**5.1. Developing compounds that selectively target individual PATs**

Existing chemicals used to inhibit palmitoylation are neither selective nor potent. The com‐ pounds used most commonly are 2-bromopalmitate (2BP), tunicamycin, and cerulenin. Each of these is a lipid-based molecule (Figure 4). 2BP has been used most frequently at a concen‐ tration of ~100 µM to block palmitoylation in spite of the fact that at least two studies have shown that the IC50 of 2-BP is ~10 µM [138, 139]. 2BP is not tolerated well by cultured cells and causes death even after a brief exposure to 100 µM. 2BP inhibits several enzymes in‐ volved in lipid metabolism, including carnitine palmitoyltransferase 1, fatty acid CoA ligase, glycerol-3-phosphate acyltransferase, and enzymes in the synthesis of triacylglycerol bio‐ synthesis [140, 141]. This high degree of promiscuity as well as the toxicity of 2BP renders it nearly useless as a tool to determine anything specific about palmitoylation related signal‐ ing issues that equally plague cerulenin and tunicamycin. The uses and effects of these three inhibitors was reviewed recently [142].

**Figure 4.** Lipid-based inhibitors of palmitoylation.

than labeling with 125I-palmitoyl-CoA. These features, especially their ability to effectively substitute for endogenous fatty acids, make them ideal for labeling palmitoyl proteins in live cells, providing a significantly more direct measure of protein palmitoylation than can be achieved in any other assay format. It is easy to imagine that use of such probes will

COOH

live cells purified proteins

**Figure 3.** Using Click chemistry and bio-orthogonal probes to label palmitoyl cysteines. A) A palmitoylated protein; the shaded box indicates the thioester bond. B) Azido-palmitate is transferred to a protein forming a thioester bond with a cysteine residue. The azide moiety of the azido-palmitate reacts, via the Staudinger reaction, with the tagged (in this case biotin) phosphine, forming an amide bond. The biotin-tagged proteins can then be affinity purified and analyzed in various ways including mass spectrometry. Tags and reporters other than biotin can be added to the phosphine

Existing chemicals used to inhibit palmitoylation are neither selective nor potent. The com‐ pounds used most commonly are 2-bromopalmitate (2BP), tunicamycin, and cerulenin. Each of these is a lipid-based molecule (Figure 4). 2BP has been used most frequently at a concen‐ tration of ~100 µM to block palmitoylation in spite of the fact that at least two studies have shown that the IC50 of 2-BP is ~10 µM [138, 139]. 2BP is not tolerated well by cultured cells

O

O

phosphine-biotin Staudinger ligation

N S <sup>3</sup> azido-fatty acid modified protein(s)

S-palmitoylation

PPh2 <sup>O</sup> <sup>O</sup>

H biotin N

<sup>N</sup> <sup>S</sup> HO

biotinylated proteins

Streptavidin blot affinity purification

mass spec analysis

come to dominate in experimental systems for studying palmitoylation.

O S <sup>O</sup> <sup>N</sup> H2N <sup>N</sup>

cysteine residue

O

palmitate

N OH <sup>3</sup> azido-fatty acids

providing a wide array of potential methods for subsequent analyses.

**5. Pharmacological modulation of Palmitoylation**

**5.1. Developing compounds that selectively target individual PATs**

**A**

270 Drug Discovery

**B**

H H

> Smith and colleagues [126] recently screened a compound library in an attempt to identify more selective and potent inhibitors of palmitoylation, in particular inhibitors of PATs. This screen identified single compounds from five chemical classes (Compounds I-V) that inhib‐ ited cellular processes associated with palmitoylation. The assays used in the screens includ‐ ed: measuring the *in vivo* and *in vitro* growth rate of an NIH/3T3 cell line that overexpressed DHHC17, displacement from the plasma membrane of myristoylated or farnesylated GFP, and *in vitro* palmitoylation of small, non-complex, myristoylated or farnesylated, synthetic, fluorescent peptides intended to mimic known palmitoylation substrates [123, 126, 127]. These assays could not discriminate a direct effect of any compound on any PAT. They could only report the activity of compounds that acted at some point (not excluding direct PAT inhibition) in any pathway that leads to or affects palmitoylation; compounds like 2BP, cerulenin, and tunicamycin. This assertion was borne out in follow-up studies on the same compounds [138] (see below). Perhaps the most intriguing finding in this report was that compounds I-IV were able to suppress the oncogenic behavior of human cells that overex‐

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‐ mining their exact mechanism of action.

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‐

Discovery of Selective and Potent Inhibitors of Palmitoylation

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

273

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‐

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

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‐

ate post-translational protein modifications, again kinases being a classic example.

pects of PAT and substrate expression.

**5.3. Primary screen for PAT inhibitors**

purified proteins.

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

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.
