Heping Shi

Diverse chemical scaffolds support direct inhibition of the membrane bound O-acyltransferase Porcupine

Background: The acyltransferase Porcupine (Porcn) is essential for active Wnt ligand production and is chemically tractable. Results: Novel small molecules targeting Porcn enables interrogation of Wnt signaling in vitro and in vivo. Conclusion: Porcn is highly druggable and supports diverse cellular responses in embryonic development and regeneration. Significance: Porcn inhibitors represent versatile chemical probes for Wnt signaling in vivo and are potential anti-cancer therapeutic agents. Secreted Wnt proteins constitute one of the largest families of intercellular signaling molecules in vertebrates with essential roles in embryonic development and adult tissue homeostasis. The functional redundancy of Wnt genes and the many forms of cellular responses they elicit, including some utilizing the transcriptional co-activator β-catenin, has limited the ability of classical genetic strategies to uncover their roles in vivo. We had previously identified a chemical compound class termed Inhibitor of Wnt Production (or IWP) that targets Porcupine (Porcn), an acyltransferase catalyzing the addition of fatty acid adducts onto Wnt proteins. Here we demonstrate that diverse chemical structures are able to inhibit Porcn by targeting its putative active site. When deployed in concert with small molecules that modulate the activity of Tankyrase enzymes and glycogen synthase kinase 3 β (GSK3β), additional transducers of Wnt/β-catenin signaling, the IWP compounds reveal an essential role for Wnt protein fatty acylation in eliciting β-catenin-dependent and -independent forms of Wnt signaling during zebrafish development. This collection of small molecules facilitates rapid dissection of Wnt gene function in vivo by limiting the influence of redundant Wnt gene functions on phenotypic outcomes and enables temporal manipulation of Wnt-mediated signaling in vertebrates.

cGAS is essential for the antitumor effect of immune checkpoint blockade

Significance The recent success of immune checkpoint blockades, such as the use of antibodies against CTLA4, PD-1, and PD-L1 in cancer therapies has reinvigorated the concept of intrinsic antitumor immunity, but how the immune system detects tumors and generates antitumor immunity is still not well understood. Here we showed that the PD-L1 blockade lost its antitumor effects in mice lacking the cytosolic DNA sensor cGAS, suggesting that cGAS is essential for intrinsic antitumor immunity. Further, we showed that the cGAS product cGAMP has strong antitumor effects, especially when it is combined with the PD-L1 antibody. These results demonstrate that cGAS plays a pivotal role in cancer immunity and that cGAMP and its derivatives may be used directly for cancer immunotherapy. cGMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates innate immune responses. cGAS catalyzes the synthesis of cGAMP, which functions as a second messenger that binds and activates the adaptor protein STING to induce type I interferons (IFNs) and other immune modulatory molecules. Here we show that cGAS is indispensable for the antitumor effect of immune checkpoint blockade in mice. Wild-type, but not cGAS-deficient, mice exhibited slower growth of B16 melanomas in response to a PD-L1 antibody treatment. Consistently, intramuscular delivery of cGAMP inhibited melanoma growth and prolonged the survival of the tumor-bearing mice. The combination of cGAMP and PD-L1 antibody exerted stronger antitumor effects than did either treatment alone. cGAMP treatment activated dendritic cells and enhanced cross-presentation of tumor-associated antigens to CD8 T cells. These results indicate that activation of the cGAS pathway is important for intrinsic antitumor immunity and that cGAMP may be used directly for cancer immunotherapy.

The development of highly potent inhibitors for porcupine.

Porcupine is a member of the membrane-bound O-acyltransferase family of proteins. It catalyzes the palmitoylation of Wnt proteins, a process required for their secretion and activity. We recently disclosed a class of small molecules (IWPs) as the first reported Porcn inhibitors. We now describe the structure-activity relationship studies and the identification of subnanomolar inhibitors. We also report herein the effects of IWPs on Wnt-dependent developmental processes, including zebrafish posterior axis formation and kidney tubule formation.

Molecular basis for the specific recognition of the metazoan cyclic GMP-AMP by the innate immune adaptor protein STING

Significance The presence of cytosolic DNA in mammalian cells signifies microbial invasions and triggers the DNA sensor protein cGAS to produce the second messenger molecule 2′3′-cGAMP, which elicits innate immune responses by binding to and activating the homodimerized adaptor protein STING. Here we show that the high affinity of the asymmetric ligand 2′3′-cGAMP to the symmetric dimer of STING originates from its unique mixed phosphodiester linkages. 2′3′-cGAMP, but not its linkage isomers, adopts an organized free-ligand conformation that resembles the STING-bound conformation and pays low energy costs in changing into the active conformation. Whereas biological structural studies have focused on analyses of protein conformations, our results demonstrate that analyses of free-ligand conformations can be equally important in understanding protein–ligand interactions. Cyclic GMP-AMP containing a unique combination of mixed phosphodiester linkages (2′3′-cGAMP) is an endogenous second messenger molecule that activates the type-I IFN pathway upon binding to the homodimer of the adaptor protein STING on the surface of endoplasmic reticulum membrane. However, the preferential binding of the asymmetric ligand 2′3′-cGAMP to the symmetric dimer of STING represents a physicochemical enigma. Here we show that 2′3′-cGAMP, but not its linkage isomers, adopts an organized free-ligand conformation that resembles the STING-bound conformation and pays low entropy and enthalpy costs in converting into the active conformation. Our results demonstrate that analyses of free-ligand conformations can be as important as analyses of protein conformations in understanding protein–ligand interactions.

Identification and characterization of β-sitosterol target proteins.

β-Sitosterol is the most abundant plant sterol in the human diet. It is also the major component of several traditional medicines, including saw palmetto and devils claw. Although β-sitosterol is effective against enlarged prostate in human clinical trials and has anti-cancer and anti-inflammatory activities, the mechanisms of action are poorly understood. Here, we report the identification of two new binding proteins for β-sitosterol that may underlie its beneficial effects.

A synthetic combinatorial approach to disabling deviant Hedgehog signaling

Mutations in components of the Hedgehog (HH) signal transduction pathway are found in the majority of basal cell carcinoma (BCC) and medulloblastoma incidents. Cancerous cells with intrinsic or acquired resistance to antagonists targeting the seven transmembrane effector Smoothened (SMO) frequently invoke alternative mechanisms for maintaining deviant activity of the GLI DNA binding proteins. Here we introduce a chemical agent that simultaneously achieves inhibition of SMO and GLI activity by direct targeting of the SMO heptahelical domain and the GLI-modifying enzymes belonging to the histone deacetylase (HDAC) family. We demonstrate a small molecule SMO-HDAC antagonist (IHR-SAHA) retains inhibitory activity for GLI transcription induced by SMO-dependent and -independent mechanisms frequently associated with cancer biogenesis. Synthetic combinatorial therapeutic agents such as IHR-SAHA that a priori disable cancer drivers and anticipated mechanisms of drug resistance could extend the duration of disease remission, and provide an alternative clinical development path for realizing combinatorial therapy modalities.