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. 2023:685:1-41.
doi: 10.1016/bs.mie.2023.03.001. Epub 2023 Apr 19.

Paracatalytic induction: Subverting specificity in hedgehog protein autoprocessing with small molecules

Daniel A Ciulla  1 , Zihan Xu  2 , John L Pezzullo  3 , Patricia Dranchak  4 , Chunyu Wang  5 , José-Luis Giner  3 , James Inglese  6 , Brian P Callahan  2
Affiliations

Paracatalytic induction: Subverting specificity in hedgehog protein autoprocessing with small molecules

Daniel A Ciulla et al. Methods Enzymol. 2023.

Abstract

Paracatalytic inducers are antagonists that shift the specificity of biological catalysts, resulting in non-native transformations. In this Chapter we describe methods to discover paracatalytic inducers of Hedgehog (Hh) protein autoprocessing. Native autoprocessing uses cholesterol as a substrate nucleophile to assist in cleaving an internal peptide bond within a precursor form of Hh. This unusual reaction is brought about by HhC, an enzymatic domain that resides within the C-terminal region of Hh precursor proteins. Recently, we reported paracatalytic inducers as a novel class of Hh autoprocessing antagonists. These small molecules bind HhC and tilt the substrate specificity away from cholesterol in favor of solvent water. The resulting cholesterol-independent autoproteolysis of the Hh precursor generates a non-native Hh side product with substantially reduced biological signaling activity. Protocols are provided for in vitro FRET-based and in-cell bioluminescence assays to discover and characterize paracatalytic inducers of Drosophila and human hedgehog protein autoprocessing, respectively.

Keywords: Autoprocessing; Cholesterol; HTS; Hedgehog; Paracatalysis; Specificity.

