A number of histone-binding domains are implicated in cancer through improper binding of chromatin. In a clinically reported case of acute myeloid leukemia (AML), a genetic fusion protein between nucleoporin 98 and the third plant homeodomain (PHD) finger of JARID1A drives an oncogenic transcriptional program that is dependent on histone binding by the PHD finger. By exploiting the requirement for chromatin binding in oncogenesis, therapeutics targeting histone readers may represent a new paradigm in drug development. In this study, we developed a novel small molecule screening strategy that utilizes HaloTag technology to identify several small molecules that disrupt binding of the JARID1A PHD finger to histone peptides. Small molecule inhibitors were validated biochemically through affinity pull downs, fluorescence polarization, and histone reader specificity studies. One compound was modified through medicinal chemistry to improve its potency while retaining histone reader selectivity. Molecular modeling and site-directed mutagenesis of JARID1A PHD3 provided insights into the biochemical basis of competitive inhibition.

Cellular metabolites act as important signaling cues, but are subject to complex unknown chemistry. Kynurenine is a tryptophan metabolite that plays a crucial role in cancer and the immune system. Despite its atypical, non-ligand-like, highly polar structure, kynurenine activates the aryl hydrocarbon receptor (AHR), a PER, ARNT, SIM (PAS) family transcription factor that responds to diverse environmental and cellular ligands. The activity of kynurenine is increased 100-1000-fold by incubation or long-term storage and relies on the hydrophobic ligand-binding pocket of AHR, with identical structural signatures for AHR induction before and after activation. We purified trace-active derivatives of kynurenine and identified two novel, closely related condensation products, named trace-extended aromatic condensation products (TEACOPs), which are active at low picomolar levels. The synthesized compound for one of the predicted structures matched the purified compound in both chemical structure and AHR pharmacology. Our study provides evidence that kynurenine acts as an AHR pro-ligand, which requires novel chemical conversions to act as a receptor agonist.

Receptor interacting protein kinase-1 and -3 (RIP1 and RIP3) are essential mediators of cell death processes and participate in inflammatory responses. Our group recently demonstrated that gene deletion of Rip3 or pharmacological inhibition of RIP1 attenuated pathogenesis of abdominal aortic aneurysm (AAA), a life-threatening degenerative vascular disease characterized by depletion of smooth muscle cells (SMCs), inflammation, negative extracellular matrix remodeling, and progressive expansion of aorta. The goal of this study was to develop drug candidates for AAA and other disease conditions involving cell death and inflammation. We screened 1141 kinase inhibitors for their ability to block necroptosis using the RIP1 inhibitor Necrostatin-1s (Nec-1s) as a selection baseline. Positive compounds were further screened for cytotoxicity and virtual binding to RIP3. A cluster of top hits, represented by GSK2593074A (GSK’074), displayed structural similarity to the established RIP3 inhibitor GSK’843. In multiple cell types including mouse SMCs, fibroblasts (L929), bone marrow derived macrophages (BMDM), and human colon epithelial cells (HT29), GSK’074 inhibited necroptosis with an IC50 of ~3 nM. Furthermore, GSK’074, but not Nec-1s, blocked cytokine production by SMCs. Biochemical analyses identified both RIP1 and RIP3 as the biological targets of GSK’074. Unlike GSK’843 which causes profound apoptosis at high doses (>3 µM), GSK’074 showed no detectable cytotoxicity even at 20 µM. Daily intraperitoneal injection of GSK’074 at 0.93 mg/kg significantly attenuated aortic expansion in two mouse models of AAA (calcium phosphate: DMSO 66.06 ± 9.17% vs GSK’074 27.36 ± 8.25%, P < 0.05; Angiotensin II: DMSO 85.39 ± 15.76% vs GSK’074 36.28 ± 5.76%, P < 0.05). Histologically, GSK’074 treatment diminished cell death and macrophage infiltration in aneurysm-prone aortae. Together, our data suggest that GSK’074 represents a new class of necroptosis inhibitors with dual targeting ability to both RIP1 and RIP3. The high potency and minimum cytotoxicity make GSK’074 a desirable drug candidate of pharmacological therapies to attenuate AAA progression and other necroptosis related diseases.

