AI-Based Discovery and CryoEM Structural Elucidation of a K_ATP Channel Pharmacochaperone

Reviewer #1 (Public review):

Summary:

Multiple compounds that inhibit ATP-sensitive potassium (KATP) channels also chaperone channels to the surface membrane. The authors used an artificial intelligence (AI)-based virtual screening (AtomNet) to identify novel compounds that exhibit chaperoning effects on trafficking-deficient disease-causing mutant channels. One compound, which they named Aekatperone, acts as a low affinity, reversible inhibitor and effective chaperone. A cryoEM structure of KATP bound to Aekatperone showed that the molecule binds at the canonical inhibitory site.

Strengths and weaknesses:

The details of the AI screening itself are inevitably opaque but appear to differ from classical virtual screening in not involving any physical docking of test compounds into the target site. The authors mention criteria that were used to limit the number of compounds so that those with high similarity to known binders and 'sequence identity' (does this mean structural identity) were excluded. The identified molecules contain sulfonylurea-like moieties. How different are they from other sulfonylure4as?

The experimental work confirming that Aekatperone acts to traffic mutant KATP channels to the surface and acts as a low affinity, reversible, inhibitor is comprehensive and clear, with very convincing cell biological and patch-clamp data, as is the cryoEM structural analysis, for which the group are leading experts. In addition to the three positive chaperone-effective molecules, the authors identified a large number of compounds that are predicted binders but apparently have no chaperoning effect. Did any of them have an inhibitory action on channels? If so, does this give clues to separating chaperoning from inhibitory effects?

The authors suggest that the novel compound may be a promising therapeutic for the treatment of congenital hyperinsulinism due to trafficking defective KATP mutations. Because they are low-affinity, reversible, inhibitors. This is a very interesting concept, and perhaps a pulsed dosing regimen would allow trafficking without constant channel inhibition (which otherwise defeats the therapeutic purpose), although it is unclear whether the new compound will offer advantages over earlier low-affinity sulfonylurea inhibitor chaperones. These include tolbutamide which has very similar affinity and effect to Aekatperone. As the authors point out this (as well as other sulfonylureas) is currently out of favor because of potential adverse cardiovascular effects, but again, it is unclear why Aekatperone should not have the same concerns.

Reviewer #2 (Public review):

Summary:

In their study 'AI-Based Discovery and CryoEM Structural Elucidation of a KATP Channel Pharmacochaperone', ElSheikh and colleagues undertake a computational screening approach to identify candidate drugs that may bind to an identified binding pocket in the SUR1 subunit of KATP channels. Other KATP channel inhibitors such as glibenclamide have been previously shown to bind in this pocket, and in addition to inhibition of KATP channel function, these inhibitors can very effectively rescue cell surface expression of trafficking deficient KATP mutations that cause excessive insulin secretion (Congenital Hyperinsulinism). However, a challenge for their utility for the treatment of hyperinsulinism has been that they are powerful inhibitors of the channels that are rescued to the channel surface. In contrast, successful therapeutic pharmacochaperones (eg. CFTR chaperones) permit the function of the channels rescued to the cell membrane. Thus, a key criterion for the authors' approach, in this case, was to identify relatively low-affinity compounds that target the glibenclamide binding site (and be washed off) - these could potentially rescue KATP surface expression but also permit KATP function.

Strengths:

The main findings of the manuscript include:

(1) Computational screening of a large virtual compound library, followed by functional screening of cell surface expression, which identified several potential candidate pharmacochaperones that target the glibenclamide binding site.

(2) Prioritization and functional characterization of Aekatperone as a low-affinity KATP inhibitor which can be readily 'washed off' in patch clamp and cell-based efflux assays. Thus the drug clearly rescues cell surface expression but can be manipulated experimentally to permit the function of rescued channels.

(3) Determination of the binding site and dynamics of this candidate drug by cryo-EM, and functional validation of several residues involved in drug sensitivity using mutagenesis and patch clamp.

The experiments are well-conceived and executed, and the study is clearly described. The results of the experiments are very straightforward and clearly support the conclusions drawn by the authors. I found the study to provide important new information about the KATP chaperone effects of certain drugs, with interesting considerations in terms of ion channel biology and human disease.

Weaknesses:

I don't have any major criticisms of the study as described, but I had some remaining questions that could be addressed in a revision.

(1) The chaperones can effectively rescue KATP trafficking mutants, but clearly not as strongly as the higher affinity inhibitor glibenclamide. Is this relationship between inhibitory potency, and efficacy of trafficking an intrinsic challenge of the approach? I suspect that it may be an intractable problem in the sense that the inhibitor-bound conformation that underlies the chaperone effect cannot be uncoupled from the inhibited gating state. But this might not be true (many partial agonist drugs with low efficacy can be strongly potent, for example). In this case, the approach is really to find a 'happy medium' of a drug that is a weak enough inhibitor to be washed away, but still strong enough to exert some satisfactory chaperone effect. Could some additional clarity be added in the discussion on whether the chaperone and gating effects can be 'uncoupled'?

(2) Based on the western blots in Figure 2B, the rescue of cell surface expression appears to require a higher concentration of AKP compared to the concentration-response of channel inhibition (~9 microM in Figure 3, perhaps even more potent in patch clamp in Figure 2C). Could the authors clarify/quantify the concentration response for trafficking rescue?

(3) A future challenge in the application of pharmacochaperones of this type in hyperinsulinism may be the manipulation of chaperone concentration in order to permit function. In experiments, it is straightforward to wash off the chaperone, but this would not be the case in an organism. I wondered if the authors had attempted to rescue channel function with diazoxide in the presence of AKP, rather than after washing off (ie. is AKP inhibition insurmountable, or can it be overcome by sufficient diazoxide).

(4) Do the authors have any information about the turnover time of KATP after the wash-off of the chaperone (how stable are the rescued channels at the cell surface)? This is a difficult question to probe when glibenclamide is used as a chaperone, but may be much simpler to address with a lower affinity chaperone like AKP.