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org/biochemistry Article

Nanomolar, Noncovalent Antagonism of Hedgehog Cholesterolysis:

Exception to the “Irreversibility Rule” for Protein Autoprocessing
Inhibition
Andrew G. Wagner, Robert T. Stagnitta, Zihan Xu, John L. Pezzullo, Nabin Kandel, José-Luis Giner,
Douglas F. Covey, Chunyu Wang, and Brian P. Callahan*
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ABSTRACT: Hedgehog (Hh) signaling ligands undergo carboxy

terminal sterylation through specialized autoprocessing, called
cholesterolysis. Sterylation is brought about intramolecularly in a
single turnover by an adjacent enzymatic domain, called HhC,
which is found in precursor Hh proteins only. Previous attempts to
identify antagonists of the intramolecular activity of HhC have
yielded inhibitors that bind HhC irreversibly through covalent
mechanisms, as is common for protein autoprocessing inhibitors.
Here, we report an exception to the “irreversibility rule” for
autoprocessing inhibition. Using a fluorescence resonance energy transfer-based activity assay for HhC, we screened a focused library
of sterol-like analogues for noncovalent inhibitors and identified and validated four structurally related molecules, which were then
used for structure−activity relationship studies. The most effective derivative, tBT-HBT, inhibits HhC noncovalently with an IC50 of
300 nM. An allosteric binding site for tBT-HBT, encompassing residues from the two subdomains of HhC, is suggested by kinetic
analysis, mutagenesis studies, and photoaffinity labeling. The inhibitors described here resemble a family of noncovalent, allosteric
inducers of HhC paracatalysis which we have described previously. The inhibition and the induction appear to be mediated by a
shared allosteric site on HhC.

■ INTRODUCTION
Hedgehog (Hh) ligands, the extracellular signaling factors
involved in embryo development and many types of
cancers,1−8 appear unique in their post-translational mod-
ification by cholesterol.9,10 Lipidation occurs during Hh
biosynthesis through an autoprocessing reaction called
cholesterolysis. A partially characterized ∼25 kDa C-terminal
domain, HhC, present in the precursor forms of Hh serves as
the intramolecular catalyst for cholesterolysis (Figure 1, lef t).
HhN-chol, the cholesterylated product, is released from HhC
and undergoes additional lipidation as it matures into the Hh
ligand. 10,11 Deactivating mutations in HhC result in
endoplasmic reticulum (ER) retention and ER-associated
protein degradation of the Hh precursor,12,13 thereby
suppressing downstream Hh signaling.12,14
We have begun pursuing selective antagonists of HhC that
mimic the effects of deactivating HhC mutations.15−17 We
view HhC inhibitors as novel directions for therapeutic
intervention in human cancers, particularly for malignancies
driven by the Hh ligand and where existing Hh blocking drugs
have lost efficacy.7,18,19 Inhibitors of HhC cholesterolysis hold
additional promise as chemical tools to understand the basic
biology of Hh signaling. HhC inhibitors may also stabilize
HhC’s dynamic structure,20 enabling the structural character-
ization of this unusual autoprocessing element.21 Of HhC’s
two subdomains, the Hedgehog intein (HINT) and the sterol
recognition region (SRR), only the HINT has its three-
dimensional structure resolved by experimental structural
methods.22−24
HhC and the protein substrate, HhN, are covalently linked
in the Hh precursor protein, and this presents a special
challenge for antagonism. There are substantial chelate-type
connectivity effects between the HhC catalyst and HhN
substrate that must be overcome.25−28 Additional challenges
arise from the limited turnover inherent to protein
autoprocessing, typically a single turnover. Suppressing a single
turnover by an intramolecular catalyst demands inhibitors that
are bound tightly; dissociation permits autoprocessing to
begin, potentially diminishing the inhibitor recognition.
Accordingly, small-molecule antagonists of Hh cholesterolysis
and related forms of protein autoprocessing nearly all depend
Received: October 20, 2021
Revised: November 29, 2021

© XXXX American Chemical Society https://doi.org/10.1021/acs.biochem.1c00697

A Biochemistry XXXX, XXX, XXX−XXX
Biochemistry pubs.acs.org/biochemistry Article

Figure 1. HhC functions as a dedicated self-cleaving, protein carboxy sterol ligase of the Hh ligand. (Left) Hh autoprocessing has two steps: (Step
1) the side-chain thiol of a conserved cysteine residue at position 1 of HhC attacks the amide bond of the preceding residue, glycine, to form an
internal thioester and (Step 2) binding of cholesterol to HhC triggers the transesterification to the lipid’s 3β-OH group, generating a
cholesterylated Hh ligand and displacing HhC. The two subdomains of HhC are indicated as follows: HINT (Hedgehog/Intein) and SRR. (Right)
Representative structures of HhC inhibitors reported here and of HhC paracatalytic inducers described previously.

