**5.3. Primary screen for PAT inhibitors**

pressed DHHC17. However, there is no direct evidence to indicate that compounds I-IV ex‐ ert these actions via inhibition of DHHC17 or through the palmitoylation of Ras proteins by DHHC17 as was speculated [53, 126]. Nevertheless, since these compounds reduced the *in vivo* growth of tumors from cells overexpressing DHHC17 [126], it would be worth deter‐

Subsequent studies by Linder, Deschenes and colleagues [138] tested four of the five com‐ pounds identified by Smith and colleagues and found that they were not selective for DHHC proteins. This report also included a wealth of information defining the mechanisms by which 2BP inhibits palmitoylation. Briefly, only one of the four compounds re-tested, compound V and 2BP, inhibited the activity of any of the four DHHC proteins tested. Nei‐ ther compound V nor 2BP was selective for any of the PATs tested, and 2BP was more po‐ tent. Both compound V and 2BP blocked autoacylation of the PATs; compound V was reversible, 2BP was not. Even though compound V was able to inhibit the activity of the four PATs tested and in the same manner as 2BP, these experiments could not determine whether compound V also blocks palmitoylation indiscriminately at steps prior to the actual

There would be no compelling reason to begin a drug discovery program to identify inhibi‐ tors of just any PAT. Rather one would choose to begin with a PAT that is linked to a dis‐ ease state—a situation where misregulated expression or function of that PAT was clearly linked to a pathological state. As discussed earlier, links between PAT expression (but not yet altered function) have been demonstrated for both neurological disorders and cancer; thus, candidate PATs that would be appropriate targets for drug development exist. Both overexpression and absence of PAT expression have been implicated in the development of cancer. Dampening the activity of an existing PAT is a conceptually and mechanistically simpler task than accurately restoring the specific activity of a PAT that is not expressed or absent. This review is concerned with PAT assays and PAT inhibitors, so we will address the case of PAT overexpression in ideal terms as well as the technical issues that surround the development and implementation of the assays designed to discover PAT inhibitors.

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

active-site, ATP-competitive inhibitors has been successful (eg, [145]).

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

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‐

palmitoylation event, as is the case with 2BP, cerulenin, and tunicamycin.

mining their exact mechanism of action.

272 Drug Discovery

**inhibitors**

The discovery and refinement of drugs to modulate PAT activity will require the use of mul‐ tiple assay types. The initial success of each can only be a matter of speculation at the begin‐ ning of the project, and the success of the primary screen will influence the choice of follow up assays. However, one unique aspect of palmitoylation suggests a logical starting point. The most dramatic visible change that can occur when a protein is palmitoylated is when it moves from the cytoplasm to the plasma (or other) membrane. The technology to measure such a translocation in living cells using high-throughput microscopy has been demonstrat‐ ed [139, 147] and along with many other such morphometric analyses, has become well es‐ tablished in drug discovery programs and the basic life sciences [148-150]. This technology is often referred to as high-content screening (HCS). To develop an assay to identify inhibi‐ tors of a single PAT using HCS, it would be ideal to have identified the most clinically rele‐ vant, cytosolic substrate of the PAT of interest and to have determined that this substrate is palmitoylated exclusively by this one PAT or, alternatively, by no other PAT expressed in the cell type that will be used for the screen. However, biological systems rarely offer ideal situations, and accommodations will inevitably need to be made. The ideal substrate would then be fused to a monomeric fluorescent protein (FP) [5, 151] to generate a fluorescent re‐ porter of palmitoylation that localizes primarily or exclusively to the PM. Cells stably ex‐ pressing this reporter would then be grown in multi-well imaging plates and exposed to a chemical compound library, and the subcellular distribution of the FP-tagged palmitoyla‐ tion substrate evaluated by HCS. Compounds that cause redistribution of the fluorescent re‐ porter from the PM to the cytoplasm are candidates (or hits) for follow up analyses that will determine if they blocked palmitoylation of the reporter by directly inhibiting the PAT of interest or indirectly by some other mechanism. Typically, compounds in a large library (tens to hundreds of thousands of compounds) are tested at a single concentration and repli‐ cated, often three times, to increase the confidence of selecting biologically active com‐ pounds. But, the relationship of replicates is solely statistical, not pharmacological. An alternative screening method for identifying hits is "titration-based screening" called qHTS [152]. This method, which has been used successfully by Jim Inglese, Doug Auld and Collea‐ gues at the NIH Chemical Genomics Center, measures the assay system response to multi‐ ple (up to seven), different concentrations of a single compound. The increased density and accuracy of the data produced by this method can provide many benefits over screening at a single concentration (for a full description of the merits of qHTS see [152]). Among the most important benefits of screening at multiple concentrations is that it alleviates the problems of false-negatives and false-positives that plague screens run at a single concentration. The nominal, additional effort required at the front end of the process is generously compensat‐ ed by a subsequent reduction in the effort required to choose which hits to pursue in followup assays.

