Serine hydrolases are an exceptionally large and diverse class of enzymes that play important roles in virtually all biological processes in mammals (Simon and Cravatt, 2010
). Many serine hydrolases, however, remain poorly characterized with respect to their biochemical and physiological functions. Using ABPP, we previously identified one such serine hydrolase, the integral membrane enzyme KIAA1363, as being highly expressed by aggressive human cancer cells (Jessani et al., 2002
) and primary tumors (Jessani et al., 2005
). We also succeeded in identifying lead inhibitors for KIAA1363, which helped to define a role for this enzyme in NEL metabolism in cancer cells (Chiang et al., 2006
). These findings, when integrated with the large historical body of work designating NEL metabolism as a prominent biochemical pathway dysregulated in cancer cells (Albert and Anderson, 1977
; Lin et al., 1978
; Roos and Choppin, 1984
; Snyder and Wood, 1969
; Wood and Snyder, 1967
), suggest that KIAA1363 might play an important role in tumorigenesis. Testing this hypothesis, however, required more advanced KIAA1363 inhibitors that possess the potency and selectivity needed for extensive pharmacological studies.
Here, we have used competitive ABPP-guided medicinal chemistry to create JW480 – a low-nM, selective, and in vivo
-active inhibitor for KIAA1363. We believe that JW480 likely acts as an irreversible inhibitor of KIAA1363 that carbamoylates the enzyme’s serine nucleophile, which is consistent with the known mechanism of action for other carbamate inhibitors of serine hydrolases (Alexander and Cravatt, 2005
; Long et al., 2009b
). Confirmation of an irreversible mode of inhibition for JW480 will require further studies, such as direct mass-spectrometry characterization of the carbamoylated inhibitor-enzyme adduct as has been measured for other serine hydrolases (Alexander and Cravatt, 2005
). But, an irreversible mechanism of action for JW480 is supported by our competitive ABPP results that show sustained inhibition of KIAA1363 in tissue extracts from JW480-treated mice. Assuming that JW480 is an irreversible inhibitor of KIAA1363, future experiments could include: 1) appending an alkyne onto JW480 to convert it into a ‘clickable’ probe for assessing proteome-wide target interactions beyond the serine hydrolases that are counterscreened by competitive ABPP, as has been shown for other enzymes (Ahn et al., 2009
; Alexander and Cravatt, 2005
), and 2) incorporation of a fluorophore into the JW480 structure for creating a probe to image KIAA1363 activity in vivo
, as has been achieved for other enzymes (Blum et al., 2005
The starting scaffold for JW480 originated from a recent ‘library-versus-library’ competitive ABPP screen where we assayed 70+ serine hydrolases against 150+ carbamate small molecules (Bachovchin et al., 2010
). The breadth of this screen pointed to areas for improvement of lead KIAA1363 inhibitors, most notably, in designating HSL and AChE as common off-targets for these compounds. Interestingly, and as noted previously (Bachovchin et al., 2010
), neither of these enzymes share much sequence homology with KIAA1363, underscoring the value of proteomic profiling methods like competitive ABPP that can identify ‘pharmacological homology’ among distantly related enzymes. We were able to minimize cross-reactivity with HSL and AChE by incorporating a bulky naphthalene group into the N
-alkyl substituent of JW480.
Eliminating AChE cross-reactivity was of obvious importance, given that potent inhibitors of this enzyme are neurotoxic (Casida and Quistad, 2005
). Removing HSL cross-reactivity may also be valuable because both KIAA1363 and HSL are expressed at high levels in macrophages, where each enzyme has been suggested to play a role in neutral cholesterol ester hydrolysis (Buchebner et al., 2010
; Igarashi et al., 2010
; Okazaki et al., 2008
). The relative contribution that KIAA1363 and HSL make to cholesterol ester metabolism in macrophages remains unclear, and we anticipate that JW480 should offer a valuable pharmacological tool to investigate this question. Among the more than 40 serine hydrolases counterscreened by competitive ABPP in our combined analyses of brain and cancer cell proteomes, only a single off-target for JW480 was detected – the carboxylesterase ES1. As has been discussed previously (Bachovchin et al., 2010
), CEs are promiscuous enzymes involved in xenobiotic metabolism in tissues such as the liver. They are common off-targets for mechanism-based serine hydrolase inhibitors, including carbamates (Alexander and Cravatt, 2005
; Bachovchin et al., 2010
; Long et al., 2009b
). We do not believe, however, that such cross-reactivity with ES1 (and possibly other CEs) is a major problem for using JW480 as a pharmacological tool to investigate KIAA1363 function, especially for studies in the nervous system and cancer, where CE expression is low. Furthermore, one can use carbamates that do not inhibit KIAA1363, but still show CE cross-reactivity as ‘negative control’ compounds, as we have shown in this study with the FAAH inhibitor URB597.