Figure 2. Library screening identifies structurally related noncovalent HhC inhibitors. (A) Tripartite HhC reporter construct, C−H−Y, where HhC
is flanked by CFP and YFP. HhC cholesterolysis results in a loss of the FRET signal as H−Y and cholesterylated C separate. (B) Preliminary
inhibitory activity with compound 1. Traces show screen data with C−H−Y in the presence of 1.5 μM cholesterol and 50 μM 1 (red), along with
controls: C−H−Y in buffer only (black, solid) and C−H−Y with cholesterol (black dotted). (C) Chemical structures and IC50 values of screen hit
compounds 1−4. (D) Dose−response curves of inhibitor compounds 1−4 (green, blue, orange, and red, respectively) against C−H−Y in the presence
of 1.5 μM cholesterol.

on binding the precursor protein irreversibly through covalent

interactions.16,17,29−32
Here, we describe an exception to the empirical “irrever-
sibility rule” for protein autoprocessing inhibition. Screening a
focused library of sterol-like analogues, we identified the first
noncovalent, tight-binding inhibitors of HhC cholesterolysis.
The most effective inhibitor has an IC50 value of 300 × 10−9
M. A mixed inhibition mode is suggested by kinetic analysis
and by photoaffinity labeling (PAL). Computational modeling
and mutagenesis of HhC suggest that inhibitors bind an
allosteric site composed of residues from HhC’s two
subdomains. We note a striking resemblance between the
inhibitors of HhC cholesterolysis described here and a family
of noncovalent, inducers of HhC paracatalysis, which we have
described previously (Figure 1, right).15 The inhibitor/
activator duality appears to be mediated by the same allosteric
site in HhC.
B https://doi.org/10.1021/acs.biochem.1c00697
■ RESULTS AND DISCUSSION
Library Screening Identifies a Family of HhC
Inhibitors. We tested 1187 steroid analogues from Chem-
Bridge as potential inhibitors of Hh cholesterolysis using our
continuous light-to-dark fluorescence resonance energy trans-
fer (FRET) activity assay.15−17 The FRET reporter construct,
C−H−Y, has the Drosophila melanogaster HhC flanked by cyan
fluorescent protein (CFP) (C) and yellow fluorescent protein
(YFP) (Y) as the FRET donor and acceptor, respectively.
FRET is measured as the ratio of emissions of 540 nm/460 nm
after excitation at 400 nm. Cholesterolysis results in the loss of
FRET as C−H−Y autoprocesses into HhC-YFP (H−Y) and
sterylated CFP (C-chol) (Figure 2A).
Standard reaction conditions for compound screening using
C−H−Y in 96-well plates have been described.15 Briefly, wells
contained Ni-NTA purified C−H−Y (0.1 μM) in cholester-
olysis buffer (Tris, 20 mM, pH 7.1; Fos-Choline 12, 1.5 mM;
Biochemistry XXXX, XXX, XXX−XXX
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Figure 3. Inhibition kinetics and photoactive sterol labeling suggest that the substrate and inhibitor can be bound by HhC simultaneously. (A)
Mixed-type inhibition kinetics apparent in the presence of inhibitor 1. Initial velocity of C−H−Y cholesterolysis with increasing concentrations of
compound 1 shows progressively lower maximum rate values. (B) Cravatt’s tSP, used here as a photoactivatable substrate analogue of HhC. (C)
Photolabeling of HhC by tSP persists with saturating concentrations of 1, consistent with noncompetitive interactions. All samples contained 5 μM
HhC and 50 μM azide-linked fluorophore; other components were varied as indicated. (Top) Photolabeling was assessed by SDS-PAGE and
fluorescent gel imaging. (Bottom) Coomassie stain for total HhC protein. Controls: Lanes 1−5, 8, and 9. Experimental: Lane 6, photolabeled HhC
by tSP in the absence of 1. Lane 7, diminished photolabeling of HhC by tSP with saturating cholesterol. Lane 10, persistent tSP PAL in the presence
of saturating compound 1.

