to higher selectivity. High quality chemical probes for intracellular targets need to be adequately permeable and should is should not be subject to active transport. The problems caused by active efflux are obvious because chemical probes need to get into cells in order to engage intracellular targets but there’s another reason that adequate permeability and minimal active transport are especially important for chemical probes. In order to interpret results, you need to know the free concentration of the probe at the site of action and active transport, whether it manifests itself as efflux or influx, leads to uncertainty the intracellular free concentration. Although it may be possible to measure intracellular free concentration (see M2013) it’s fiddly to do so if you’re trying to measure target engagement at the same time and it’s not generally possible to do so in vivo. It's much better to be in a position to invoke the FDH with confidence and this point is well made in the Smith and Rowland perspective:
“Many misleading assumptions about drug concentrations and access to drug targets are based on total drug. Correction, if made, is usually by measuring tissue binding, but this is limited by the lack of homogenicity of the organ or compartment. Rather than looking for technology to measure the unbound concentration it may be better to focus on designing high lipoidal permeable molecules with a high chance of achieving a uniform unbound drug concentration.”
If the intention is to use a chemical probe for in vivo studies then you’ll need to be confident that adequate exposure at the site of action can be achieved. My view is that it would be difficult to perform a meaningful assessment of the suitability of a chemical probe for in vivo studies without relevant experimental in vivo measurements. You might, however, be able to perform informative in vivo experiments with a chemical probe in the absence of existing pharmacokinetic measurements (provided that you monitor plasma levels and know how tightly the probe is bound by plasma proteins) although you’ll still need to invoke the FDH for intracellular targets.
If you’re only going to use a chemical probe in cell-based experiments then you really don’t need to worry about achieving oral exposure and this has implications for probe design. The requirement for a chemical probe to have acceptable pharmacokinetic characteristics imposes constraints on design (which may make it more difficult to achieve the desired degree of selectivity) while pharmacokinetic optimization is likely to consume significant resources. As is the case for chemical probes intended for in vivo use, you’ll want to be in a position to invoke the FDH.
In this post, I’ve argued that you need to be thinking very carefully about passive permeability and active transport (whether it leads to efflux or influx) when designing, using or assessing chemical probes. In particular, having experimental measurements available that show that a chemical probe exhibits acceptable passive permeability and is not actively transported will greatly increase confidence that the chemical probe is indeed fit for purpose. It’s not my intention to review methods for measuring passive permeability or active transport in this post although I’ll point you to the B2018, S2021, V2011 and X2021 articles in case any of these are helpful.
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