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Holoprosencephaly (HPE) is a failure of the forebrain to bifurcate and is the most common structural malformation of the embryonic brain. Mutations in SHH underlie most familial (17%) cases of HPE; and, consistent with this, Shh is expressed in midline embryonic cells and tissues and their derivatives that are affected in HPE. It has long been recognized that a graded series of facial anomalies occurs within the clinical spectrum of HPE, as HPE is often found in patients together with other malformations such as acrania, anencephaly, and agnathia. However, it is not known if these phenotypes arise through a common etiology and pathogenesis. Here we demonstrate for the first time using mouse models that Hedgehog acyltransferase (Hhat) loss-of-function leads to holoprosencephaly together with acrania and agnathia, which mimics the severe condition observed in humans. Hhat is required for post-translational palmitoylation of Hedgehog (Hh) proteins; and, in the absence of Hhat, Hh secretion from producing cells is diminished. We show through downregulation of the Hh receptor Ptch1 that loss of Hhat perturbs long-range Hh signaling, which in turn disrupts Fgf, Bmp and Erk signaling. Collectively, this leads to abnormal patterning and extensive apoptosis within the craniofacial primordial, together with defects in cartilage and bone differentiation. Therefore our work shows that Hhat loss-of-function underscrores HPE; but more importantly it provides a mechanism for the co-occurrence of acrania, holoprosencephaly, and agnathia. Future genetic studies should include HHAT as a potential candidate in the etiology and pathogenesis of HPE and its associated disorders.
Itraconazole (ITZ) is an FDA-approved member of the triazole class of anti-fungal agents. Two recent drug repurposing screens identified ITZ as a promising anti-cancer chemotherapeutic that inhibits both angiogenesis and the hedgehog (Hh) signaling pathway. We have synthesized and evaluated first and second generation ITZ analogues for their anti-Hh and anti-angiogenic activities to more fully probe the structural requirements for these anti-cancer properties. Our overall results suggest that the triazole functionality is required for ITZ-mediated inhibition of angiogenesis, but that it is not essential for inhibition of Hh signaling. The synthesis and evaluation of stereochemically defined des-triazole ITZ analogues also provides key information as to the optimal configuration around the dioxolane ring of the ITZ scaffold. Finally, the results from our studies suggest that two distinct cellular mechanisms of action govern the anti-cancer properties of the ITZ scaffold.
A growing body of evidence suggests that autophagy inhibition enhances the effectiveness of chemotherapy, especially in difficult-to-treat cancers. Existing autophagy inhibitors are primarily lysosomotropic agents. More specific autophagy inhibitors are highly sought-after. The microtubule-associated protein 1A/1B light chain 3B protein, LC3B, is an adapter protein that mediates key protein-protein interactions at several points in autophagy pathways. In this work, we used a known peptide ligand as a starting point to develop improved LC3B inhibitors. We obtained structure-activity relationships that quantify the binding contributions of peptide termini, individual charged residues, and hydrophobic interactions. Based on these data, we used artificial amino acids and diversity-oriented stapling to improve affinity and resistance to biological degradation, while maintaining or improving LC3B affinity and selectivity. These peptides represent the highest-affinity LC3B-selective ligands reported to date, and they will be useful tools for further elucidation of LC3B's role in autophagy and in cancer.
Peptides constrained by intramolecular cross-links, especially stapled α-helices, have emerged as versatile scaffolds for drug development. However, there are fewer examples of similarly constrained scaffolds for other secondary structures. Here, we used a novel computational strategy to identify an optimal staple for antiparallel β-strands, and then we incorporated that staple within a β-hairpin peptide. The hairpin uses 4-mercaptoproline as a novel staple component, which contributes to a unique, kinked structure. The stapled hairpins show a high degree of structure in aqueous solution, excellent resistance to degradation in cell lysates, and cytosolic penetration at micromolar concentrations. They also overlay with a unique subset of kinked hairpin motifs at protein− protein interaction interfaces. Thus, these scaffolds represent promising starting points for developing inhibitors of cellular protein−protein interactions.
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