Diversity oriented clicking delivers β-substituted alkenyl sulfonyl fluorides as covalent human neutrophil elastase inhibitors

Diversity Oriented Clicking (DOC) is a discovery method geared toward the rapid synthesis of functional libraries. It combines the best attributes of both classical and modern click chemistries. DOC strategies center upon the chemical diversification of core "SuFExable" hubs-exemplified by 2-Substituted-Alkynyl-1-Sulfonyl Fluorides (SASFs)-enabling the modular assembly of compounds through multiple reaction pathways. We report here a range of stereoselective Michael-type addition pathways from SASF hubs including reactions with secondary amines, carboxylates, 1H-1,2,3-triazole, and halides. These high yielding conjugate addition pathways deliver unprecedented β-substituted alkenyl sulfonyl fluorides as single isomers with minimal purification, greatly enriching the repertoire of DOC and holding true to the fundamentals of modular click chemistry. Further, we demonstrate the potential for biological function - a key objective of click chemistry - of this family of SASF-derived molecules as covalent inhibitors of human neutrophil elastase

Synthetic development of a broadly neutralizing antibody against snake venom long-chain α-neurotoxins

Snakebite envenoming is a major global public health concern for which improved therapies are urgently needed. The antigenic diversity present in snake venom toxins from various species presents a considerable challenge to the development of a universal antivenom. Here, we used a synthetic human antibody library to find and develop an antibody that neutralizes long-chain three-finger α-neurotoxins produced by numerous medically relevant snakes. Our antibody bound diverse toxin variants with high affinity, blocked toxin binding to the nicotinic acetylcholine receptor in vitro, and protected mice from lethal venom challenge. Structural analysis of the antibody-toxin complex revealed a binding mode that mimics the receptor-toxin interaction. The overall workflow presented is generalizable for the development of antibodies that target conserved epitopes among antigenically diverse targets, and it offers a promising framework for the creation of a monoclonal antibody–based universal antivenom to treat snakebite envenoming

Structure/Function analysis of the Staphylococcus aureus extracellular adherence protein and the human innate immune system

Doctor of PhilosophyBiochemistry and Molecular Biophysics Interdepartmental ProgramBrian V. GeisbrechtThe pathogenic bacterium Staphylococcus aureus actively evades many aspects of human innate immunity by expressing a series of secreted inhibitory proteins. A number of these proteins have been shown to specifically bind to and inhibit components of the complement system. Since complement is known to play a significant role in the pathophysiology of human inflammatory diseases, our long-term goal is to understand the structure, function, and mechanism of Staphylococcal immune evasion proteins to develop complement-targeted therapeutics. Since its discovery, the extracellular adherence protein (Eap) has been shown to be a crucial component in the pathogenesis and survival of S. aureus through its ability to interact and inhibit multiple aspects of the innate immune system. We have shown that Eap inhibits the classical and lectin pathways of complement by a previously undescribed mechanism. Specifically, Eap binds with nanomolar affinity to complement protein C4b, and thereby blocks binding of the classical and lectin pathway pro-protease C2 to C4b. This effectively eliminates formation of the CP/LP C3 proconvertase, which is required for amplification of downstream complement activity and subsequent inflammatory events. The full-length, mature Eap protein from S. aureus strain Mu50 consists of four ~97 residue domains, each of which adopt a similar beta-grasp fold, and are connected to one another through short linker regions that give rise to an elongated, but structured protein. Through multiple structural and functional assays, we have identified the 3rd and 4th domains of Eap as being critical for interacting with C4b and subsequent inhibition of the complement cascade. Alternative approaches to a standard co-crystal structure of Eap34 bound to C4b provided evidence that Eap domains 3 and 4 both contain a low affinity, but saturable binding site for C4b; we were able to map these sites to the α-chain and γ-chain, specifically the metal-ion-dependent adhesion site of the C345c domain, of C4b, both of which have been previously shown to be required for pro-protease binding. To provide higher resolution information, we took advantage of the abundance of surface exposed lysines in Eap34, and employed a lysine-acetylation foot printing mass spectrometry technique. This identified seven lysines in Eap34 that undergo changes in solvent exposure upon C4b binding and confirmation of these residues was done through site-directed mutagenesis, followed by direct binding and functional assays. Together, these results provide structural and functional insight into one of the many ways that Staphylococcus aureus can evade the killing powers of the innate immune system. Future plans are directed at conducting site-specific screens to identify small molecule/peptide compounds that target the Eap34 binding site on C4b. Such molecules would constitute attractive lead compounds in the search for specific inhibitors of the classical and lectin complement pathways