ethylenediaminetetraacetic acid (EDTA), 5 mM; and dithio-
threitol (DTT), 1 mM) in a total volume of 100 μl. Library
compounds were added from 10 mM dimethyl sulfoxide
(DMSO) stocks to a final concentration of 50 μM (0.5%
DMSO v/v). Prior to initiating the reaction with cholesterol,
plates were incubated for 30 min at 30 °C while FRET
readings were recorded every 2 min. Data collected during this
delay were monitored to flag compounds that interfered with
the C−H−Y signal. Cholesterolysis was initiated by adding
cholesterol from an ethanol stock (1.5 μM final, 4% ethanol v/
v), and FRET was recorded every 2 min for 1.5 h. All plates
contained 16 controls wells, 8 with C−H−Y plus cholesterol,
representing 100% activity, and 8 wells with C−H−Y minus
cholesterol, representing 100% inhibition. A total of five
compounds from the library displayed inhibitory activity
toward C−H−Y. In Figure 2B, we show kinetic traces for
C−H−Y in the presence of 1, the most effective inhibitor from
the screen along with intraplate controls. The remaining hit
molecules possessed structural features similar to that of
compound 1: N-tert butyl pyrrole, aliphatic cyclic amine, and
an aryl group (Figure 2C).
Four of the five preliminary hits 1−4 were available for
repurchase and were retested by dose−response inhibition
experiments using C−H−Y. The compounds were also
evaluated by end-point kinetic analysis using sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig-
ure S1). For the dose−response experiments, compounds were
dissolved in DMSO to 10 mM and then diluted from 100 to
0.015 μM into wells containing C−H−Y (0.1 μM) over a 9-
point titration. To initiate the assay, cholesterol was added at
its KM value of 1.5 μM,33 and FRET readings were collected
every 2 min. Initial velocity values were calculated as the slope
of the linear phase of the FRET decay at each inhibitor
concentration. Relative velocity (RV) was calculated by
dividing the initial velocity with the inhibitor by the initial
velocity in the absence of the inhibitor. Plots of the RV values
C https://doi.org/10.1021/acs.biochem.1c00697
as a function of inhibitor concentration (Figure 2D) were used
to calculate the IC50 values (Supporting Information). IC50
values for 1−4, obtained from ≥3 independent experiments,
are as follows: 1, 2.0 μM; 2, 4.0 μM; 3, 12 μM; and 4, 81 μM.
Kinetic Evidence of Mixed-Type Inhibition: Com-
pound 1 Diminishes Catalysis through Effects on Both
kmax and KM. We assessed the changes in the kinetic
parameters, kmax and KM, for cholesterolysis using C−H−Y
in the presence of 1 to understand the mode of inhibition. The
above dose−response experiments indicated an IC50 value of
2.0 μM. In the kinetic experiments described here, 1 was added
to C−H−Y at concentrations of 0.8, 2.5, 5.0, 10, and 25 μM,
corresponding to 0.4−12.5 × the IC50 value. To those wells,
we titrated substrate cholesterol from 0.2 to 100 μM. In Figure
3A, the initial velocity is plotted as a function of increasing
cholesterol concentration. Initial velocity data were fit to a
standard Michaelis−Menten equation (Supporting Informa-
tion). Analysis of the changing kinetic values ±1 suggested
mixed-type inhibition, where both KM and kmax values are
depressed by the inhibitor. Calculated KM values under the
conditions tested were as follows: no inhibitor, KM 1.2 μM;
with 0.8 μM 1, KM 1.1 μM; with 2.5 μM 1, KM 1.6 μM; with 10
μM 1, KM 2.6 μM; and with 25 μM 1, KM 4.0 μM. The
calculated KM values appear modestly sensitive to increasing
concentrations of 1, with an increase in KM of 3-fold following
a 25-fold increase in the concentration of 1. There was a
clearer concentration-dependent suppression of kmax with the
addition of 1. In the absence of an inhibitor, kmax for
cholesterolysis is 1.9 × 10−3 s−1. With 0.8 μM 1, kmax is reduced
to 1.3 × 10−3 s−1, and at 2.5 μM 1, kmax is reduced further to
0.9 × 10−3 s−1. Increasing the concentration of 1 to 10 and 25
μM decreases kmax to 0.48 × 10−3 s−1 and 0.29 × 10−3 s−1,
respectively. In summary, inhibitor 1 interferes mainly with the
turnover rate along with a lesser but reproducible increase in
the KM for substrate cholesterol. These results are consistent
with mixed-type inhibition.
Biochemistry XXXX, XXX, XXX−XXX
Biochemistry pubs.acs.org/biochemistry Article