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Figures

Fig. 1
Fig. 1
Catalytic fidelity vs paracatalytic induction. (Left) Molecular specificity at the ground state and at the transition state exhibited by a native enzyme reduces side reactions. Nonnative reactions of the physiological substrate (red path) and transformations involving alternative substrate (purple path) are energetically disfavored. (Right) Native enzyme in complex with a paracatalytic inducer shows altered specificity. Allosteric or orthosteric binding of the antagonist (*) cause the enzyme to take up and transform an alternative substrate (*ES2) resulting in the generation of a side product (*EP2) and diminished yield of the physiological product.
Fig. 2
Fig. 2
Native and paracatalytic hedgehog precursor protein autoprocessing. (Top) Cholesterolysis of Hh precursor is self-catalyzed by the C-terminal enzymatic domain (HhC, green) through an N—S acyl shift (step 1) followed by transesterification to substrate cholesterol (step 2). (Bottom) Cholesterol-independent autoprocessing using water in place of cholesterol as the substrate. This ordinarily slow side reaction is sped up substantially when HhC is bound by a paracatalytic inducer (purple).
Fig. 3
Fig. 3
In vitro Hh autoprocessing assay with FRET-active C—H—Y precursor. (A) Reporter Construct. Arabinose inducible promoter regulates E. coli expression of the His-tagged chimeric precursor, C—H—Y. (B) Assay principle. Spatial proximity of C and Y proteins in precursor allows for FRET; autoprocessing separates C from H—Y resulting in a loss of FRET. (C.) Kinetics of C—H—Y cholesterol-driven auto-processing. FRET signal from C—H—Y samples in the absence and presence of added cholesterol over 2h. FRET is 540nm/460nm emission ratio after excitation at 400nm.
Fig. 4
Fig. 4
Kinetic and SDS-PAGE results consistent with paracatalytic induction of Hh autoprocessing. (A) FRET assay. FRET monitored as a function of time for C—H—Y (0.1μM) incubated in reaction buffer with added HAC-8 (50μM) (left); in reaction buffer only (middle); in reaction buffer with added cholesterol (50μM). (B) Gel-based assay of Hh autoprocessing. Chimeric precursor protein (SHhN-DHhC) (Owen et al., 2015) was incubated overnight in reaction buffer only (set 1); in reaction buffer with cholesterol (50μM) (set 2); in reaction buffer with HAC-8 (50μM) (set 3).
Fig. 5
Fig. 5
Analytical Reverse Phase HPLC results consistent with paracatalytic Hh autoprocessing activity induced by HAC-8. Chromatograms represent samples of C—H—Y after incubation in reaction buffer (Top); after incubation in reaction buffer with HAC-8 (50μM) (Middle); and after incubation in reaction buffer with cholesterol (50μM) (Bottom). Sample preparation and RP-HPLC gradient conditions are described herein.
Fig. 6
Fig. 6
Kinetic analysis of paracatalytic induction of Hh autoprocessing by HAC-8. (A) Structure of HAC-8. (B) Representative kinetic traces of C—H—Y ± HAC-8. FRET values plotted as a function of time from C—H—Y in reaction buffer only (top left) and with the indicated final concentration of added HAC-8. (C.) Concentration response plot for HAC-8 with C—H—Y indicate saturable binding with single digit μM affinity. FRET data from kinetic traces (B) were used to calculate apparent first order rate constants (kobs) at the indicated concentration of HAC-8 (table). Those rate constants were then plotted as a function of HAC-8 concentration to calculate an AC50 value of 5 μM and kmax value 9 × 10−4 s−1 using a modified Michaelis Menten equation.
Fig. 7
Fig. 7
Two-step synthesis of HAC-8.
Fig. 8
Fig. 8
Annotated 1H NMR spectrum of HAC-8 (15mg/mL) in deuterated chloroform.
Fig. 9
Fig. 9
Cell-based bioluminescent reporter for inducible Hh autoprocessing. (A) Reporter Construct. CMV promoter for constitutive transcription of HA-tagged nanoluciferase (HA-NLuc) fused to human Sonic HhC(D46A) with IgK leader peptide. (B) D46A mutant lacks the catalytic aspartate residue required for the substrate activity of cholesterol. (C) Autoprocessing of SHhC(D46A) is rescued by substrate assisted catalysis using cholesterol analog, 2-ACC. (D) NLuc-SHhC(D46A) functions as a gain-of-signal extracellular reporter, in which activation of Hh by 2-ACC or by paracatalytic compounds secretes NLuc into the culture media which is then detected by luminescence.
Fig. 10
Fig. 10
Application of assay to qHTS. (A) Interplate qHTS configuration where the compound library is titrated down a series of microtiter plates, typically using a 384 or 1536 well plate format. (B) A 1536-well plate containing D46A cells treated with the high concentration of a compound library detected by CLZ-2P. Column 1 represents 32 replicate controls treated with DMSO only. Column 2 contains 32 replicates of cells treated with high concentration (28.75μM) of 2-ACC. Column 3 shows a 16-pt titration of 2-ACC from 57.7μM to 1.75nM, 1:2, in duplicate (C, solid circles). Column 4 shows a 16-pt, 1:3 titration of cycloheximide in duplicate from 57.5μM to 4.01pM (C, open circles). Columns 5 to 48 are treated with small molecule library compounds. Bright wells (e.g., green circle) indicate potential activation of SHhC(D46A) autoprocessing, while dark wells (e.g., red circle) may be autoprocessing inhibited. (D) Digitonin titration used to illustrate the assay response to a cytotoxic compound.
Fig. 11
Fig. 11
Western blot analysis of HA-NLuc-SHhC(D46A) cell lysates after treatment with control compound 2-ACC or potential paracatalytic inducers. Hh activation is apparent by loss of precursor (52kDa) and by the accumulation of HA-NLuc product (24kDa). Compounds 1 and 2 are included as examples of possible activators. Compound 1 demonstrated activation by increased bioluminescence in the primary qHTS screen but failed to show autoprocessing activation by this follow-up Western blot, suggesting off-target effects. Compound 2 showed activity in the primary qHTS screen as well as by Western blot consistent with on-target autoprocessing induction. GAPDH (37kDa) is included as a loading control.
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