NLRP3 is a key regulator of the innate immune system involved in sensing a variety of pathogen and danger signals. Priming and activation of NLRP3 leads to the release and maturation of pro-inflammatory cytokines, as well as gasdermin D-mediated cell death. Inhibition of dysregulated NLRP3 activity has been associated with promising therapeutic opportunities for a variety of systemic and neurological diseases including atherosclerosis and Parkinson's disease. Herein, we discuss how a high-throughput screen (HTS) allowed us to discover new chemical scaffolds that specifically bind to NLRP3 and inhibit its function in a selective manner. We also describe how an enantiomer of HTS hit 5, compound 11, demonstrated in vivo inhibition of NLRP3.

Using a high-throughput screening (HTS) approach, a new GTP-site binding pyridine-carboxylate series of cGAS inhibitors was discovered. The biochemical potency of this new pyridine carboxylate series was improved 166-fold from the original hit to double-digit nanomolar levels using structure-based design insights, but the series was found to suffer from low permeability and low bioavailability. A structure-based hybridization of the metal-binding motifs of the pyridine carboxylate series and our previously disclosed tetrahydrocarboline GTP-site ligand 23 identified pyrimidine amide compound 36. Compound 36 is potent against both human and mouse cGAS isoforms and has a favorable pharmacokinetic (PK) profile in mice. Additionally, compound 36 displayed a dose-dependent reduction in cGAMP production in a ConA pharmacodynamic mouse model of acute liver injury, demonstrating potential utility as an in vivo tool compound for further investigation of the cGAS pathway.

A property-focused optimization strategy was employed to modify the carboxylic acid head group of a class of EP4 agonists in order to minimize its absorption upon oral administration. The resulting oxalic acid monohydrazide-derived carboxylate isostere demonstrated utility as a class of prodrug showing colon-targeted delivery of parent agonist 2, with minimal exposure observed in the plasma. Oral administration of NXT-10796 demonstrated tissue specific activation of the EP4 receptor through modulation of immune genes in the colon, without modulation of EP4 driven biomarkers in the plasma compartment. Although further in depth understanding of the conversion of NXT-10796 is required for further assessment of the developability of this series of prodrugs, using NXT-10796 as a tool molecule has allowed us to confirm that tissue-specific modulation of an EP4-modulated gene signature is possible, which allows for further evaluation of this therapeutic modality in rodent models of human disease.

Novel prostaglandin E2 receptor 4 (EP4) agonists featuring a pyridone core and an allylic alcohol ω-chain were discovered. These agonists were shown to be selective over EP1, EP2 and EP3. Analogs harboring a 4-carboxylic acid phenethyl α-chain displayed improved potency over those containing an n-heptanoic acid chain. Key SAR relationships were also identified.

Bicyclo[1.1.1]pentanes are effective bioisoteres for aromatic rings, tert-butyl groups, and alkynes. Here we report the first method to synthesize 3-alkylbicyclo[1.1.1]pentan-1-amines directly from [1.1.1]propellane via sequential addition of magnesium amides and alkyl electrophiles. The mild reaction conditions tolerate a variety of important functional groups and enable efficient incorporation of several pharmaceutically relevant amines onto the bicyclo[1.1.1]pentane scaffold. This method's utility is highlighted by its ability to significantly streamline the syntheses of several important bicyclo[1.1.1]pentan-1-amine building blocks.

An enantioselective synthesis of the previously-disclosed ITK inhibitor GNE-6688 is described. Synthesis of the nitropyrazole fragment is highlighted by a Ru-catalyzed transfer hydrogenation using the Wills tethered ligand system. Synthesis of the pyrazole carboxylic acid fragment features an allylboration catalyzed by a chiral diol-SnCl4 complex, followed by a highly diastereoselective directed cyclopropanation. (C) 2019 Elsevier Ltd. All rights reserved.

NF-κB-inducing kinase (NIK) is a protein kinase central to the noncanonical NF-κB pathway downstream from multiple TNF receptor family members, including BAFF, which has been associated with B cell survival and maturation, dendritic cell activation, secondary lymphoid organ development, and bone metabolism. We report herein the discovery of lead chemical series of NIK inhibitors that were identified through a scaffold-hopping strategy using structure-based design. Electronic and steric properties of lead compounds were modified to address glutathione conjugation and amide hydrolysis. These highly potent compounds exhibited selective inhibition of LTβR-dependent p52 translocation and transcription of NF-κB2 related genes. Compound 4f is shown to have a favorable pharmacokinetic profile across species and to inhibit BAFF-induced B cell survival in vitro and reduce splenic marginal zone B cells in vivo.