Figure 4. Nanomolar HhC inhibitor and provisional binding site by computational modeling and point mutagenesis. (A) Chemical structures of
HhC inhibitor analogues, along with IC50 values toward cholesterolysis. (B) Proposed ternary complex of the substrate, the inhibitor, and HhC.
Color key: inhibitor 8 or tBT-HBT, yellow; cholesterol, magenta; HINT subdomain, cyan; and SRR, purple. Point mutations and corresponding KD
values are shown along with each mutant’s cholesterolysis activity (active/inactive). We note that mutations have been made at several homologous
positions by Ondrus and her associates (refs 20 and 21) in human Sonic HhC (C1, H72, L73, L128, C143, Y144, H154, R161, H193, W194, and
Y195), and the resulting effects on cholesterolysis activity accord well with the behavior of the mutants evaluated here. Mutation color key: red,
complete loss of inhibitor binding; orange, 60−300 fold loss of inhibitor binding; and gray, <10-fold loss of inhibitor binding affinity.

Photolabeling of HhC by a Diazirine-Modified
Substrate Analogue Persists with Saturating Concen-
trations of 1, Suggesting a Ternary HhC−Substrate−
Inhibitor Complex. Mixed-type inhibitors can be bound by
enzymatic catalysts both in the presence and in the absence of
the substrate. To further probe for ternary “E-S-I” complex
formation, suggested by the above kinetic analysis, we applied
PAL with the trans-sterol probe (tSP)34 as an active-site
modifier of HhC (Figure 3B). We reasoned that if inhibitor 1
and tSP were bound by HhC at separate sites, PAL of HhC by
tSP would likely be insensitive to added 1; if, however,
inhibitor 1 and tSP bind HhC competitively, the addition of
saturating 1 should diminish the PAL of HhC. tSP contains a
diazirine group at C-6 of the sterol nucleus. Alkyl diazirine
groups decompose under UV light to produce N2 and a
reactive diazo intermediate or, alternatively, a carbene.35
Reaction with nearby protein residues generates a covalent
protein/probe adduct.20,36,37 The sterol side chain in tSP
includes an alkyne group to facilitate the analysis of PAL
through the use of copper click chemistry.34,38
We established the utility of tSP as a cholesterol analogue for
HhC in two ways. First, we tested tSP as an alternative HhC
substrate using the C−H−Y system. The results demonstrated
that tSP was readily accepted as a substrate, with a KM value of
6.8 μM and a kmax value of 4.2 × 10−3 s−1 (Figure S2A,B),
differing by < 2-fold in kmax/KM compared with cholesterol.
Next, we mapped the tSP binding site on HhC using tandem
mass spectrometry (MS/MS) analysis of an HhC/tSP adduct.
Earlier truncation studies of HhC indicated that the SRR
resides in the final 70−90 amino acids of HhC.22 To simplify
the mapping study, we used a product form of HhC, post-
autoprocessing. Details on the preparation of product HhC
and photolabeling to form the HhC/tSP adduct are provided
in the Supporting Information (Figure S2C). Preliminary MS/
MS analysis of purified HhC/tSP indicated the tSP attachment
to HhC peptide EQLHSSPK (HhC residues 174−181). This
D https://doi.org/10.1021/acs.biochem.1c00697
site maps to the SRR of HhC. In summary, tSP furnishes an
active-site PAL probe for HhC.
Confident in the utility of tSP, we carried out an
experimental test for HhC-tSP-1 ternary complex formation.
We sought to determine whether the addition of 1 would
diminish the active-site photolabeling of HhC by tSP. We
mixed HhC (5 μM) and tSP (10 μM) ± saturating inhibitor 1
(200 μM), followed by UV irradiation to activate PAL and
then click chemistry labeling to attach an azide-modified
fluorophore (50 μM) to the alkyne group on tSP. A covalent
HhC/tSP/fluorophore complex was detected by SDS-PAGE
with UV gel imaging. In Figure 3C, the results of the control
reactions with tSP, HhC, and the azide-modified fluorophore
are shown first in lanes 1−7. Out of this set, a fluorescence
signal is apparent in lane 6 only. The sample in lane 6 was
subjected to UV irradiation and click chemistry. The covalent
HhC/tSP/fluorophore species detected in lane 6 is absent in
