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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, 1 H -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.

Effector T cells comprise the cellular arm of the adaptive immune system and are essential for mounting immune responses against pathogens and cancer. To reach effector status, costimulation through CD28 is required. Here, we report that sialic acid-containing glycans on the surface of both T cells and APCs are alternative ligands of CD28 that compete with binding to its well-documented activatory ligand CD80 on the APC, resulting in attenuated costimulation. Removal of sialic acids enhances antigen-mediated activation of naïve T cells and also increases the revival of effector T cells made hypofunctional or exhausted via chronic viral infection. This occurs through a mechanism that is synergistic with antibody blockade of the inhibitory PD-1 axis. These results reveal a previously unrecognized role of sialic acid ligands in attenuation of CD28-mediated costimulation of T cells., T cells require multiple signals to activate, including costimulation via CD28. We report that sialoglycans are alternative ligands for CD28 and attenuate costimulation by competing with CD80.

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.

Members of the CA class of cysteine proteases have multifaceted roles in physiology and virulence for many bacteria. Streptococcal pyrogenic exotoxin B (SpeB) is secreted by Streptococcus pyogenes and implicated in the pathogenesis of the bacterium through degradation of key human immune effector proteins. Here, we developed and characterized a clickable inhibitor, 2S-alkyne, based on X-ray crystallographic analysis and structure-activity relationships. Our SpeB probe showed irreversible enzyme inhibition in biochemical assays and labeled endogenous SpeB in cultured S. pyogenes supernatants. Importantly, application of 2S-alkyne decreased S. pyogenes survival in the presence of human neutrophils and supports the role of SpeB-mediated proteolysis as a mechanism to limit complement-mediated host defense. We posit that our SpeB inhibitor will be a useful chemical tool to regulate, label, and quantitate secreted cysteine proteases with SpeB-like activity in complex biological samples and a lead candidate for new therapeutics designed to sensitize S. pyogenes to host immune clearance.

Optimization of small-molecule probes or drugs is a synthetically 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 sulfur(VI) fluoride exchange (SuFEx) click chemistry. A high-throughput screening hit benzyl (cyanomethyl)carbamate (Ki = 8 μM) 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 were directly screened to yield drug-like inhibitors with 480-fold higher potency (Ki = 18 nM). We showed that the improved molecule is active in a bacteria-host coculture. Since this SuFEx linkage reaction succeeds on picomole scale for direct screening, we anticipate our methodology can accelerate the development of robust biological probes and drug candidates.

The emergence of SARS-CoV-2 underscores the need for strategies to rapidly develop neutralizing monoclonal antibodies that can function as prophylactic and therapeutic agents and to help guide vaccine design. Here, we demonstrate that engineering approaches can be used to refocus an existing neutralizing antibody to a related but resistant virus. Using a rapid affinity maturation strategy, we engineered CR3022, a SARS-CoV-1 neutralizing antibody, to bind SARS-CoV-2 receptor binding domain with >1000-fold improved affinity. The engineered CR3022 neutralized SARS-CoV-2 and provided prophylactic protection from viral challenge in a small animal model of SARS-CoV-2 infection. Deep sequencing throughout the engineering process paired with crystallographic analysis of an enhanced antibody elucidated the molecular mechanisms by which engineered CR3022 can accommodate sequence differences in the epitope between SARS-CoV-1 and SARS-CoV-2. The workflow described provides a blueprint for rapid broadening of neutralization of an antibody from one virus to closely related but resistant viruses.

Effector T cells comprise the cellular arm of the adaptive immune system and are essential for mounting immune responses against pathogens and cancer. To reach effector status, co-stimulation through CD28 is required. Here, we report that sialic acid-containing glycans on the surface of both T cells and APCs are alternative ligands of CD28 that compete with binding to its well-documented activatory ligand CD80 on the APC, resulting in attenuated co-stimulation. Removal of sialic acids enhances T cell activation and enhances the activity of effector T cells made hypofunctional via chronic viral infection through a mechanism that is synergistic with antibody blockade of the inhibitory PD-1 axis. These results reveal a previously unrecognized role for sialic acids in attenuation of CD28 mediated co-stimulation of T cells.One Sentence SummarySialic acids attenuate the second signal required for T cell activation.

Author(s): Rogers, Thomas F; Zhao, Fangzhu; Huang, Deli; Beutler, Nathan; Burns, Alison; He, Wan-Ting; Limbo, Oliver; Smith, Chloe; Song, Ge; Woehl, Jordan; Yang, Linlin; Abbott, Robert K; Callaghan, Sean; Garcia, Elijah; Hurtado, Jonathan; Parren, Mara; Peng, Linghang; Ricketts, James; Ricciardi, Michael J; Rawlings, Stephen A; Smith, Davey M; Nemazee, David; Teijaro, John R; Voss, James E; Andrabi, Raiees; Briney, Bryan; Landais, Elise; Sok, Devin; Jardine, Joseph G; Burton, Dennis R | Abstract: The development of countermeasures to prevent and treat COVID-19 is a global health priority. In under 7 weeks, we enrolled a cohort of SARS-CoV-2-recovered participants, developed neutralization assays to interrogate serum and monoclonal antibody responses, adapted our high throughput antibody isolation, production and characterization pipeline to rapidly screen over 1000 antigen-specific antibodies, and established an animal model to test protection. We report multiple highly potent neutralizing antibodies (nAbs) and show that passive transfer of a nAb provides protection against high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs define protective epitopes to guide vaccine design.

