Shh is a secreted signaling protein that is essential for proper embryonic development1,2
. In adults, aberrant Shh signaling drives initiation and maintenance of medulloblastoma and basal cell carcinoma, and has been implicated in the progression of prostate cancer, gastrointestinal tumors, and pancreatic cancer3
. The mature Shh signaling protein is formed via a series of post-translational processing reactions. Following removal of the signal peptide, Shh undergoes autocleavage to produce a 19 kDa N-terminal product, ShhN. During this reaction, cholesterol is attached to the C terminus of ShhN4
. In a separate reaction, Hhat catalyzes attachment of palmitate to the N-terminal cysteine of ShhN via an amide bond4,5
. Palmitoylation of Shh plays a critical role in regulating the signaling potency of Shh in cells6,7
. Hhat knockout mice and palmitoylation-deficient Shh transgenic mice exhibit developmental defects similar to those observed in Shh knockout mice7
. Thus, Hhat presents an attractive, novel target to block Shh signaling.
Hhat is a member of the membrane bound O-acyl transferase (MBOAT) family of proteins8
. Due to the presence of multiple transmembrane domains, molecular and structural characterization of this family in general, and Hhat in particular, has been limited5,9
. In an effort to discover a small-molecule inhibitor of Hhat, we conducted a high-throughput screen using a peptide-based assay to monitor Hhat-mediated Shh palmitoylation. We screened a library of 63,885 unique structures (Supplementary Results
, Supplementary Table 1
). A secondary screen was performed on 648 molecules, using the peptide-based assay and an orthogonal cell viability assay, to yield 95 confirmed hits. Four compounds, RU-SKI 39 (1
), 41 (2
), 43 (3
) and 50 (4
), were selected based on their low IC50
values and drug-like scaffold (, Supplementary Figs. 1 and 2
Structures and IC50 values of the Hhat inhibitor hit compounds.
The four Hhat inhibitors were re-tested in an in vitro
palmitoylation assay using ShhN protein. Each compound at 12.5 μM inhibited Hhat-mediated palmitoylation of ShhN by 40–80% (). ShhN C24A, a mutant Shh protein that cannot incorporate palmitate, and Hhat D339A, an inactive Hhat mutant9
, served as negative controls. Inhibition of ShhN palmitoylation was specific to the RU-SKI compounds, since two structurally related molecules, C-1 (5
) and C-2 (6
; Supplementary Fig. 3
), did not affect ShhN palmitoylation (). We next analyzed the kinetics of RU-SKI 43 inhibition of ShhN palmitoylation in vitro
using purified Hhat and ShhN. RU-SKI 43 behaved as an uncompetitive inhibitor (Ki
=7.4 μM) with respect to Shh, and as a noncompetitive inhibitor (Ki
=6.9 μM) with respect to 125
Hhat inhibition was then analyzed using COS-1 cells expressing HA-tagged Hhat and Shh. Cells were pre-treated for 16 h with 25 μM compound or DMSO, then labeled with 125
I- iodo-palmitate, a radiolabeled palmitate analog5,10,11
. RU-SKI 39, 41 and 43 strongly inhibited radiolabel incorporation into ShhN; RU-SKI 50 was less effective in cells (, Supplementary Fig. 4a
). Based on its ability to function as an Hhat inhibitor in vitro
and in cells, we focused on RU-SKI 43. Dose-dependent inhibition of Shh palmitoylation was observed following only 5 h of treatment (, Supplementary Fig. 4c
). Importantly, no effect on Shh palmitoylation was observed when cells were incubated with 10 μM C-2 (Supplementary Fig. 4 b,c
Several lines of evidence suggest that inhibition by RU-SKI 43 is specific to Shh palmitoylation. Neither palmitoylation of H-Ras and Fyn nor myristoylation of c-Src was affected by treatment of cells with the compound (). Treatment of cells with RU-SKI 43 had no effect on fatty acylation of Wnt3a12
by Porcupine, another member of the MBOAT family, whereas Wnt C59 (a Porcupine inhibitor) blocked radiolabel incorporation (). Overexpression of Hhat reduced the ability of RU-SKI 43 to inhibit Shh palmitoylation in transfected COS-1 cells, whereas overexpression of Porcupine had no effect (Supplementary Fig. 5
). Moreover, RU-SKI 43 inhibited palmitoylation of Shh by endogenous Hhat in COS-1 cells (Supplementary Fig. 6
). Finally, RU-SKI 43 did not alter Shh autoprocessing, steady-state levels of Shh and Hhat, or subcellular localization of Shh and Hhat (, Supplementary Fig. 7
). Taken together, these data support the contention that RU-SKI 43 specifically inhibits Hhat but not other fatty acyl transferases.