lane 3. The lane 3 sample, which lacked UV irradiation, shows
that HhC in complex with tSP dissociates without PAL. The
control sample in lane 7 is identical to the lane 6 sample except
that it also included cholesterol (200 μM). The absence of the
HhC/tSP/fluorophore complex in lane 7 demonstrates that
active site binding by cholesterol competitively blocks the PAL
by the substrate analogue, tSP. In the second sample set, Figure
3C lane 8−10, we examined the influence of inhibitor 1 on
HhC/tSP photolabeling. Samples in lane 8−9 are controls to
test for nonspecific fluorophore labeling promoted by added 1;
no evidence was observed. The key experimental sample is in
lane 10, containing HhC, tSP, plus saturating 1, and an azide-
modified fluorophore. Despite saturating 1, this inhibitor of
HhC cholesterolysis did not significantly diminish active site
photolabeling by tSP. The persistence of photolabeling of HhC
by substrate analogue tSP in the presence of inhibitor 1 is
consistent with the mode-of-inhibition analysis above:
cholesterolysis antagonism involves a catalytically inert ternary
complex of HhC, sterol, and 1.
Biochemistry XXXX, XXX, XXX−XXX
Biochemistry pubs.acs.org/biochemistry
Analogue Testing Identifies tBT-HBT as the First
Nanomolar Inhibitor of HhC Cholesterolysis. To explore
the structure−activity relationship (SAR) with this novel class
of HhC inhibitors, we prepared and tested several analogues of
1−4 (Figure 4A). The first analogue, 5, incorporates the
benzothiazole and N-t-butyl pyrrole groups of 1 and the
homopiperazine of 2. As an inhibitor of cholesterolysis, the
molecule was improved 2−3 fold over 1 and 2, with an IC50 of
1.2 μM toward C−H−Y in the FRET assay. In analogue 6, we
replaced the t-butyl group of 5 with a hydrogen atom; this
truncation resulted in a substantially weakened inhibitor, with
an IC50 of 61 μM. The t-butyl group was restored in analogues
7 and 8, while the heterocycle was switched from the pyrrole to
1,3 oxazole 7 or thiophene 8. Compound 7 was indistinguish-
able from 5 as a cholesterolysis inhibitor. Thiophene 8
provided a ∼ 5-fold increase in binding compared with
analogue 6. Analogue 8 is thereby the first submicromolar
noncovalent inhibitor of HhC cholesterolysis, with an IC50 of
300 nM. Four additional analogues, 9−12, based on 8 were
prepared next. The t-butyl group was truncated in 9 or
substituted in 10−12. Each change diminished the inhibitory
activity to varying degrees, reinforcing the importance of the t-
butyl group for binding by HhC. Analogue 8, hereafter called
tBT-HBT, emerged from this study as the best inhibitor,
improving the IC50 by ∼10-fold from the top hit of the library
screen.
Inhibitor Binding by HhC Requires Residues from the
SRR and HINT Subdomains. We used the nM inhibitor tBT-
HBT (8) along with a panel of HhC mutants to probe for the
inhibitor binding site. To guide the mutagenesis, a computa-
tional model of the ternary complex, HhC−substrate−
inhibitor, was prepared with D. melanogaster using the program
MODELLER (Supporting Information).39 Results of virtual
docking40 of substrate cholesterol and tBT-HBT into the
predicted D. melanogaster HhC structure are shown in Figure
4B. Single-point mutations in HhC were introduced based in
part on this predicted ternary complex as well as on residue
conservation across HhC sequences.
The binding affinity of tBT-HBT by HhC and HhC mutants
was characterized from an inhibitor-dependent enhancement
in FRET with the C−H−Y reporter. By using ΔFRET to
report the inhibitor binding, we could evaluate a greater variety
of HhC mutants, including mutants that lack catalytic activity.
In two previous studies involving irreversible inhibitors of
HhC, we observed that the covalent adduct formation with C−
H−Y caused a FRET quenching.16,17 The structural changes in
HhC that underlie these small molecule induced FRET
changes in C−H−Y are not yet known. The FRET
enhancement observed here from C−H−Y with added tBT-
HBT was not apparent in C−Y, a FRET control construct