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.

Neutrophils contain high levels of chymotrypsin-like serine proteases (NSPs) within their azurophilic granules that have a multitude of functions within the immune system. In response, the pathogen Staphylococcus aureus has evolved three potent inhibitors (Eap, EapH1, and EapH2) that protect the bacterium as well as several of its secreted virulence factors from the degradative action of NSPs. We previously showed that these so-called EAP domain proteins represent a novel class of NSP inhibitors characterized by a non-covalent inhibitory mechanism and a distinct target specificity profile. Based upon high levels of structural homology amongst the EAP proteins and the NSPs, as well as supporting biochemical data, we predicted that the inhibited complex would be similar for all EAP/NSP pairs. However, we present here evidence that EapH1 and EapH2 bind the canonical NSP, Neutrophil Elastase (NE), in distinct orientations. We discovered that alteration of EapH1 residues at the EapH1/NE interface caused a dramatic loss of affinity and inhibition of NE, while mutation of equivalent positions in EapH2 had no effect on NE binding or inhibition. Surprisingly, mutation of residues in an altogether different region of EapH2 severely impacted both the NE binding and inhibitory properties of EapH2. Even though EapH1 and EapH2 bind and inhibit NE and a second NSP, Cathepsin G, equally well, neither of these proteins interacts with the structurally related, but non-proteolytic granule protein, azurocidin. These studies expand our understanding of EAP/NSP interactions and suggest that members of this immune evasion protein family are capable of diverse target recognition modes.

The extracellular adherence protein (Eap) plays a crucial role in pathogenesis and survival of Staphylococcus aureus by inhibiting the classical and lectin pathways of complement. We have previously shown that Eap binds with nanomolar affinity to complement C4b and disrupts the initial interaction between C4b and C2, thereby inhibiting formation of the classical and lectin pathway C3 pro-convertase. Although an underlying mechanism has been identified, the structural basis for Eap binding to C4b is poorly understood. Here, we show that Eap domains 3 and 4 each contain a low-affinity, but saturable binding site for C4b. Taking advantage of the high lysine content of Eap, we used a zero-length crosslinking approach to map the Eap binding site to both the α'- and γ-chains of C4b. We also probed the C4b/Eap interface through a chemical footprinting approach involving lysine modification, proteolytic digestion, and mass spectrometry. This identified seven lysines in Eap that undergo changes in solvent exposure upon C4b binding. We found that simultaneous mutation of these lysines to either alanine or glutamate diminished C4b binding and complement inhibition by Eap. Together, our results provide insight into Eap recognition of C4b, and suggest that the repeating domains that comprise Eap are capable of multiple ligand-binding modes.

Sulfur fluoride exchange (SuFEx) has emerged as the new generation of click chemistry. We report here a SuFEx-enabled, agnostic approach for the discovery and optimization of covalent inhibitors of human neutrophil elastase (hNE). Evaluation of our ever-growing collection of SuFExable compounds toward various biological assays unexpectedly revealed a selective and covalent hNE inhibitor: benzene-1,2-disulfonyl fluoride. Synthetic derivatization of the initial hit led to a more potent agent, 2-(fluorosulfonyl)phenyl fluorosulfate with IC 50 0.24 μM and greater than 833-fold selectivity over the homologous neutrophil serine protease, cathepsin G. The optimized, yet simple benzenoid probe only modified active hNE and not its denatured form.

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.

The pathogenic bacterium Staphylococcus aureus has evolved to actively evade many aspects of the human innate immune system by expressing a series of secreted inhibitory proteins. Among these, the extracellular adherence protein (Eap) has been shown to inhibit the classical and lectin pathways of the complement system. By binding to complement component C4b, Eap is able to inhibit formation of the CP/LP C3 pro-convertase. Secreted full-length, mature Eap consists of four ~98 residue domains, all of which adopt a similar beta-grasp fold, and are connected through a short linker region. Through multiple biochemical approaches, it has been determined that the third and fourth domains of Eap are responsible for C4b binding. Here we report the backbone and side-chain resonance assignments of the 11.3 kDa fourth domain of Eap. The assignment data has been deposited in the BMRB database under the accession number 26726.

