Bump and hole

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.

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.

[3] 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 (S99T/F113A) cyclophilin mutant.

Conserved ATP binding sites and similar catalytic mechanisms pose a challenge to selectively inhibiting a particular kinase to determine its function.

In early work, v-Src kinase I338A/G mutants were shown to accept [γ-32P]-labeled bumped N6-cyclopentyl and N6-benzyl ATP analogs as alternative cofactors to radiolabel its substrates.

[15] In order to probe bromodomain-specific functions of members of the BET family, small-molecule inhibitors JQ1 and I-BET were developed, but they lacked inter- and intra-BET (between BDs on the same protein) selectivity.

[16] The lab of Alessio Ciulli produced bump-and-hole pairs consisting of ET, a derivative of I-BET with an ethyl bump, and different members of the BET family with an L94A mutation in their BD1.

Recently, the Ciulli group developed a new bump-and-hole pair consisting of BET mutants with a Leu to Val mutation in a BD and the bumped small-molecule inhibitor 9-ME-1.

Jingli Hou and colleagues sought to deliver nitric oxide, an important messenger for promoting tissue growth processes like angiogenesis and vasculogenesis, in a spatiotemporally controlled manner.

However, non-tissue-specific systemic release of NO, which can reduce therapeutic efficiency and cause harmful side effects, from these pro-drugs was evident due to widespread distribution of endogenous glycosidases.

The bumped pro-drug evaded cleavage by wild type β-galactosidase due to the methylated O6 of the galactose moiety and strict regioselectivity of glycosidases.

Hou et al. found markedly increased therapeutic efficiency of NO delivery via the bump-and-hole engineered system, compared to the unmodified pro-drug, in rat hindlimb ischemia and mouse acute kidney injury models.

[18] The N-Acetylgalactosaminyl transferase (GalNac Ts) family transfers N-Acetylgalactosamine to the Ser/Thr side chains (O-linked glycosylation) of its substrates, using UDP-GalNac as a cofactor.

Schematic of bump-and-hole method. [ 1 ]
The first reported bump-and-hole pair. Hole-modified S99T/F113A mutant cyclophilin has an expanded hydrophobic pocket to accept a methyl bump in cyclosporin A analog MeIle11CsA. [ 4 ]
The bumped ATP analog N6-cyclopentyl ATP cannot bind wild type v-Src kinase, but can bind its bump-and-hole pair, I338G v-Src kinase. [ 8 ]
The bumped ET inhibitor has selectivity for L94A BET BD due to steric complementarity. The un-bumped I-BET inhibitor would promiscuously bind BDs. [ 13 ]
Schematic of bumped pro-drug and hole-modified enzyme, releasing the drug only in the presence of the bump-and-hole pair. [ 18 ]
Hole-modified BH GalNac-Ts paired with UDP-GalNac analogs to tag GalNac T substrates to be visualized with click chemistry. [ 19 ]