Discovery of Inhibitors that Elucidate the Role of UCH-L1 Activity in the H1299 Lung Cancer Cell Line

Abstract

Neuronal ubiquitin C-terminal hydrolase (UCH-L1) has been linked to Parkinson's disease (PD), the progression of certain nonneuronal tumors, and neuropathic pain. Certain lung tumor-derived cell lines express UCH-L1 but it is not expressed in normal lung tissue, suggesting that this enzyme plays a role in tumor progression, either as a trigger or as a response. Small-molecule inhibitors of UCH-L1 would be helpful in distinguishing between these scenarios. By utilizing high-throughput screening (HTS) to find inhibitors and traditional medicinal chemistry to optimize their affinity and specificity, we have identified a class of isatin O-acyl oximes that selectively inhibit UCH-L1 as compared to its systemic isoform, UCH-L3. Three representatives of this class (30, 50, 51) have IC(50) values of 0.80-0.94 micro M for UCH-L1 and 17-25 micro M for UCH-L3. The K(i) of 30 toward UCH-L1 is 0.40 micro M and inhibition is reversible, competitive, and active site directed. Two isatin oxime inhibitors increased proliferation of the H1299 lung tumor cell line but had no effect on a lung tumor line that does not express UCH-L1. Inhibition of UCH-L1 expression in the H1299 cell line using RNAi had a similar proproliferative effect, suggesting that the UCH-L1 enzymatic activity is antiproliferative and that UCH-L1 expression may be a response to tumor growth. The molecular mechanism of this response remains to be determined.

Deubiquitinases in cancer: new functions and therapeutic options

“…Nevertheless, there are small-molecule inhibitors against UCHs and USPs that could be useful as anticancer drugs (Supplementary Table 2). Thus, UCHL1 was the first DUB reported to be neutralized by small-molecule inhibitors, which belong to two different classes: isatin O-acyl oximes and 3-amino-2-oxo-7H-thieno [2,3-b]pyridin-6-one derivatives (Liu et al, 2003;Colland, 2010). The treatment of lung carcinoma cell lines with these inhibitors promotes cell proliferation, which confirms the antiproliferative role of UCHL1 in these cells (Liu et al, 2003).…”

“…Thus, UCHL1 was the first DUB reported to be neutralized by small-molecule inhibitors, which belong to two different classes: isatin O-acyl oximes and 3-amino-2-oxo-7H-thieno [2,3-b]pyridin-6-one derivatives (Liu et al, 2003;Colland, 2010). The treatment of lung carcinoma cell lines with these inhibitors promotes cell proliferation, which confirms the antiproliferative role of UCHL1 in these cells (Liu et al, 2003). In the case of UCHL3, small-molecule inhibitors have been identified by virtual screening using crystal structure data for this enzyme and a virtual compound library (Hirayama et al, 2007).…”

“…In the case of UCHL3, small-molecule inhibitors have been identified by virtual screening using crystal structure data for this enzyme and a virtual compound library (Hirayama et al, 2007). There are three competitive inhibitors against UCHL3 with similar dihydro-pyrrole skeletons and several isatin derivatives identified in the previous high-throughput screening of UCHL1 inhibitors (Liu et al, 2003). Regarding USPs, 2-cyano-pyrimidine and triazine derivatives have been identified as inhibitors of USP2 (Guedat and Colland, 2007), whereas a small-molecule compound that inhibits USP7 (HBX 41,108) stabilizes and activates p53 in a nongenotoxic manner, inhibiting cancer cell growth (Colland et al, 2009).…”

Ubiquitin C-terminal hydrolase 1: A novel functional marker for liver myofibroblasts and a therapeutic target in chronic liver disease

“…LDN 57444 is a reversible, competitive, active-site directed inhibitor of UCHL1 [20]. To investigate whether the inhibition of UCHL1 would reduce liver fibrogenesis we established an experimental therapeutic regime in mice (Fig.…”

Invited Perspective: High-Throughput Screening in Academia: The Harvard Experience

“…Since it became fully operational in January of 2002, LDDN has successfully screened nearly a dozen targets, and about as many are currently being screened. Despite the relative youth of the LDDN, a number of projects have yielded publishable studies [7][8][9] as well as starting points for medicinal chemistry projects and molecules that can be used in animal models of disease.…”