A Selection of Macrocyclic Peptides That Bind STING From an mRNA-Display Library With Split Degenerate Codons.

Recent improvements in mRNA display have enabled the selection of peptides that incorporate non-natural amino acids, thus expanding the chemical diversity of macrocycles beyond what is accessible in nature. Such libraries have incorporated non-natural amino acids at the expense of natural amino acids by reassigning their codons. Here we report an alternative approach to expanded amino-acid diversity that preserves all 19 natural amino acids (no methionine) and adds 6 non-natural amino acids, resulting in the highest sequence complexity reported to date. We have applied mRNA display to this 25-letter library to select functional macrocycles that bind human STING, a protein involved in immunoregulation. The resulting STING-binding peptides include a 9-mer macrocycle with a dissociation constant (KD ) of 3.4 nM, which blocks binding of cGAMP to STING and induces STING dimerization. This approach is generalizable to expanding the amino-acid alphabet in a library beyond 25 building blocks.

Smoothened is a fully competent activator of the heterotrimeric G protein G(i).

Smoothened (Smo) is a 7-transmembrane protein essential to the activation of Gli transcription factors (Gli) by hedgehog morphogens. The structure of Smo implies interactions with heterotrimeric G proteins, but the degree to which G proteins participate in the actions of hedgehogs remains controversial. We posit that the G(i) family of G proteins provides to hedgehogs the ability to expand well beyond the bounds of Gli. In this regard, we evaluate here the efficacy of Smo as it relates to the activation of G(i), by comparing Smo with the 5-hydroxytryptamine(1A) (5-HT(1A)) receptor, a quintessential G(i)-coupled receptor. We find that with use of [(35)S]guanosine 5'-(3-O-thio)triphosphate, first, with forms of G(i) endogenous to human embryonic kidney (HEK)-293 cells made to express epitope-tagged receptors and, second, with individual forms of Gα(i) fused to the C terminus of each receptor, Smo is equivalent to the 5-HT(1A) receptor in the assay as it relates to capacity to activate G(i). This finding is true regardless of subtype of G(i) (e.g., G(i2), G(o), and G(z)) tested. We also find that Smo endogenous to HEK-293 cells, ostensibly through inhibition of adenylyl cyclase, decreases intracellular levels of cAMP. The results indicate that Smo is a receptor that can engage not only Gli but also other more immediate effectors.

The alpha subunit of the G protein G13 regulates activity of one or more Gli transcription factors independently of smoothened.

Smoothened (Smo) is a seven-transmembrane (7-TM) receptor that is essential to most actions of the Hedgehog family of morphogens. We found previously that Smo couples to members of the G(i) family of heterotrimeric G proteins, which in some cases are integral although alone insufficient in the activation of Gli transcription factors through Hedgehog signaling. In response to a report that the G(12/13) family is relevant to Hedgehog signaling as well, we re-evaluated the coupling of Smo to one member of this family, G(13), and investigated the capacity of this and other G proteins to activate one or more of forms of Gli. We found no evidence that Smo couples directly to G(13). We found nonetheless that Gα(13) and to some extent Gα(q) and Gα(12) are able to effect activation of Gli(s). This capacity is realized in some cells, e.g. C3H10T1/2, MC3T3, and pancreatic cancer cells, but not all cells. The mechanism employed is distinct from that achieved through canonical Hedgehog signaling, as the activation does not involve autocrine signaling or in any other way require active Smo and does not necessarily involve enhanced transcription of Gli1. The activation by Gα(13) can be replicated through a G(q)/G(12/13)-coupled receptor, CCK(A), and is attenuated by inhibitors of p38 mitogen-activated protein kinase and Tec tyrosine kinases. We posit that G proteins, and perhaps G(13) in particular, provide access to Gli that is independent of Smo and that they thus establish a basis for control of at least some forms of Gli-mediated transcription apart from Hedgehogs.

Identification of RIPK3 Type II Inhibitors Using High-Throughput Mechanistic Studies in Hit Triage.

Necroptosis has been implicated in a variety of disease states, and RIPK3 is one of the kinases identified to play a critical role in this signaling pathway. In an effort to identify RIPK3 kinase inhibitors with a novel profile, mechanistic studies were incorporated at the hit triage stage. Utilization of these assays enabled identification of a Type II DFG-out inhibitor for RIPK3, which was confirmed by protein crystallography. Structure-based drug design on the inhibitors targeting this previously unreported conformation enabled an enhancement in selectivity against key off-target kinases.

Structure of M11L: A myxoma virus structural homolog of the apoptosis inhibitor, Bcl-2.

Apoptosis of virally infected cells is an innate host mechanism used to prevent viral spread. However, viruses have evolved a number of proteins that function to modulate the apoptotic cascades and thereby favor productive viral replication. One such antiapoptotic protein, myxoma virus M11L, has been shown to inhibit mitochondrial-dependent apoptosis by binding to and blocking the two executioner proteins Bak and Bax. Since M11L has no obvious sequence homology with Bcl-2 or Bcl-x(L), the normal cellular inhibitors for Bak and Bax, and the structure of M11L has not been solved, the mode of binding to Bak and Bax is not known. In order to understand how M11L functions, the crystal structure of M11L was solved to 2.91 A. Despite the lack of sequence similarity, M11L is a structural homolog of Bcl-2. Studies using a peptide derived from Bak indicate that M11L binds to Bak with a similar affinity (4.9 +/- 0.3 microM) to the published binding affinities of Bcl-2 and Bcl-x(L) to the same peptide (12.7 microM and 0.5 microM, respectively), indicating that M11L inhibits apoptosis by mimicking and competing with host proteins for the binding of Bak and Bax. The structure provides important insight into how myxoma virus and other poxviruses facilitate viral dissemination by inhibiting mitochondrial dependent apoptosis.