**6. Conclusion**

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 follow-

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

> Palmitoyl Acyltransferase CoA SH

O

CoA S

Palmitoylated protein

Palmitoyl CoA

S

Protein

O

O N

N

Cysteine +

O N

O

<sup>N</sup> SH

Protein

O

and sensitivity in a high throughput manner (Figure 5) [153].

up assays.

274 Drug Discovery

**Figure 5.**

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

The number of signaling networks in which palmitoylation plays a pivotal role is large and growing. The relationship between PAT gene expression and cancer is perhaps the most evi‐ dent as the fraction of PAT genes implicated in metastasis and tumorigenesis is notably high. There is also a remarkable confluence between our increasing understanding of palmi‐ toylation and our increasing awareness of the importance of lipid rafts, one of the primary residences of palmitoylated proteins in cancer [154, 155]. In instances where there is a rela‐ tionship between aberrant expression of PATs and cancer, the critical questions relate to the substrates of these PATs and their associated signaling networks. Identification of these sig‐ naling networks will potentially provide new therapeutic targets for the prevention or rever‐ sal of cancer progression. Given the preponderance of palmitoylated proteins resident in the neuronal synapse (which is itself a lipid raft of sorts), it is clearly another area of research that deserves (and has already received) a great deal of attention.

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While there has been some progress made in identifying pharmacological modulators of palmitoylation [125, 126, 138], there is nothing yet known about how to specifically target individual PATs. From a practical standpoint, inhibiting specific PATs may be a simpler process than developing specific PAT agonists. The advent of assays with the ability to measure changes in the activity of a single PAT along with the identification of PAT-sub‐ strate associations should enable further development of new assays to identify specific pharmacological modulators of individual PATs as well as providing important information on the signaling networks associated with specific PATs.

Our ability to understand palmitoylation and its importance to human health and disease is only as good as the technological methods we use to make accurate and valid measure‐ ments. Our ability to investigate the basic mechanisms of how PATs work, of PAT/substrate relationships, and how palmitoylation affects signaling processes related to disease would be improved significantly by the development of selective and potent pharmacological tools. Until such tools are available, we should be mindful that using compounds such as 2-BP, cerulenin, and tunicamycin may lead to erroneous conclusions. Developing non-lipid, selec‐ tive inhibitors that target the PAT active site is feasible. The challenges that exist are concep‐ tually similar in some aspects to those faced during the development of selective, smallmolecule inhibitors of kinases that do not resemble ATP. Based on current knowledge, the most logical PATs to target first are those for which overexpression is oncogenic. However, the motivation to initiate drug discovery programs on a large scale will probably remain be‐ low the required threshold until more conclusive data are available from more sophisticat‐ ed, whole-animal experiments that link PATs to oncogenesis.
