Bump and hole
The bump-and-hole method is a tool in chemical genetics for studying a specific isoform in a protein family without perturbing the other members of the family. The unattainability of isoform-selective inhibition due to structural homology in protein families is a major challenge of chemical genetics. With the bump-and-hole approach, a protein–ligand interface is engineered to achieve selectivity through steric complementarity while maintaining biochemical competence and orthogonality to the wild-type pair. Typically, a "bumped" ligand/inhibitor analog is designed to bind a corresponding "hole-modified" protein. Bumped ligands are commonly bulkier derivatives of a cofactor of the target protein. Hole-modified proteins are recombinantly expressed with an amino acid substitution from a larger to smaller residue, e.g., glycine or alanine, at the cofactor binding site. The designed ligand/inhibitor has specificity for the engineered protein due to steric complementarity, but not the native counterpart due to steric interference.
History
Inspiration for the bump-and-hole method was drawn from mutant E. coli strains that carried an A294S mutant version of phenylalanine tRNA synthetase and survived exposure to p-fluoroPhe, which has a slight "bump" and is cytotoxic when incorporated during translation. The A294S mutant strain was able to incorporate Phe, but not the bumped p-fluoroPhe due to steric crowding from the hydroxyl group of S294. Conversely, a "hole" created by a T45G mutation in ribonuclease A expanded its substrate specificity from pyrimidine nucleobases to a larger purine, adenine. Subsequent work in the laboratories of Peter G. Schultz and David A. Tirrell showed that a hole-modified A294G phenylalanine tRNA synthetase mutant was able to incorporate the bumped p-fluoroPhe in translation, demonstrating that steric manipulation can successfully broaden substrate scope, even for the highly specific aminoacyl synthetase.The first bump-and-hole pair, developed by Stuart Schreiber and colleagues, was a bumped cyclosporin A small-molecule with an Ile replacing Val at position 11, and a hole-modified cyclophilin mutant. Cyclosporin A is a chemical inducer of dimerization (CID) of cyclophilin. This first bump-and-hole pair was engineered to improve the binding efficiency between wild-type cyclosporin A and cyclophilin, thereby giving more efficient CID. The bumped cyclosporin A was found to interact efficiently with the hole-modified cyclophilin mutant, but not endogenous cyclophilin. The orthogonal CID pair was used to inhibit calcineurin-mediated dephosphorylation of nuclear factor of activated T cells in a cell- and tissue-specific manner. More recently, this first bump-and-hole pair was used to induce the assembly of ten-eleven translocation 2 dioxygenase in cells for temporally controlled DNA demethylation.
Applications
As structural information about protein-ligand interfaces has become available, bump-and-hole pairs have been used to elucidate the substrates of specific proteins from various protein classes, as well as to develop orthogonal neoenzyme-neosubstrate therapeutics.Kinases
Human protein kinases use ATP as a cofactor to phosphorylate substrate proteins. Kinases play critical roles in complex cell signaling networks. Conserved ATP binding sites and similar catalytic mechanisms pose a challenge to selectively inhibiting a particular kinase to determine its function. Kevan Shokat's lab has developed bump-and-hole pairs using kinase mutants with bulky "gatekeeper" residues in the ATP-binding pocket replaced by Gly or Ala, and bulky ATP analogs. In early work, v-Src kinase I338A/G mutants were shown to accept -labeled bumped N6-cyclopentyl and N6-benzyl ATP analogs as alternative cofactors to radiolabel its substrates. Only the mutant kinase was able to bind the bumped ATP analogs, allowing labeling of substrates specific to the engineered v-Src kinase. Purification and MS-based proteomics yielded the substrates of v-Src kinase. Hole-modified kinase and bumped ATP analog pairs enabled substrate profiling of several other kinases, including CDK1, Pho85, ERK2, and JNK.Whereas bumped ATM analogs can help deconvolute kinase substrate profiles, one drawback of this strategy is the cell impermeability of the bumped analogs. To get around this, the Shokat group demonstrated that a bumped ATP analog, kinetin ATP or KTP, could be synthesized endogenously in cells cultured with kinetin. Once synthesized, it can activate a PINK1 kinase mutant, which is otherwise inactive in the absence of the bumped analog. Inactive PINK1 is implicated in Parkinson's disease. In the context of PD, the mutant PINK1-KTP pair represents an orthogonal neoenzyme-neosubstrate therapeutic.
The Shokat group also applied the bump-and-hole approach to develop selective, cell-permeable bumped inhibitors of mutant kinases. For the I338G v-Src kinase, a 4-amino-l-tert-butyl-3-pyrazolopyrimidine derivative called p-tButPhe-PP1 was developed for selective inhibition; steric bulk precluded binding to the wild-type v-Src kinase. In mammalian cell lines, active v-Src kinase is required for transformation by Rous sarcoma virus. In cell lines expressing I338G v-Src kinase and transfected with RSV, treatment with p-tButPhe-PP1 caused the reversal of transformation, suggesting inhibition of the kinase mutant. Later, the group developed bumped inhibitors 1-naphthyl PP1 and 1-methylnaphthyl PP1, which inhibited hole-modified yeast kinases with IC50 values in low nanomolar concentrations.