The pathogenic bacterium Staphylococcus aureus actively evades many aspects of human innate immunity by expressing a series of small inhibitory proteins. A number of these proteins inhibit the complement system, which labels bacteria for phagocytosis and generates inflammatory chemoattractants. Although the majority of staphylococcal complement inhibitors act on the alternative pathway to block the amplification loop, only a few proteins act on the initial recognition cascades that constitute the classical pathway (CP) and lectin pathway (LP). We screened a collection of recombinant, secreted staphylococcal proteins to determine whether S. aureus produces other molecules that inhibit the CP and/or LP. Using this approach, we identified the extracellular adherence protein (Eap) as a potent, specific inhibitor of both the CP and LP. We found that Eap blocked CP/LP-dependent activation of C3, but not C4, and that Eap likewise inhibited deposition of C3b on the surface of S. aureus cells. In turn, this significantly diminished the extent of S. aureus opsonophagocytosis and killing by neutrophils. This combination of functional properties suggested that Eap acts specifically at the level of the CP/LP C3 convertase (C4b2a). Indeed, we demonstrated a direct, nanomolar-affinity interaction of Eap with C4b. Eap binding to C4b inhibited binding of both full-length C2 and its C2b fragment, which indicated that Eap disrupts formation of the CP/LP C3 proconvertase (C4b2). As a whole, our results demonstrate that S. aureus inhibits two initiation routes of complement by expression of the Eap protein, and thereby define a novel mechanism of immune evasion.

Neutrophils contain high levels of chymotrypsin‐like serine proteases (NSPs) within their azurophilic granules that have a multitude of functions within the immune system. In response, the pathogen Staphylococcus aureus has evolved three potent inhibitors (Eap, EapH1, and EapH2) that protect the bacterium as well as several of its secreted virulence factors from the degradative action of NSPs. We previously showed that these so‐called EAP domain proteins represent a novel class of NSP inhibitors characterized by a non‐covalent inhibitory mechanism and a distinct target specificity profile. Based upon high levels of structural homology amongst the EAP proteins and the NSPs, as well as supporting biochemical data, we predicted that the inhibited complex would be similar for all EAP/NSP pairs. However, we present here evidence that EapH1 and EapH2 bind the canonical NSP, Neutrophil Elastase (NE), in distinct orientations. We discovered that alteration of EapH1 residues at the EapH1/NE interface caused a dramatic loss of affinity and inhibition of NE, while mutation of equivalent positions in EapH2 had no effect on NE binding or inhibition. Surprisingly, mutation of residues in an altogether different region of EapH2 severely impacted both the NE binding and inhibitory properties of EapH2. Even though EapH1 and EapH2 bind and inhibit NE and a second NSP, Cathepsin G, equally well, neither of these proteins interacts with the structurally related, but non‐proteolytic granule protein, azurocidin. These studies expand our understanding of EAP/NSP interactions and suggest that members of this immune evasion protein family are capable of diverse target recognition modes.

The extracellular adherence protein (Eap) plays a crucial role in pathogenesis and survival of Staphylococcus aureus by inhibiting the classical and lectin pathways of complement. We have previously shown that Eap binds with nanomolar affinity to complement C4b and disrupts the initial interaction between C4b and C2, thereby inhibiting formation of the classical and lectin pathway C3 pro‐convertase. Although an underlying mechanism has been identified, the structural basis for Eap binding to C4b is poorly understood. Here, we show that Eap domains 3 and 4 each contain a low‐affinity, but saturable binding site for C4b. Taking advantage of the high lysine content of Eap, we used a zero‐length crosslinking approach to map the Eap binding site to both the α′‐ and γ‐chains of C4b. We also probed the C4b/Eap interface through a chemical footprinting approach involving lysine modification, proteolytic digestion, and mass spectrometry. This identified seven lysines in Eap that undergo changes in solvent exposure upon C4b binding. We found that simultaneous mutation of these lysines to either alanine or glutamate diminished C4b binding and complement inhibition by Eap. Together, our results provide insight into Eap recognition of C4b, and suggest that the repeating domains that comprise Eap are capable of multiple ligand‐binding modes.

The complement system plays a central role in a number of human inflammatory diseases, and there is a significant need for development of complement-directed therapies. The discovery of an arsenal of anti-complement proteins secreted by the pathogen Staphylococcus aureus brought with it the potential for harnessing the powerful inhibitory properties of these molecules. One such family of inhibitors, the SCINs, interact with a functional “hot-spot” on the surface of C3b. SCINs not only stabilize an inactive form of the alternative pathway (AP) C3 convertase (C3bBb), but also overlap the C3b binding site of complement factors B and H. Here we determined that a conserved Arg residue in SCINs is critical for function of full-length SCIN proteins. Despite this, we also found SCIN-specific differences in the contributions of other residues found at the C3b contact site, which suggested that a more diverse repertoire of residues might be able to recognize this region of C3b. To investigate this possibility, we conducted a phage display screen aimed at identifying SCIN-competitive 12-mer peptides. In total, seven unique sequences were identified and all exhibited direct C3b binding. A subset of these specifically inhibited the AP in assays of complement function. The mechanism of AP inhibition by these peptides was probed through surface plasmon resonance approaches, which revealed that six of the seven peptides disrupted C3bBb formation by interfering with factor B/C3b binding. To our knowledge this study has identified the first small molecules that retain inhibitory properties of larger staphylococcal immune evasion proteins.