https://www.sciencedirect.com/science/article/pii/S2451945618302289?via%3Dihub

The Bump-and-Hole Tactic: Expanding the Scope of Chemical Genetics

Key figures

• [Figure 1]: Defines bump-and-hole allele-specific chemical genetics as paired steric engineering of a protein pocket and complementary ligand/cofactor analog.
  
• [Figure 2]: Shows foundational orthogonal systems for PheRS amino acid incorporation, CypA-cyclosporin induced proximity, and chemically rescued hGH-hGHR interaction.
  
• [Figure 3]: Demonstrates analog-sensitive kinase and RNA helicase engineering through gatekeeper mutations paired with bumped ATP, PP1, or DDX3 inhibitors.
  
• [Figure 4]: Maps bump-and-hole methyltransferase systems using SAM analogs, including in-cell Hey-SAM synthesis and CliEn-Seq-style chromatin profiling.
  
• [Figure 5]: Extends the approach to epigenetic writers, readers, and erasers through GCN5, BRD4 bromodomain, and KDM4A engineering.
  
• [Figure 6]: Shows PARP analog-sensitive NAD+ systems that enable proteome-wide substrate identification and genome-wide PARP activity profiling.
  
• [Figure 7]: Demonstrates protein-protein interaction engineering by sterically reprogramming chorismate mutase dimerization and strengthening KIX-MLL binding with bumped peptides.
  

1) Thesis (one sentence)

To address the inability of conventional genetics or small-molecule inhibitors to acutely and isoform-specifically perturb conserved protein families, in allele-specific chemical genetics across kinases, methyltransferases, acetyltransferases, bromodomains, demethylases, PARPs, and engineered protein-protein interactions, complementary bump-and-hole engineering causes orthogonal activation, inhibition, or substrate labeling of selected protein variants by pairing hole-forming mutations at ligand/cofactor or interaction interfaces with sterically matched bumped cofactors, inhibitors, or peptides, supported by review synthesis of structural, biochemical, cellular, proteomic, and genome-wide case studies.

2) Evidence card (three bullets only)

• Strongest result: (Fig. 3; Fig. 4; Fig. 6) Analog-sensitive kinase technology was extended to more than 100 proteins, while methyltransferase and PARP bump-and-hole systems enabled enzyme-specific substrate labeling, enrichment, mass spectrometry, and genome-wide chromatin activity profiling.
  
• Method enabler: (Fig. 1; Fig. 2; Fig. 3; review + chemical genetics tools including gatekeeper mutagenesis, bumped cofactor/inhibitor synthesis, click chemistry, mass spectrometry, ChIP-seq, and engineered PPIs) Orthogonality is created by removing a bulky residue from the protein interface and restoring productive binding only for a ligand, cofactor, or peptide bearing a complementary steric bump.
  
• Critical limitation: (Fig. 3; Fig. 4; Fig. 5) Successful pair construction depends on structural knowledge, a well-defined ligand/cofactor pocket, and chemically accessible bumped analogs; specific caveats include limited cell permeability of bumped ATP analogs and incomplete DNMT/RNMT orthogonality because wild-type enzymes can still process some SAM analogs.
  

Optional

Quote bank (2–4 short excerpts)

• Quote 1: “Chemical genetics, however, is not free from limitations.” (Introduction, page 1171)
  
• Quote 2: “PPIs operate over a large surface area and can be highly dynamic in nature.” (Allele-Specific Engineering at the PPI, page 1180)
  

Key comparisons (1–3 lines)

• Compared to: RNAi, CRISPR knockout, temperature-sensitive alleles, and broad-spectrum small-molecule inhibitors.
  
• Win: Provides rapid, conditional, tunable, allele-specific perturbation while preserving most native interactions and avoiding loss of the entire gene product.
  
• Tradeoff: Requires engineered alleles and customized chemistry, and performance can collapse if the hole mutation weakens activity, the bumped ligand lacks permeability, or endogenous wild-type proteins still bind the analog.
  

Methods I might copy (protocol hooks)

• Construct design / Models: Engineer holes by replacing bulky gatekeeper/interface residues with smaller residues, as shown for CypA S99T/F113A, v-Src I338G, DDX3 F182A/F182G, G9a Y1154A, GLP1 Y1211A, MAT1 I117A, BRD4 BD1 L94A, KDM4A F185G, PARP1 K903A or L877A, ARTD10 I987G, ARTD11 I313G, and chorismate mutase BzF72 with L76T/I80’G suppressor mutations.
  
• Conditions / Instruments: Treat NIH3T3 cells expressing I338G v-Src with 100 µM p-tButPhe-PP1; inject analog-sensitive myc-prkciI316A mRNA and N6-benzyl ATPγS into zebrafish embryos and extract proteins at 8 h postfertilization; use HEK293T lysates or cells for GCN5, MAT1/G9a, and PARP substrate-labeling workflows; use HeLa nuclear extracts for PARP-selective ADP-ribosylation mapping.
  
• Readout / Analysis: Track orthogonal kinase substrates by γ-32P or ATPγS labeling plus enrichment; profile methyltransferase, acetyltransferase, and PARP substrates by clickable cofactors, pull-down, in-gel fluorescence, and quantitative mass spectrometry; map chromatin activity with CliEn-Seq or Click-ChIP-seq; validate PPI engineering by cellular localization, cell growth, or binding assays.
  

Open questions / Theoretical implications (2–5 bullets)

• Can bump-and-hole design be generalized from enzymes with obvious cofactor pockets to dynamic PPIs whose binding surfaces are broad, shallow, and conformationally heterogeneous?
  
• Can cell-permeable bumped cofactors be made broadly enough to support acute intracellular perturbation without relying on in-cell cofactor biosynthesis?
  
• Can CRISPR knock-in of silent hole-modified alleles become the default way to avoid overexpression artifacts in allele-specific chemical genetics?
  
• Can disease-associated mutations that naturally create or remove steric volume be treated as endogenous bump-and-hole opportunities for precision pharmacology?
  
• Can orthogonal protein-ligand engineering be coupled to induced proximity, activity-based profiling, and genome editing to perturb and read out specific protein states in living cells?