Using JW480 (and an shRNA probe that targets KIAA1363), we found that disrupting KIAA1363 reduces MAGE levels in human prostate cancer cells and impairs several of their pro-tumorigenic properties, including migration, invasion, and serum-free survival. These metabolic and cell biological effects were correlated with significant reductions in tumor growth in mouse xenograft models treated with JW480 (or using shKIAA1363 prostate cancer cells). These data collectively support a pro-tumorigenic function for KIAA1363. We should note, however, that tumors treated with JW480 or from shKIAA1363 prostate cancer cells continued to grow in vivo
, indicating that blockade of KIAA1363 slows, but does not completely block tumor progression. This outcome was not due to incomplete inhibition of KIAA1363, as we were able to confirm full inactivation of KIAA1363 in explanted tumors from JW480-treated mice. In future studies, it would be interesting to test whether KIAA1363 inhibitors show additive or synergistic anti-tumor activity when combined with other chemotherapeutic agents. Also, the strong anti-invasive effects of JW480 suggest that blockade of KIAA1363 could impede cancer metastasis in vivo
. Finally, more extensive biochemical and cell biological studies are required to understand the mechanism by which KIAA1363-MAGE pathway supports prostate cancer pathogenicity. Previous work showed that this pathway is coupled to the production of pro-tumorigenic lipids, such as alkyl-LPA, in ovarian cancer cells (Chiang et al., 2006
). Broader metabolomic experiments should reveal whether changes in LPA or other bioactive lipids are also observed in JW480-treated prostate cancer cells.
In closing, we believe that JW480 possesses an impressive array of features that qualify it as a frontline pharmacological probe for KIAA1363, including high potency against both the human and mouse orthologues of this enzyme, minimal cross-reactivity with other serine hydrolases, and excellent activity in living cells and mice. We anticipate that future studies with JW480 will help to illuminate the role that KIAA1363 plays in many (patho)physiological processes, including cancer, macrophage biology, and the nervous system. From a methodological perspective, our success in converting lead carbamates originating from a large-scale screen (Bachovchin et al., 2010
) into a KIAA1363 inhibitor that displays greatly improved potency and selectivity can be attributed, at least in part, to the information content garnered by competitive ABPP, which assays inhibitors against numerous enzymes in parallel directly in native proteomes. This type of “proteomic medicinal chemistry”, which has also impacted other inhibitor development programs (Arastu-Kapur et al., 2008
; Deu et al.; Li et al., 2007
; Long et al., 2009a
; Staub and Sieber, 2009
), should continue to provide an efficient means to create versatile pharmacological probes for a wide range of enzymes.
Mapping dysregulated metabolic pathways in cancer cells has the potential to uncover biochemical mechanisms that support tumorigenesis and identify new drug targets. The serine hydrolase KIAA1363 has been found to be highly elevated in aggressive cancer cells from multiple tumors of origin, where it plays a role in ether lipid metabolism. Attempts to further characterize KIAA1363 function in cancer have, however, been thwarted by a lack of pharmacological tools to study this enzyme. Here, we have addressed this challenge by creating a potent, selective, and in vivo-active inhibitor of KIAA1363. Key to the development of this compound (termed JW480) was the use of competitive activity-based profiling assays to simultaneously refine potency for KIAA1363 and selectivity across the larger serine hydrolase class. JW480 reduces monoalkylglycerol ether levels in androgen-independent prostate cancer cells and impairs their migration, invasion, survival, and in vivo tumor growth. These studies thus support a pro-tumorigenic function for KIAA1363 and report a versatile inhibitor for pharmacological characterization of this enzyme in living systems.