The human body hosts a variety of microorganisms including bacteria and fungi as a part of the human microbiome. Whether as a commensal or pathogenic interaction, the process in which these eukaryotic and prokaryotic cells sense the presence of each other and respond accordingly is a key aspect of understanding the biology of the human microbiome and immune system. A wide range of fungal cells are known to reside in different sites in the human body. Some of these cells, as opportunistic microorganisms, can be the cause of major health complications in immunocompromised patients. Recent studies have shown that specific fragments associated with microorganisms can be recognized by these fungi and cause morphological changes. Some of these Pathogens‐Associated Molecular Patterns (PAMPs) are known to be recognized by the human immune system as well and lead to further activation of immune responses. Understanding the role of these fragments interacting with fungal cells is crucial for studying the pathogenicity of these microorganisms. In this regard, we investigated the Candida albicans, the most common pathogenic fungus in humans. Going from a budding yeast morphology to an invasive hyphae form, C. albicans can invade epithelial cells and cause further infection. This transformation is mediated by the adenylyl cyclase, Cyr1, and different triggers, including bacterial cell wall fragments, have been suggested to activate the Cyr1 protein and induce the hyphal growth of the cells. Interestingly, a host of bacterial peptidoglycan fragments have been shown to be recognized by the human immune system as well and bind to nucleotide‐binding oligomerization domain‐like receptors, or NOD‐like receptors. Structural similarities between Cyr1 protein and NOD proteins suggest that these peptidoglycan fragments of the bacterial cell wall can directly bind to Cyr1 protein and initiate the downstream pathway causing pathogenic morphological transformation of C. albicans. In this study, a small library of bacterial derived carbohydrates, including chemically synthesized peptidoglycan derivatives alongside with anthracyclines produced by Streptomyces was prepared and tested for their ability to induce hyphal growth in C. albicans cells. The binding between these compounds and the leucine‐rich repeat (LRR) domain of Cyr1 protein was further investigated via Surface Plasmon Resonance assay. These results demonstrate that Cyr1 can bind to peptidoglycan derivatives of bacterial cell wall with high affinity and further activate the hyphal growth of the C. albicans. These findings can lead us to a more detailed investigation of the binding process at molecular level and to study the Cyr1 protein as a potential target for inhibition of C. albicans pathogenicity via small molecule inhibitors.Support or Funding InformationThe authors are thankful for support from the Delaware COBRE program, supported by a grant from the National Institute of General Medical Sciences (NIGMS 1 P30 GM110758 and 1 P20 GM104316‐01A1) from the National Institutes of Health. Acknowledgement is gratefully made to the National Science Foundation (CAREER CHE 1554967) for support of this research. We thank Daniel Scanlon, James Melnyk, Amy Schaefer, Kristen DeMeester for their support.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Within the human body are trillions of microorganisms which must maintain a proper balance with the human host as well as amongst each other. These organisms must be able to accurately identify and respond to the bacterial cells that surround them. Pattern recognition receptors (PRRs) can bind pathogen associated molecular patterns (PAMPs) and enable these microorganisms to recognize their surrounding microbiome. Similarly, in yeast species Candida albicans the adenylyl cyclase CYR1p is found to bind muramyl dipeptide (MDP) a fragment of the bacterial cell wall or peptidoglycan. Bacterial cell wall fragments like MDP are interestingly found to trigger a phenotypical change in C. albicans to enter pathogenic hyphal growth through a cascade initiated by the binding of MDP to CYR1p. Another member of the Candida family, Candida glabrata has been found to not experience the same phenotypical switch to hyphal growth observed in C. albicans in the presence of MDP despite both species being of the same family and both commonly involved in yeast infections. The Leucine Rich Repeat (LRR) domain specifically has been shown to be the site of MDP binding within the protein CYR1p in C. albicans. To understand the differences in the mechanism of peptidoglycan recognition by C. albicans and C. glabrata, the LRR domain of C. glabrata was expressed and purified. Analysis of the binding or lack thereof between MDP and the LRR domain of CYR1p from C. glabrata could help to elucidate why C. albicans has hyphae growth triggered by MDP but C. glabrata does not.Support or Funding InformationUniversity of Delaware Department of Chemistry and BiochemistryThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The current standard of care for HIV is a combination of drugs, known as highly active antiretroviral therapy (HAART). A frequent component of HAART are non-nucleoside reverse transcriptase inhibitors (NNTRI) which prevent conversion of viral RNA into DNA. Herein we discuss the discovery, early and late development of doravirine, an investigational drug for the treatment of HIV. Key structure activity relationships, as well as critical optimized pharmacokinetic and physico-chemical parameters are presented. The evolution of the synthesis methodologies used throughout the lifetime of the program will also be elaborated, including the early development supply route and our efforts toward the establishement of the manufacturing process.

Every organism, from the complex to the single cellular, is surrounded by trillions of microorganisms. In order to survive an organism must be able to recognize and adapt to the microbiome that surrounds it. The ability to recognize these microorganisms occurs when the appropriate pathogen associated molecular pattern (PAMPs) binds to the proper pattern recognition receptor (PRR). PAMPs are molecules associate with microorganisms such as lipopolysaccharide, viral RNA, and peptidoglycan. Interestingly, yeast respond to small molecules generated by bacteria. Recognition of the bacterial cell wall fragment muramyl dipeptide (MDP) by the adenylyl cyclase, CYR1p, triggers C. albicans to enter pathogenic hyphal growth. While not characterized as a PRR, CYR1p contains a Leucine Rich Repeat (LRR) domain, which is a common protein motif found in many PRRs, including toll‐like receptors and nod‐like receptors in human, and is often the site of PAMP binding. Using a surface plasmon resonance (SPR) assay, we were able to determine the binding affinity for MDP to the LRR domain of CYR1p. Similar SPR assays were used to determine which portions of MDP is necessary for binding CYR1p. The molecules found to bind CYR1p will be tested in vivo, to determine which, if any, are able to trigger hyphal growth in C. albicans.