A cell-based bioluminescence reporter assay of human Sonic Hedgehog protein autoprocessing to identify inhibitors and activators.

The Sonic Hedgehog (SHh) precursor protein undergoes biosynthetic autoprocessing to cleave off and covalently attach cholesterol to the SHh signaling ligand, a vital morphogen and oncogenic effector protein. Autoprocessing is self-catalyzed by SHhC, the SHh precursor's C-terminal enzymatic domain. A method to screen for small molecule regulators of this process may be of therapeutic value. Here, we describe the development and validation of the first cellular reporter to monitor human SHhC autoprocessing noninvasively in high-throughput compatible plates. The assay couples intracellular SHhC autoprocessing using endogenous cholesterol to the extracellular secretion of the bioluminescent nanoluciferase enzyme. We developed a WT SHhC reporter line for evaluating potential autoprocessing inhibitors by concentration response-dependent suppression of extracellular bioluminescence. Additionally, a conditional mutant SHhC (D46A) reporter line was developed for identifying potential autoprocessing activators by a concentration response-dependent gain of extracellular bioluminescence. The D46A mutation removes a conserved general base that is critical for the activation of the cholesterol substrate. Inducibility of the D46A reporter was established using a synthetic sterol, 2-α carboxy cholestanol, designed to bypass the defect through intramolecular general base catalysis. To facilitate direct nanoluciferase detection in the cell culture media of 1536-well plates, we designed a novel anionic phosphonylated coelenterazine, CLZ-2P, as the nanoluciferase substrate. This new reporter system offers a long-awaited resource for small molecule discovery for cancer and for developmental disorders where SHh ligand biosynthesis is dysregulated.

Specificity Distorted: Chemical Induction of Biological Paracatalysis.

We define paracatalysis as the acceleration of a reaction that appears abnormal or nonphysiological. With the high specificity of enzymes, side reactivity of this kind is typically negligible. However, enzyme paracatalysis can be amplified to levels that are biologically significant through interactions with a special class of small molecule "antagonist", here termed a paracatalytic inducer. Compounds with this unusual mode of action tend to be natural products, identified by chance through phenotypic screens. In this Perspective, we suggest two general types of paracatalytic inducer. The first type promotes substrate ambiguity, where the enzyme's ground state selectivity is compromised, enabling the transformation of non-native substrates. The second type involves transition state ambiguity, where the paracatalytic inducer changes the enzyme's interactions with the activated substrate, giving rise to non-native bond making. Although they are unusual, small molecules that induce paracatalysis have established value as hypothesis-generating probes and a few substances, i.e., aspirin and the aminoglycosides, have proven to be translatable as medicines.

General Base Swap Preserves Activity and Expands Substrate Tolerance in Hedgehog Autoprocessing.

Hedgehog (Hh) autoprocessing converts Hh precursor protein to cholesterylated Hh ligand for downstream signaling. A conserved active-site aspartate residue, D46, plays a key catalytic role in Hh autoprocessing by serving as a general base to activate substrate cholesterol. Here we report that a charge-altering Asp-to-His mutant (D46H) expands native cholesterylation activity and retains active-site conformation. Native activity toward cholesterol was established for D46H in vitro using a continuous FRET-based autoprocessing assay and in cellulo with stable expression in human 293T cells. The catalytic efficiency of cholesterylation with D46H is similar to that with wild type (WT), with kmax/KM = 2.1 × 103 and 3.7 × 103 M-1 s-1, respectively, and an identical pKa = 5.8 is obtained for both residues by NMR. To our knowledge this is the first example where a general base substitution of an Asp for His preserves both the structure and activity as a general base. Surprisingly, D46H exhibits increased catalytic efficiency toward non-native substrates, especially coprostanol (>200-fold) and epicoprostanol (>300-fold). Expanded substrate tolerance is likely due to stabilization by H46 of the negatively charged tetrahedral intermediate using electrostatic interactions, which are less constrained by geometry than H-bond stabilization by D46. In addition to providing fundamental insights into Hh autoprocessing, our findings have important implications for protein engineering and enzyme design.