SuFEx-Enabled High-Throughput Medicinal Chemistry

Optimization of small-molecule probes or drugs is a lengthy, challenging and resource-intensive process. Lack of automation and reliance on skilled medicinal chemists is cumbersome in both academic and industrial settings. Here, we demonstrate a high-throughput hit-to-lead process based on the biocompatible SuFEx click chemistry. A modest high-throughput screening hit against a bacterial cysteine protease SpeB was modified with a SuFExable iminosulfur oxydifluoride [RN=S(O)F2] motif, rapidly diversified into 460 analogs in overnight reactions, and the products directly screened to yield drug-like inhibitors with 300-fold higher potency. We showed that the improved molecule is drug-like and biologically active in a bacteria-host coculture. Since these reactions can be performed on a picomole scale to conserve reagents, we anticipate our methodology can accelerate the development of robust biological probes and drug candidates.</p

"Sleeping Beauty" Phenomenon: SuFEx-Enabled Discovery of Selective Covalent Inhibitors of Human Neutrophil Elastase

Sulfur-Fluoride Exchange (SuFEx) has emerged as the new generation of click chemistry. We report here a SuFEx-enabled approach exploiting the "Sleeping beauty" phenomenon of sulfur fluoride compounds in the context of the serendipitous discovery of selective covalent human neutrophil elastase (hNE) inhibitors. Evaluation of an ever-growing collection of SuFExable compounds toward various biological assays unexpectedly yielded a selective and covalent hNE inhibitor, benzene-1,2-disulfonyl fluoride. Derivatization of the initial hit led to a better agent, 2- triflyl benzenesulfonyl fluoride, itself made through a SuFEx trifluoromethylation process, with IC50 = 1.1 μM and ~200-fold selectivity over the homologous neutrophil serine protease, cathepsin G. The optimized probe only modified active hNE and not its denatured form, setting another example of the "sleeping beauty" phenomenon of sulfur fluoride capturing agents for the discovery of covalent medicines

Diversity Oriented Clicking: Synthesis of beta-Substituted Alkenyl Sulfonyl Fluorides as Covalent Human Neutrophil Elastase Inhibitors

Diversity Oriented Clicking (DOC) is a discovery method geared towards the rapid synthesis of functional libraries. It combines the best attributes of both classical and modern click chemistries. DOC strategies center upon the chemical diversification of core “SuFExable” hubs – exemplified by 2-Substituted-Alkynyl-1-Sulfonyl Fluorides (SASFs) – enabling the modular assembly of compounds through multiple reaction pathways. We report here a range of stereoselective Michael-type addition pathways from SASF hubs including reactions with secondary amines, carboxylates, 1H-1,2,3-triazole, and halides. These high yielding conjugate addition pathways deliver unprecedented beta-substituted alkenyl sulfonyl fluorides as single isomers with minimal purification, greatly enriching the repertoire of DOC and holding true to the fundamentals of modular click chemistry. Further, we demonstrate the biological function – another key objective of click chemistry – of this new scaffold as covalent inhibitors of human neutrophil elastase (hNE). The ease of diversification of SASFs through click pathways, enabling rapid access to biologically important molecules, further validates Diversity Oriented Clicking as an effective and robust method for lead discovery