Retesting hits from the primary screen in this secondary, enzymatic assay would determine if the compounds directly inhibited palmitoylation of the substrate in the reaction. It would also identify compounds that inhibit palmitoylation by competing with palmitoyl-CoA for access to the PAT active site, as well as allosteric inhibitors. Structural analysis of the com‐ pounds would provide information about how they inhibit palmitoylation. A binding assay in which compounds are tested for their ability to compete directly with palmitoyl-CoA for binding to the PAT would more conclusively determine the mechanism by which the com‐

Discovery of Selective and Potent Inhibitors of Palmitoylation

http://dx.doi.org/10.5772/52503

275

It is likely that the inhibitors identified will represent multiple classes of compounds distin‐ guished by their chemical structures. However, the chemicals identified in these screens are unlikely to represent the most potent or selective compounds that exist. Further refinement by probing and refining the chemical space of each compound in a medicinal chemistry ef‐ fort will be required to achieve both objectives. Generating higher affinity analogs of the hit compounds for the PAT of interest will improve the selectivity and potency of the com‐ pounds for an individual PAT. However, the question of how selective any of these com‐ pounds is for a single PAT must be answered by determining their ability to inhibit other PATs—counterscreening. Since the family of PATs is relatively small (23 genes), it would not be unreasonable to measure the effects of the compounds on each of the other PATs in

The assay development pathway proposed above is outlined only in very general and ideal terms, glossing over inevitable technical challenges that must be overcome for the project to be successful. However, even as brief as this description is, it exceeds by far the complexity of any published attempt to identify selective PAT inhibitors to date. A valuable, practical guide to choosing, developing, and validating assays including those proposed above is

The discovery of the molecular identity of PATs was a pivotal event that has fostered sub‐ stantial progress in the field of lipidation, having a profoundly positive effect on many fields of biology. Many long-standing questions have been greeted with answers as well as a clear‐ er direction in which new inquiries should proceed. While sometimes criticized as being stamp collecting, defining the palmitoyl proteome of specific cells and tissues would pro‐ vide new and unforeseen insight into many cellular processes. The methods described here provide the technical foundation for defining the palmitoyl proteome. Defining the intrinsic and extrinsic mechanisms and factors that regulate PAT activity will also be crucial and challenging. Future assays to investigate such details will certainly benefit from the demon‐ strated usefulness of bioorthagonal probes that appear to be treated by cells as if they were palmitate. These probes may provide a more direct measure of palmitoylation than the ex‐

pounds were inhibiting substrate palmitoylation.

the enzymatic assay described above.

**6. Conclusion**

available at: http://ncgc.nih.gov/guidance/manual\_toc.html.

change of cysteine-reactive probes for palmitate on purified proteins.

Displacement or translocation of the fluorescent palmitoylation reporter from the PM to the cytoplasm in response to a compound cannot provide evidence that the compound has this effect by direct inhibition of a PAT. Secondary screens designed to determine which of the hits works by direct inhibition of PAT activity will be required. One option would be to de‐ termine the effects of each hit on the enzymatic activity of the PAT of interest. Jennings et al have demonstrated that a PAT can be purified from a membrane environment and retain its enzymatic function i.e., transfer of palmitate to a substrate. The metabolically active form of palmitate in a living system is palmitoyl-CoA. Transfer of palmitate to a substrate results in the liberation of CoA from palmitate, a chemical species that can be measured with accuracy and sensitivity in a high throughput manner (Figure 5) [153].

**Figure 5.**

Retesting hits from the primary screen in this secondary, enzymatic assay would determine if the compounds directly inhibited palmitoylation of the substrate in the reaction. It would also identify compounds that inhibit palmitoylation by competing with palmitoyl-CoA for access to the PAT active site, as well as allosteric inhibitors. Structural analysis of the com‐ pounds would provide information about how they inhibit palmitoylation. A binding assay in which compounds are tested for their ability to compete directly with palmitoyl-CoA for binding to the PAT would more conclusively determine the mechanism by which the com‐ pounds were inhibiting substrate palmitoylation.

It is likely that the inhibitors identified will represent multiple classes of compounds distin‐ guished by their chemical structures. However, the chemicals identified in these screens are unlikely to represent the most potent or selective compounds that exist. Further refinement by probing and refining the chemical space of each compound in a medicinal chemistry ef‐ fort will be required to achieve both objectives. Generating higher affinity analogs of the hit compounds for the PAT of interest will improve the selectivity and potency of the com‐ pounds for an individual PAT. However, the question of how selective any of these com‐ pounds is for a single PAT must be answered by determining their ability to inhibit other PATs—counterscreening. Since the family of PATs is relatively small (23 genes), it would not be unreasonable to measure the effects of the compounds on each of the other PATs in the enzymatic assay described above.

The assay development pathway proposed above is outlined only in very general and ideal terms, glossing over inevitable technical challenges that must be overcome for the project to be successful. However, even as brief as this description is, it exceeds by far the complexity of any published attempt to identify selective PAT inhibitors to date. A valuable, practical guide to choosing, developing, and validating assays including those proposed above is available at: http://ncgc.nih.gov/guidance/manual\_toc.html.