The histone deacetylase sirtuin 6 (SIRT6) regulates numerous biological functions, including transcriptional repression, DNA repair, and telomere maintenance. Recombinant SIRT6 displays catalytic efficiencies 2 orders of magnitude greater for long-chain deacylation than deacetylation against peptide substrates; however, deacetylation can be enhanced by allosteric small-molecule activators. Here, we investigated the mechanisms of activated lysine deacetylation and enhanced long-chain acyl-group removal by SIRT6. Activity-based screening identified compounds that activated histone peptide deacetylation 18-48-fold. Chemical optimization based on structure–activity relationships yielded an activator with improved potency and selectivity for SIRT6. Using this novel activator, we conducted biochemical and kinetic analyses revealing that SIRT6 is activated via acceleration of a catalytic step occurring after substrate binding but before NAD+ cleavage. We identified a SIRT6 variant, R65A, that maintains basal deacetylase activity but cannot be activated and failed to enhance long-chain deacylation. Additional biochemical studies revealed that Arg-65 is critical for activation by facilitating a conformational step that initiates chemical catalysis. This work suggests that SIRT6 activation of deacetylation involves a similar mechanism to improved catalysis as that of long-chain deacylation. The identification of novel SIRT6 activators and the molecular insights into activation and catalysis presented here provide a foundational understanding for physiological SIRT6 activation and for rational design of activating molecules.

Benzyne-[2 + 2] cycloadditions with enamides are described. This effort led to the development of a highly stereoselective tandem [2 + 2] cycloaddition-pericyclic ring-opening-intramolecular-Ntethered-[4 + 2] cycloaddition for rapid assembly of nitrogen heterocycles.Chemistry of benzynes 1 has reemerged as evident by an elegant array of work that has appeared in the recent literature covering a number of different synthetic transformations. [2][3][4] As Larock 4g pointed out, this new surge is in part related to the usage of Kobayashi's 5 mild fluoride-based conditions for generating benzynes in situ from ortho-(trimethylsilyl)aryl triflates. In particular, Stoltz's 6 beautiful studies on annulations of enamides with benzynes en route to indolines and iso-quinolines provoked us to envision another transformation also involving enamides but through a [2 + 2] cycloaddition as shown in Scheme 1. While benzyne-[2 + 2] cycloaddition is well-known, 1,7 there are very few examples involving enamines 8 with no systematic examinations of enamides in this capacity. 1,9,10 While conceptually simple, this endeavor is timely and significant because enamides represents an increasingly more accessible substrate 11-14 and a useful functional group in modern organic synthesis. 15,16 Correspondence to: Richard P. Hsung, rhsung@wisc. edu. Supporting Information Available: Experimental procedures as well as NMR spectra and characterizations are available for all new compounds and free of charge via Internet http://pubs.acs.org. Although amido-benzocyclobutane products 14b-e were isolated as an inseparable mixture of diastereomers with ratios of 1:1 to 2:1, to control this diastereoselectivity is not essential because the major application of these amido-benzocyclobutanes pertains to a pericyclic ringopening, which would lose the stereochemical information en route to amido-oquinonedimethides. In accord with this understanding, we recognized that while these new amido-benzocyclobutanes are synthetically useful, the most attractive feature here is to employ enamide-benzyne- NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript olefin geometry is preserved. 1,19 In addition, enamide 27 with a tri-substituted olefin was also feasible for the tandem sequence, although the yield for the desired aza-tetracycle 28 is lower. We note here that even with the more electron-rich di -and tri-substituted olefins, the benzyne-[2 + 2] cycloaddition still favored the enamide motif.The second facet features an asymmetric variant of this tandem sequence [Scheme 4]. Chiral enamide 29, derived from (S)-aspartic acid, could be transformed into enamide 31 tethered to a trans-acrylate motif through Swern oxidation and Wittig olefination. Subjecting 31 to the tandem benzyne-[2 + 2]-ring-opening-intramolecular [4 + 2] process afforded aza-tetracycle 34 in a highly stereoselective manner. This exercise underscores the timeliness and significance of this strategy because chiral enamides are readily accessible thro...