where HhC is omitted. A FRET enhancement was also absent
with C-HINT-Y and C-SRR-Y, truncation mutants of HhC
where one of the two subdomains of HhC is deleted. The
insensitivity of C−Y, C-HINT-Y, and C-SRR-Y to added tBT-
HBT (Figure S3) suggests that inhibitor recognition requires
full-length HhC.
Results of our mutagenesis study on tBT-HBT binding by
HhC are summarized in Figure 4B. Experimental data of
ΔFRET plotted as a function of the increasing tBT-HBT
concentration for wild-type and HhC point mutants is found in
Figure S4. All 13 point mutations weakened the tBT-HBT
affinity for HhC. We grouped the mutational effects into three
general categories: <10-fold loss of the binding affinity (Figure
E https://doi.org/10.1021/acs.biochem.1c00697
Article
4B, gray); between 60- and 300-fold loss of the binding affinity
(Figure 4B, orange); and abolished inhibitor binding (Figure
4B, red). The most severe loss in tBT-HBT binding mapped to
mutations in the HINT domain (L73, F86, C143, and Y144,
colored red) lining the predicted tBT-HBT binding site in our
computational model. Mutations that produced a large but not
total loss of tBT-HBT binding mapped to the SRR subdomain
(H154A, R161A, H193Y, and W194A, colored orange). Two
mutations in HINT (C1A and H72A, colored gray) and two in
the SRR (G191D and Y195F, colored gray) resulted in smaller
losses of the tBT-HBT affinity. The compromised binding in
H154A, R161A, and H193A, which are distal to the predicted
tBT-HBT binding site, lack clear explanation, indicating that
the computational model needs further improvement.
Collectively, the results support the idea that tBT-HBT
binding by HhC involves SRR and HINT subdomain
interactions.
■ CONCLUDING REMARKS AND
INHIBITOR/ACTIVATOR DUALITY
HhC, the C-terminal half of Hedgehog precursor proteins,
possesses intramolecular cholesterolysis activity that is
necessary to release and sterylate the adjacent Hh signaling
ligand, HhN. The significance of this initiating step in the Hh
signaling pathway is three-fold: the transformation appears
unique to Hh family proteins; it lies upstream of all known
receptor interactions; and lastly, sterylation provides a vital
membrane anchor that regulates Hh ligand diffusion and
physiological signaling. Small molecules that manipulate HhC
autoprocessing could help unravel the complex biology of Hh
signaling by providing an off switch near the pathway’s origin.
The promise of HhC antagonists/agonists extends to
therapeutics in treating sporadic tumors and congenital
disorders where Hh ligand signaling is dysregulated.
Because of their unique action, protein autoprocessing
domains represent intrinsically hard-to-hit targets for non-
covalent antagonism. There is a large entropic advantage
associated with intramolecular catalysis that must be overcome,
and the biological function of protein autoprocessing events
such as Hh cholesterolysis requires a single turnover only.
Published examples of autoprocessing inhibitors are dominated
by irreversible antagonism, and the few examples of non-
covalent inhibitors show modest binding affinity.
The findings reported here represent an important exception
to the “irreversibility rule” for autoprocessing inhibition. We
speculate that the most effective compound, tBT-HBT (8), is
bound by HhC near the interface of the SRR and HINT,
trapping these two subdomains in a conformation that is
capable of binding cholesterol but incapable of catalyzing
cholesterolysis. To our knowledge, tBT-HBT is the tightest
binding noncovalent inhibitor of HhC cholesterolysis, and it
appears to be unrivaled among the reported noncovalent
inhibitors of related forms of protein autoprocessing.
In an earlier report, we described reversibly bound small
molecules targeting HhC that activate cholesterol-independent
autoproteolysis, a nonnative paracatalytic reaction. The most
effective HhC activator, HAC8, had an AC50 of 9 μM and
accelerated the rate of Hh precursor autoproteolysis by 225-
fold.15 The structures of HAC8 and tBT-HBT are strikingly