Protein-Nucleic Acid Conjugation with Sterol Linkers Using Hedgehog Autoprocessing.

Hedgehog (Hh) precursor proteins contain an autoprocessing domain called HhC whose native function is protein cleavage and C-terminal glycine sterylation. The transformation catalyzed by HhC occurs in cis from a precursor protein and exhibits wide tolerance toward both sterol and protein substrates. Here, we repurpose HhC as a 1:1 protein-nucleic acid ligase, with the sterol serving as a molecular linker. A procedure is described for preparing HhC-active sterylated DNA, called steramers, using aqueous compatible chemistry and commercial reagents. Steramers have KM values of 7-11 µM and reaction t1/2 values of ∼10 min. Modularity of the HhC/steramer method is demonstrated using four different proteins along with structured and unstructured sterylated nucleic acids. The resulting protein-DNA conjugates retain the native solution properties and biochemical function. Unlike self-tagging domains, HhC does not remain fused to the conjugate; rather, enzymatic activity is mechanistically coupled to conjugate release. That unique feature of HhC, coupled with efficient kinetics and substrate tolerance, may ease access and open new applications for these suprabiological chimeras.

Chemical Bypass of General Base Catalysis in Hedgehog Protein Cholesterolysis Using a Hyper-Nucleophilic Substrate.

Proteins in the hedgehog family undergo self-catalyzed endoproteolysis involving nucleophilic attack by a molecule of cholesterol. Recently, a conserved aspartate residue (D303, or D46) of hedgehog was identified as the general base that activates cholesterol during this unusual autoprocessing event; mutation of the catalyzing functional group (D303A) reduces activity by >104-fold. Here we report near total rescue of this ostensibly dead general base mutant by a synthetic substrate, 3ß-hydroperoxycholestane (3HPC) in which the sterol -OH group is replaced by the hyper nucleophilic -OOH group. Other hedgehog point mutants at D303, also unreactive with cholesterol, accepted 3HPC as a substrate with the rank order: WT > D303A ≈ D303N ≫ D303R, D303E. We attribute the revived activity with 3-HPC to the α-effect, where tandem electronegative atoms exhibit exceptionally high nucleophilicity despite relatively low basicity.

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

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.

Sterol A-ring plasticity in hedgehog protein cholesterolysis supports a primitive substrate selectivity mechanism.

Cholesterolysis of Hedgehog family proteins couples endoproteolysis to protein C-terminal sterylation. The transformation is self-catalyzed by HhC, a partially characterized enzymatic domain found in precursor forms of Hedgehog. Here we explore spatial ambiguity in sterol recognition by HhC, using a trio of derivatives where the sterol A-ring is contracted, fused, or distorted. Sterylation assays indicate that these geometric variants react as substrates with relative activity: cholesterol, 1.000 > A-ring contracted, 0.100 > A-ring fused, 0.020 > A-ring distorted, 0.005. Experimental results and computational sterol docking into the first HhC homology model suggest a partially unstructured binding site with substrate recognition governed in large part by hydrophobic interactions.

Hedgehog Proteins Consume Steroidal CYP17A1 Antagonists: Potential Therapeutic Significance in Advanced Prostate Cancer.

Abiraterone, a potent inhibitor of the human enzyme CYP17A1 (cytochrome P450c17), provides a last line of defense against ectopic androgenesis in advanced prostate cancer. Herein we report an unprecedented off-target interaction between abiraterone and oncogenic hedgehog proteins. Our experiments indicate that abiraterone and its structural congener, galeterone, can replace cholesterol as a substrate in a specialized biosynthetic event of hedgehog proteins, known as cholesterolysis. The off-target reaction generates covalent hedgehog-drug conjugates. Cell-based reporter assays indicate that these conjugates activate hedgehog signaling when present in the low nanomolar range. Because hedgehog signaling is implicated in prostate cancer progression, and abiraterone is administered to treat advanced stages of the disease, this off-target interaction may have therapeutic significance.