Interaction Energetics and Druggability of the Protein-Protein Interaction between Kelch-like ECH-Associated Protein 1 (KEAP1) and Nuclear Factor Erythroid 2 Like 2 (Nrf2).

Development of small molecule inhibitors of protein-protein interactions (PPIs) is hampered by our poor understanding of the druggability of PPI target sites. Here, we describe the combined application of alanine-scanning mutagenesis, fragment screening, and FTMap computational hot spot mapping to evaluate the energetics and druggability of the highly charged PPI interface between Kelch-like ECH-associated protein 1 (KEAP1) and nuclear factor erythroid 2 like 2 (Nrf2), an important drug target. FTMap identifies four binding energy hot spots at the active site. Only two of these are exploited by Nrf2, which alanine scanning of both proteins shows to bind primarily through E79 and E82 interacting with KEAP1 residues S363, R380, R415, R483, and S508. We identify fragment hits and obtain X-ray complex structures for three fragments via crystal soaking using a new crystal form of KEAP1. Combining these results provides a comprehensive and quantitative picture of the origins of binding energy at the interface. Our findings additionally reveal non-native interactions that might be exploited in the design of uncharged synthetic ligands to occupy the same site on KEAP1 that has evolved to bind the highly charged DEETGE binding loop of Nrf2. These include π-stacking with KEAP1 Y525 and interactions at an FTMap-identified hot spot deep in the binding site. Finally, we discuss how the complementary information provided by alanine-scanning mutagenesis, fragment screening, and computational hot spot mapping can be integrated to more comprehensively evaluate PPI druggability.

Metal Ions Effectively Ablate the Action of Botulinum Neurotoxin A.

Botulinum neurotoxin serotype A (BoNT/A) causes a debilitating and potentially fatal illness known as botulism. The toxin is also a bioterrorism threat, yet no pharmacological antagonist to counteract its effects has reached clinical approval. Existing strategies to negate BoNT/A intoxication have looked to antibodies, peptides, or organic small molecules as potential therapeutics. In this work, a departure from the traditional drug discovery mindset was pursued, in which the enzyme's susceptibility to metal ions was exploited. A screen of a series of metal salts showed marked inhibitory activity of group 11 and 12 metals against the BoNT/A light chain (LC) protease. Enzyme kinetics revealed that copper (I) and (II) cations displayed noncompetitive inhibition of the LC (Ki ≈ 1 µM), while mercury (II) cations were 10-fold more potent. Crystallographic and mutagenesis studies elucidated a key binding interaction between Cys165 on BoNT/A LC and the inhibitory metals. As potential copper prodrugs, ligand-copper complexes were examined in a cell-based model and were found to prevent BoNT/A cleavage of the endogenous protein substrate, SNAP-25, even at low µM concentrations of complexes. Further investigation of the complexes suggested a bioreductive mechanism causing intracellular release of copper, which directly inhibited the BoNT/A protease. In vivo experiments demonstrated that copper (II) dithiocarbamate and bis(thiosemicarbazone) complexes could delay BoNT/A-mediated lethality in a rodent model, indicating their potential for treating the harmful effects of BoNT/A intoxication. Our studies illustrate that metals can be therapeutically viable enzyme inhibitors; moreover, enzymes that share homology with BoNT LCs may be similarly targeted with metals.

Improved synthesis of 15-deoxyspergualin analogs using the Ugi multi-component reaction.

Spergualin is a natural product that exhibits immunosuppressive, anti-tumor and anti-bacterial activities. Its derivatives, such as 15-deoxyspergualin (15-DSG), have been clinically approved for acute allograft rejection. However, the reported syntheses are cumbersome (>10 steps) and they suffer from low overall yields (∼0.3% to 18%). Moreover, spergualin and its derivatives are chemically unstable and rapidly hydrolyzed in aqueous buffer. Here, we have re-explored these issues and report a modified synthetic route with significantly improved overall yield (∼31% to 47%). The key transformation is a microwave-accelerated Ugi multi-component reaction that is used to generate the peptoid core in a single step. Using the products of this route, we found that modifications of the hemiaminal significantly increased chemical stability. Thus, we anticipate that this synthetic route will improve access to biologically active 15-DSG derivatives.

Bifunctional ligands allow deliberate extrinsic reprogramming of the glucocorticoid receptor.

Therapies based on conventional nuclear receptor ligands are extremely powerful, yet their broad and long-term use is often hindered by undesired side effects that are often part of the receptor's biological function. Selective control of nuclear receptors such as the glucocorticoid receptor (GR) using conventional ligands has proven particularly challenging. Because they act solely in an allosteric manner, conventional ligands are constrained to act via cofactors that can intrinsically partner with the receptor. Furthermore, effective means to rationally encode a bias for specific coregulators are generally lacking. Using the (GR) as a framework, we demonstrate here a versatile approach, based on bifunctional ligands, that extends the regulatory repertoire of GR in a deliberate and controlled manner. By linking the macrolide FK506 to a conventional agonist (dexamethasone) or antagonist (RU-486), we demonstrate that it is possible to bridge the intact receptor to either positively or negatively acting coregulatory proteins bearing an FK506 binding protein domain. Using this strategy, we show that extrinsic recruitment of a strong activation function can enhance the efficacy of the full agonist dexamethasone and reverse the antagonist character of RU-486 at an endogenous locus. Notably, the extrinsic recruitment of histone deacetylase-1 reduces the ability of GR to activate transcription from a canonical GR response element while preserving ligand-mediated repression of nuclear factor-κB. By providing novel ways for the receptor to engage specific coregulators, this unique ligand design approach has the potential to yield both novel tools for GR study and more selective therapeutics.