similar (Figure 1, right). The compounds emerged from the
same chemical screen, and not surprisingly, HAC8 and tBT-
HBT seem to share a binding site on HhC (Figure S5). Thus,
depending on the bound effector, HhC can accelerate the
Biochemistry XXXX, XXX, XXX−XXX
Biochemistry pubs.acs.org/biochemistry
paracatalytic autoproteolysis of the precursor, as with HAC8,
or suppress the rate of native cholesterolysis, as with tBT-HBT.
A single allosteric site that can toggle between activation and
inhibition depending on the effector is intriguing and finds
precedent in selected metabolic and signaling enzymes.41−43
Despite the descriptions of protein autoprocessing domains as
primitive or protozymes, left over from an earlier age, we
continue to find close parallels with extant enzymes in terms of
catalytic mechanisms and inhibition strategies.
■
*
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.biochem.1c00697.
Compound screening; protein purification; kinetic
analysis; creation of HhC mutants; chemical synthesis;
and compound NMR and liquid chromatography mass
spectroscopy (PDF)
Accession Codes
C-terminal domain of Drosophila melanogaster Hedgehog
protein: UniProtKBQ0293
■
AUTHOR INFORMATION
Corresponding Author
Brian P. Callahan − Department of Chemistry, Binghamton
University, State University of New York, Binghamton, New
York 13902, United States; orcid.org/0000-0003-4826-
0162; Email: callahan@binghamton.edu
Authors
Andrew G. Wagner − Department of Chemistry, Binghamton
University, State University of New York, Binghamton, New
York 13902, United States; orcid.org/0000-0003-4786-
7747
Robert T. Stagnitta − Department of Chemistry, Binghamton
University, State University of New York, Binghamton, New
York 13902, United States
Zihan Xu − Department of Chemistry, Binghamton University,
State University of New York, Binghamton, New York 13902,
United States
John L. Pezzullo − Department of Chemistry, SUNY-ESF,
Syracuse, New York 13210, United States
Nabin Kandel − Department of Biological Sciences, Center for
Biotechnology and Interdisciplinary Studies, Rensselaer
Polytechnic Institute, Troy, New York 12180, United States
José-Luis Giner − Department of Chemistry, SUNY-ESF,
Syracuse, New York 13210, United States; orcid.org/
0000-0002-8092-5139
Douglas F. Covey − Department of Developmental Biology,
Taylor Family Institute for Innovative Psychiatric Research,
St. Louis, Missouri 63110, United States; orcid.org/0000-
0002-9316-8683
Chunyu Wang − Department of Biological Sciences, Center for
Biotechnology and Interdisciplinary Studies, Rensselaer
Polytechnic Institute, Troy, New York 12180, United States;
orcid.org/0000-0001-5165-7959
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.biochem.1c00697
F https://doi.org/10.1021/acs.biochem.1c00697
Article
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
National Cancer Institute (grant R01 CA206592); Department
of Defense (grant W81XWH-14-1-0155); NSF (grant CHE-
1048516); and NIH grant (S10 OD012254).
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
We acknowledge the generous support from the NIH:
CA206592 to B.P.C. and C.W.; GM143714 to JLG;
HL067773 to D.F.C.; and a CURE fellowship from the NCI
to A.G.W. We thank Callahan lab members Xiaoyu Zhang and
Dan Ciulla for help and encouragement and Dr. Juergen
Schulte for expert advice with NMR. We are grateful to Dr.
Alistair Lees and Dr. Lynn Schmitt for access to the
photolabeling equipment. We thank Carl Smith and Polina
Holubovska for help with initial compound screening. We
acknowledge the aid of the Proteomics and Metabolomics
Core Facility at Weill Cornell Medicine for proteomics MS/
MS experiments.
■ ABBREVIATIONS
Hh, Hedgehog; HhC, Hedgehog C-terminal domain; HhN,
Hedgehog N-terminal domain; HINT, Hedgehog intein
subdomain; SRR, sterol recognition region; C−H−Y, CFP−
HhC−YFP; RV, relative velocity; PAL, photoaffinity labeling;
tSP, trans sterol probe; SAR, structure−activity relationship;
tBT-HBT, compound 8; DMSO, dimethyl sulfoxide
■
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