Discovering new biology with drug-resistance alleles

Abstract

Small molecule drugs form the backbone of modern medicine's therapeutic arsenal. Often less appreciated is the role that small molecules have had in advancing basic biology. In this Review, we highlight how resistance mutations have unlocked the potential of small molecule chemical probes to discover new biology. We describe key instances in which resistance mutations and related genetic variants yielded foundational biological insight and categorize these examples on the basis of their role in the discovery of novel molecular mechanisms, protein allostery, physiology and cell signaling. Next, we suggest ways in which emerging technologies can be leveraged to systematically introduce and characterize resistance mutations to catalyze basic biology research and drug discovery. By recognizing how resistance mutations have propelled biological discovery, we can better harness new technologies and maximize the potential of small molecules to advance our understanding of biology and improve human health.

Fig. 1 |. Resistance mutations identify small molecule targets and reveal new biology.

a, Schematic illustrating the themes outlined in this piece. PDB: 5HXB, 5MO4. b, Top, chemical structure of rapamycin. Bottom, structural view of the ternary complex containing rapamycin (yellow), Homo sapiens FKBP (gray) and the H. sapiens FRB domain of mTOR (green). The FKBP residues homologous to those identified in Heitman et al. are shown in blue. PDB: 1FAP. c, Left, chemical structure of indisulam. Right, structural view of the ternary complex containing an analog of indisulam (E7820, yellow), RBM39 (gray) and DCAF15 (green). The RBM39 residues identified in Han et al. are shown in blue. PDB: 6Q0R.

Fig. 2 |. Resistance mutations unveil protein structural information and mechanisms of protein allosteric regulation.

a, Top, chemical structure of TTX. Bottom, structural view of TTX (yellow) engaging the Periplaneta americana Na_vPaS channel (gray). The Na_vPaS residues homologous to those identified as resistance mutations to TTX in Noda et al. and Terlau et al. are shown in blue^,. These residues are responsible for the selectivity of the channel for sodium over other cations. PDB: 6A95. b, Left, chemical structure of imatinib. Right, a structural view of ABL kinase is shown with resistance mutations identified by Azam et al. highlighted (red, active residues; blue, distal site residues). PDB: 5MO4. c, Left, chemical structure of palbociclib. Right, a structural view of CDK6 kinase is shown with resistance mutations identified by Persky et al. highlighted (red, active site residues; blue, distal site residue). PDB: 2EUF.

Fig. 3 |. Resistance mutations elucidate physiology and provide insight into species differences in small molecule mechanism of action.

a, Left, chemical structure of alprazolam. Right, structural view of alprazolam (yellow) engaging the H. sapiens GABA_A receptor (gray). The resistance mutation used in Rudolph et al. is shown in blue. PDB: 6HUO. b, Brain regions expressing α₁ and α₂ GABA_A receptor subtypes responsible for the physiological effects of benzodiazepine action. The location of the α₁ subtype receptors in the thalamus and cortex are highlighted in blue, whereas the location of the α₂ subtype receptors in the limbic system are highlighted in red. c, Left, chemical structure of abacavir. Right, structural view of abacavir binding the protein encoded by the HLA-B*57:01 allele. Sites of natural genetic variation in an individual’s HLA locus that block abacavir binding are shown in blue. PDB: 3VRJ. d, Left, chemical structure of thalidomide and its structural analog, pomalidomide. Right, structural view of pomalidomide (yellow) forming a ternary complex with H. sapiens CrBN (gray) and H. sapiens SALL4 zinc-finger 2 (purple). The locations of Mus musculus Sall4 residues that differ from those found in H. sapiens SALL4 are highlighted in light blue. The location of the M. musculus CRBN (mCRBN) Ile391 residue is highlighted in dark blue. Bottom, the sequences of M. musculus Sall4 and H. sapiens SALL4. Variation is highlighted in light blue. PDB: 3WX2, 6UML.

Fig. 4 |. Allele-selective kinase inhibitors and resistance mutations uncover the importance of BRAF dimerization in the MAPK signaling pathway.

a, Left, chemical structure of vemurafenib. Middle, schematic illustrating the MAPK signaling pathway in cells with wild-type BRAF. In these cells, BRAF is inactive in its monomeric form. Upon activation of Ras by upstream growth factor receptors, BRAF is phosphorylated, which leads to its dimerization. BRAF dimers represent the active form of the kinase and mediate downstream MAPK pathway activation through phosphorylation of the MEK and ERK kinases. Right, schematic illustrating the MAPK signaling pathway in cells with BRAF (V600E). BRAF (V600E) is active as a monomer. Thus, constitutive MAPK pathway activation through phosphorylation of MEK and ERK occurs in the absence of active Ras. b, Top, chemical structure of JAB34. The phenylbromide highlighted in green acts as a bump, complementary to the hole mutation engineered into SRC kinase. The acrylamide highlighted in blue is the cysteine-reactive portion of the molecule. Bottom, wild-type (WT) and mutant SRC kinases and their sensitivity to inhibition by JAB34. Half-maximal inhibitory concentration (IC₅₀) values are from Blair et al.. c, Schematic illustrating the use of JAB34 to uncover the mechanism of BRAF inhibitor-mediated dimer transactivation. d, Schematic showing the mechanism underlying resistance of the BRAF (V600E) p61 isoform to vemurafenib inhibition.

Fig. 5 |. Resistance mutations uncover the mechanism of action of LSD1 inhibitors in AML.

a, Schematic summarizing the prior and revised models of LSD1 inhibition in AML. PDB: 1AOI. b, Top, schematic illustrating the mechanism of the drug-complementary GFI1B allele. Bottom, structural views of the LSD1 catalytic site labeled by GSK-LSD1 adduct bound to wild-type Snail/Gfi-1 (SNAG) peptide (left) or a SNAG(F5A) peptide (right). PDB: 2UXX, 2Y48.

Fig. 6 |. Mutagenesis strategies enhance resistance mutation identification and enable novel methods to investigate the small molecule–protein interface.

a, Schematic illustrating the workflow of mutagenesis strategies that use variant overexpression approaches. Refs.^,– describe the use of error-prone cloning methods; refs.^,,,– describe the use of massively parallel synthesis methods. CDS, coding sequence. b, Schematic illustrating the workflow of mutagenesis strategies that use CRISPR-based technologies. Refs.^,– describe the use of CRISPR-NHEJ approaches; refs.^–, describe the use of CRISPR-HDR approaches and prime editing; and refs.^, describe the use of CRISPR base editing. c, Schematic illustrating the potential application of a resistance mutation profile to functionally classify small molecule analogs in a high-throughput manner. d, Schematic detailing the use of RADD to inform small molecule design. SAR, structure–activity relationship.