**5.2. Considerations for development of high-throughput screens to discover PAT inhibitors**

The DHHC motif in PATs defines the active site and is highly conserved in all mammalian PATs [3]. The regions of highest diversity are primarily in the N- and C-termini of the PAT. Mutation of the cysteine in the DHHC motif abolishes PAT autoacylation and palmitoyla‐ tion of the substrate, a property of all DHHC proteins studied so far. This high degree of homology in the active site sequence among PATs could give the impression that develop‐ ing highly specific, active-site inhibitors for palmitoylation will be impossible. However, this same issue exists with kinases [143, 144], and yet the development of selective and potent active-site, ATP-competitive inhibitors has been successful (eg, [145]).

The specificity of palmitoylation must be derived in part from the unique physical interac‐ tions of individual PATs with their substrates. The sequence of amino acids surrounding a substrate cysteine partially defines the potential for that cysteine to be palmitoylated. How‐ ever, the physical determinants for substrate recognition will likely extend throughout the accessible portions of the PAT and substrate as was elegantly demonstrated for DHHC17 [146]. Other factors that are likely to regulate palmitoylation are the temporal and spatial as‐ pects of PAT and substrate expression.

There are many more palmitoylated proteins than there are PATs; therefore, modulating the activity of a single PAT, even with complete compound selectivity, will likely yield a change in the palmitoylation of multiple substrates. This conundrum is common to the develop‐ ment of highly selective and potent pharmacological modulators of all enzymes that medi‐ ate post-translational protein modifications, again kinases being a classic example.

Another challenge is that each PAT traverses the plasma membrane multiple times. A con‐ servative guess would suggest that the membrane environment is important for determin‐ ing PAT structure and substrate recognition. However, Jennings et al.,[138] demonstrated that at least four PATs can be purified from a membrane environment and remain enzymati‐ cally active. These findings are both remarkable and encouraging evidence that enzyme ac‐ tivity-based and drug-binding screens for selective PAT inhibitors can be accomplished with purified proteins.
