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Bruton tyrosine kinase (BTK) is expressed in B cells and innate immune cells, acting as an essential signaling element in multiple immune cell pathways. Selective BTK inhibition has the potential to target multiple immune-mediated disease pathways. Rilzabrutinib is an oral, reversible, covalent BTK inhibitor designed for immune-mediated diseases. We examined the pharmacodynamic profile of rilzabrutinib and its preclinical mechanisms of action. In addition to potent and selective BTK enzyme and cellular activity, rilzabrutinib inhibited activation and inflammatory activities of B cells and innate cells such as macrophages, basophils, mast cells, and neutrophils, without cell death (in human and rodent assay systems). Rilzabrutinib demonstrated dose-dependent improvement of clinical scores and joint pathology in a rat model of collagen-induced arthritis and demonstrated reductions in autoantibody-mediated FcγR signaling in vitro and in vivo, with blockade of rat Arthus reaction, kidney protection in mouse Ab-induced nephritis, and reduction in platelet loss in mouse immune thrombocytopenia. Additionally, rilzabrutinib inhibited IgE-mediated, FcεR-dependent immune mechanisms in human basophils and mast cell-dependent mouse models. In canines with naturally occurring pemphigus, rilzabrutinib treatment resulted in rapid clinical improvement demonstrated by anti-inflammatory effects visible within 2 wk and all animals proceeding to complete or substantial disease control. Rilzabrutinib is characterized by reversible covalent BTK binding, long BTK residence time with low systemic exposure, and multiple mechanistic and biological effects on immune cells. Rilzabrutinib's unique characteristics and promising efficacy and safety profile support clinical development of rilzabrutinib for a broad array of immune-mediated diseases.

Reversible covalency, achieved with, for instance, highly electron-deficient olefins, offers a compelling strategy to design chemical probes and drugs that benefit from the sustained target engagement afforded by irreversible compounds, while avoiding permanent protein modification. Reversible covalency has mainly been evaluated for cysteine residues in individual kinases and the broader potential for this strategy to engage cysteines across the proteome remains unexplored. Herein, we describe a mass-spectrometry-based platform that integrates gel filtration with activity-based protein profiling to assess cysteine residues across the human proteome for both irreversible and reversible interactions with small-molecule electrophiles. Using this method, we identify numerous cysteine residues from diverse protein classes that are reversibly engaged by cyanoacrylamide fragment electrophiles, revealing the broad potential for reversible covalency as a strategy for chemical-probe discovery.

Background and purposeFollowing inflammatory stimuli, neutrophils are recruited to sites of inflammation and exert effector functions that often have deleterious effects on tissue integrity, which can lead to organ failure. Bruton's tyrosine kinase (Btk) is expressed in neutrophils and constitutes a promising pharmacological target for neutrophil-mediated tissue damage. Here, we evaluate a selective reversible inhibitor of Btk, PRN473, for its ability to dampen neutrophil influx via inhibition of adhesion receptor signalling pathways.Experimental approachIn vitro assays were used to assess fMLP receptor 1 (Fpr-1)-mediated binding of ligands to the adhesion receptors macrophage antigen-1 (Mac-1) and lymphocyte function antigen-1. Intravital microscopy of the murine cremaster was used to evaluate post-adhesion strengthening and endoluminal crawling. Finally, neutrophil influx was visualized in a clinically relevant model of sterile liver injury in vivo. Btk knockout animals were used as points of reference for Btk functions.Key resultsPharmacological inhibition of Btk by PRN473 reduced fMLP-induced phosphorylation of Btk and Mac-1 activation. Biochemical experiments demonstrated the specificity of the inhibitor. PRN473 (20 mg·kg-1 ) significantly reduced intravascular crawling and neutrophil recruitment into inflamed tissue in a model of sterile liver injury, down to levels seen in Btk-deficient animals. A higher dose did not provide additional reduction of intravascular crawling and neutrophil recruitment.Conclusions and implicationsPRN473, a highly selective inhibitor of Btk, potently attenuates sterile liver injury by inhibiting the activation of the β2 -integrin Mac-1 and subsequently neutrophil recruitment into inflamed tissue.

Drugs with prolonged on-target residence times often show superior efficacy, yet general strategies for optimizing drug-target residence time are lacking. Here we made progress toward this elusive goal by targeting a noncatalytic cysteine in Bruton's tyrosine kinase (BTK) with reversible covalent inhibitors. Using an inverted orientation of the cysteine-reactive cyanoacrylamide electrophile, we identified potent and selective BTK inhibitors that demonstrated biochemical residence times spanning from minutes to 7 d. An inverted cyanoacrylamide with prolonged residence time in vivo remained bound to BTK for more than 18 h after clearance from the circulation. The inverted cyanoacrylamide strategy was further used to discover fibroblast growth factor receptor (FGFR) kinase inhibitors with residence times of several days, demonstrating the generalizability of the approach. Targeting of noncatalytic cysteines with inverted cyanoacrylamides may serve as a broadly applicable platform that facilitates 'residence time by design', the ability to modulate and improve the duration of target engagement in vivo.

The study of steady state kinetics of spleen tyrosine kinase (Syk) that plays in important signaling role in several types of immune cells is presented. The studies provide insight into enzymology and activation mechanism Syk.