Inhibition of Hhat is predicted to block Shh signaling in cells. We used three cell-based systems to test the specificity of RU-SKI 43 for the Shh pathway. First, NIH 3T3 cells were cotransfected with plasmids encoding Shh, a Gli-responsive Firefly luciferase reporter, and Renilla luciferase as a control. Increased luciferase production was observed, compared to cells transfected with a mutant Gli-luciferase plasmid, indicative of Gli1 activation (). Importantly, addition of 10 μM RU-SKI 43 or LDE225, a Smoothened (Smo) inhibitor13
, blocked luciferase activation, consistent with Shh pathway inhibition, whereas C-2 had no effect (). These data suggest that RU-SKI 43 blocks autocrine Shh signaling in cells.
We then utilized Shh Light II cells that stably express a Gli1 reporter plasmid14
. RU-SKI 43 had no effect on the ability of the Smo agonist SAG or Shh (C42II), a recombinant, hydrophobic variant of Shh, to activate Gli1 in Shh Light II cells (). Moreover, RU-SKI 43 had no effect on Shh signaling in SuFu−/−
cells, where Shh signaling is activated downstream of Smo (). These findings imply that RU-SKI 43 inhibits Shh signaling at the level of the Shh ligand.
The effects of Hhat inhibition on paracrine Shh signaling were examined using C3H10T1/2 cells, a Shh-reporter cell line that produces alkaline phosphatase (AP) in response to Shh15
. Upon coculture with COS-1 cells expressing Shh and Hhat, C3H10T1/2 cells produced AP (). AP activity was reduced to baseline when cocultured cells were treated with 10 μM RU-SKI 43 (). The effect of RU-SKI 43 was not due to inhibition of BMPs, which can cooperate with Shh16
, as the BMP inhibitor Noggin had no effect on AP activity. These findings provide proof of concept that Hhat inhibitors block Shh signaling.
In this study, we describe the first example of a small-molecule that inhibits the activity of Hhat in vitro
and blocks Hhat-mediated Shh palmitoylation in cells. RU-SKI 43 exhibits specificity for Hhat in cells: it did not affect fatty acylation mediated by other acyltransferases and its inhibitory effect was reversed by overexpression of Hhat (Supplementary Fig. 5
). RU-SKI 43 decreased Gli1 activation in a Shh-driven cell-based reporter system, suggesting that Hhat inhibition accounts, at least in part, for the reduction of Shh signaling in cells, although we cannot exclude the possibility that off-target effects also occur. We were not able to rescue the inhibitory effect of RU-SKI 43 in Shh-transfected cells with SAG or Shh (C24II; Supplementary Fig. 8
). Even in untreated Shh-expressing cells, neither SAG (Supplementary Fig. 8
) nor overexpression of Smo17
activated Gli1 to levels higher than those achieved with Shh alone. Since these cells continuously produce Shh, it is likely that the Shh receptor Patched is saturated with ligand (Patched can bind both palmitoylated and nonpalmitoylated Shh18
), and further stimulation with exogenous Shh (C24II) or SAG cannot occur. However, RU-SKI 43 had no effect on Shh signaling in SuFu−/−
cells, nor did it affect the ability of either SAG or Shh (C24II) to activate Gli1 in Shh Light II cells (). These findings position Hhat inhibition as a novel method to turn off hedgehog signaling.
There is great interest in Shh signaling as a clinically relevant target in human malignancies3
. Several compounds that block Shh signaling have been described, including Robotnikin, which binds to Shh, and inhibitors of Smo and Gli, which are downstream signaling components of the Shh pathway19,20
. The Hhat inhibitor is unique, as it hits upstream, directly at palmitoylation of the Shh ligand. As a result, Hhat inhibition could offer a novel treatment modality for pancreatic and gastric cancers and a subset of sarcomas, cancers characterized by Shh overexpression and extremely poor prognoses3
. The short half-life of RU-SKI 43 in vivo
= 17 min in mouse plasma after IV administration) limits our ability to perform toxicity studies in animals. However, in most “normal” adult cells, Shh expression and signaling is turned off. Thus, Hhat inhibition could be a valid option for treatment of cancers characterized by Shh overexpression, and offers several potential advantages. Hhat inhibition would block all downstream Shh signaling, including noncanonical, Smo-independent pathways21,22
. Moreover, Hhat is intolerant to a wide variety of mutations9
. It is thus possible that drug resistance might be less of a concern, although long-term exposure of cancer cells has not yet been evaluated. Furthermore, Hhat inhibition could be used in combination with Smo inhibition to attack both Shh-producing and Shh-responding cells. This could increase the efficiency of therapies, and potentially delay the development of drug resistance by patients treated with Smo inhibitors23
. The work described in this study suggests that there is great potential for producing additional, optimized derivatives of Hhat inhibitors for future therapeutic development.