Substrate-selective small-molecule modulators of enzymes: Mechanisms and opportunities

Abstract

Small-molecule inhibitors of enzymes are widely used tools in reverse chemical genetics to probe biology and explore therapeutic opportunities. They are often compared with genetic knockdown or knockout and are expected to produce phenotypes similar to the genetic perturbations. This review aims to highlight that small molecule inhibitors of enzymes and genetic perturbations may not necessarily produce the same phenotype due to the possibility of substrate-selective or substrate-dependent effects of the inhibitors. Examples of substrate-selective inhibitors and the mechanisms for the substrate-selective effects are discussed. Substrate-selective modulators of enzymes have distinct advantages and cannot be easily replaced with biologics. Thus, they present an exciting opportunity for chemical biologists and medicinal chemists.

Keywords: COX-2; Chemical genetics; Gamma-secretase; Insulin degrading enzyme; Reverse chemical genetics; SIRT2; SIRT6; Sirtuins; Substrate-dependent inhibition; Substrate-selective inhibition.

Figure 1.

Scheme showing the concepts of forward and reverse genetics as well as forward and reverse chemical genetics. The topic of this review is more related to reverse genetics and reverse chemical genetics. The juxtaposition of reverse genetics and reverse chemical genetics make it easy to equalize the effect of small molecule inhibitors to that of genetic knockdown or knockout. This review emphasizes that this is not necessarily the case and small molecule inhibitors of proteins may produce phenotypes that are different from that of genetic knockout/knockdown.

Figure 2.

Exosite binding leading to substrate-selective inhibition. (A) Cartoon showing how targeting an exosite could lead to substrate-selective inhibition of an enzyme. An inhibitor (blue triangle) targeting the exosite of the shown enzyme selectively blocks substrate 2 (gold) from binding, leading to the selective inhibition of the enzyme on substrate 2. (B) Comparison of human IDE structures with inhibitor 63 (cyan) and glucagon (purple) bound (PDB 6EDS) and IDE with insulin (gold) bound (PDB 2WBY) shows that inhibitor 63 binds to a site that does not clash with glucagon, but would clash with insulin. Inhibitor 63 in the IDE-insulin structure on the right is from the superimposed PDB 6EDS structure.

Figure 3.

Allosteric site binding leading to substrate-selective inhibition. (A) Cartoon showing how targeting an allosteric site could lead to substrate-selective inhibition of an enzyme. An inhibitor binding to the allosteric site of the shown enzyme lead to slight changes in the active site and this structural change selectively blocks substrate 1 from binding, while promotes or does not inhibit substrate 2 from binding/reacting. (B) Superimposed mTOR structures from PDB 5FLC (mTORC1 complex with rapamycin and FKBP12) and 4JSX (truncated mTOR with mLST8 and Torin-2) showing that rapamycin binds to an allosteric site that is distinct from the mTOR kinase active site that is occupied by Torin-2. (C) Superimposed SIRT6 structures from PDB 3ZG6 (SIRT6 with ADP-ribose and myritoyl-lysine substrate) and 6XVG (SIRT6 with MDL-801) showing that MDL-801 occupies the myristoyl binding pocket, which inhibits myritoyl-lysine substrate from binding to SIRT6, but promote acetyl-lysine substrate binding to SIRT6. The MDL-801 binding site can thus be considered an allosteric site for the acetyl-lysine substrate and an exosite for myristoyl-lysine. ADPR: ADP-ribose. Myr-Lys: myristoyl-lysine.

Figure 4.

Cartoon showing how active-site targeting inhibitors could lead to substrate-selective inhibition of an enzyme. An inhibitor binding to the active site of the shown enzyme prevent the low-affinity substrate from binding, but does not prevent the high-affinity substrate from binding to the enzyme, leading to selective inhibition of the low affinity substrate.

Figure 5.

Substrate-selective inhibition due to interaction of inhibitor and substrates at the enzyme active site. (A) Cartoon showing how active-site targeting modulators could have different interactions with substrates, leading to substrate-selective inhibition of an enzyme. The modulator competes with substrate 1 and promote substrate 2. (B) A structure of CYP3A4 in complex with a testosterone covalent dimer (PDB 7LXL) showing that the active site is large, which could allow substrate and inhibitor binding at the same time and thus enable substrate-inhibitor interaction that could affect catalysis in a substrate-dependent manner.