Isothermal titration calorimetry (ITC) is used to compare the binding thermodynamics of calmodulin (CaM) to peptides calmodulin-dependent protein kinase II(CaMKII). The obtained value subpicomolar [K.sub.d] value suggest that the final conformational change accompanying CaM trapping buries little additional surface area, but also involves formation of new hydrogen bonds and van der Waals contacts that are responsible for CaM-trapped state.

The strength of hippocampal synapses can be persistently increased by signals that activate C[a.sup.2+] / calmodulin-dependent protein kinase II (CaMKII). This CaMKII-dependent long-term potentiation is important for hippocampal learning and memory. In this work we show that CaMKII exhibits an intriguing switch-like activation that likely is important for changes in synaptic strength. We found that autophosphorylation of CaMKII by itself showed a steep dependence on C[a.sup.2+] concentration [Hill coefficient ([n.sub.h) [approximately equal to] 5]. However, an even steeper C[a.sup.2+] dependence ([n.sub.h] [approximately equal to] 8) was observed when autophosphorylation is balanced by the dephosphorylation activity of protein phosphatase 1 (PP1). This autophosphorylation-dephosphorylation switch was found to be reversible because PP1 dephosphorylates CaMKII when C[a.sup.2+] is lowered to a basal level. The switch-like response of a CaMKII-PP1 system suggests that CaMKII and PP1 may function together as a simple molecular device that specifically translates only strong C[a.sup.2+] signals into all-or-none potentiation of individual hippocampal synapses.

Salt dependence of binding of tyrosyl-phosphorylated peptide binding to the tandem SH2 domain of the Syk kinase and the single SH2 domain of the Src kinase have been studied. The findings show that electrostatic interactions have important roles in mediating pY recognition and binding and also in the specificity of the interactions between tyrosyl phosphopeptides and SH2 domains.

A study on Src homology 2 (SH2) domains and their functions investigated the specificity of the phosphopeptide interaction involving the Src SH2 domain and a peptide containing the sequence PQpYEEIPI (pYEEI). The study also probed the energetic coupling between the +1 Glu, +2 Glu and +3 Ile phosphopeptide residues. Among other results, it was found that the Src SH2 domain is quite permissive of conservative substitutions in the EEI motif at all three peptide positions.

Isothermal titration calorimetry was used to investigate the proton linkage involved in the interaction between phosphopeptide and SRC homology 2 (SH2) binding domains. Two independent but complementary series of experiments were undertaken to probe this interaction. The method can be used in the thermodynamic evaluation of the function of ionizable groups in the domain recognition during SH2 interaction. The energetic linkage between phosphotyrosine protonation and peptide binding is described.

A study on the thermodynamic characteristics of the interaction of Src homology 2 (SH2) domain with several phosphopeptides is presented. SH2 are protein modules that bind specifically to tyrosyl phosphorylated peptides or signaling proteins. Isothermal titration calorimetric measurements reveal that peptides with smaller hydrophobic side chains at the +3 position showed moderate loss of binding affinity. The temperature-independent heat capacity change upon association is almost the same for all peptides.

p75NTR is a neurotrophin receptor that can mediate either survival or death of neurons depending on the cell context. Modulation of p75NTR is a promising strategy to promote neuronal survival for treatment of cognitive disorders such as Alzheimer's disease. Despite years of investigation into the signaling mechanisms of p75NTR, no p75NTR signaling assay has yet been developed that is compatible with efficient screening of small-molecule modulators. In this work, we developed a homogeneous cell-based assay for screening p75NTR modulators and studying p75NTR function. Stimulation of p75NTR-transfected cells using either nerve growth factor (NGF) or Pro-NGF resulted in an enhanced caspase-3 activity as assessed by cleavage of a fluorescent caspase-3 substrate. Optimization of the assay with respect to time, cell density, NGF and Pro-NGF concentration, and other factors provided a twofold increase in the caspase-3 activity compared to background. Withdrawal of serum during the NGF or Pro-NGF treatment period was found to be essential for p75NTR-dependent caspase-3 activation. We validated the method by demonstrating that a signaling-incompetent p75NTR mutant could not substitute for wild-type p75NTR in mediating caspase-3 activation. A focused library screen identified new inhibitors of p75NTR signaling. This method will be useful for identifying small- molecule modulators of p75NTR as well as further characterizing downstream signaling events. [ABSTRACT FROM AUTHOR]

The strength of hippocampal synapses can be persistently increased by signals that activate Ca[sup 2+]/calmodulin-dependent protein kinase II (CaMKII). This CaMKII-dependent long-term potentiation is important for hippocampal learning and memory. In this work we show that CaMKII exhibits an intriguing switch-like activation that likely is important for changes in synaptic strength. We found that autophosphorylation of CaMKII by itself showed a steep dependence on Ca[sup 2+] concentration [Hill coefficient (n[sub H]) ≈ 51. However, an even steeper Ca[sup 2+] dependence (n[sub H] ≈ 8) was observed when autophosphorylation is balanced by the dephosphorylation activity of protein phosphatase 1 (PP1). This autophosphorylationdephosphorylation switch was found to be reversible because PP1 dephosphorylates CaMKII when Ca[sup 2+] is lowered to a basal level. The switch-like response of a CaMKII-PP1 system suggests that CaMKII and PP1 may function together as a simple molecular device that specifically translates only strong Ca[sup 2+] signals into all-or-none potentiation of individual hippocampal synapses. [ABSTRACT FROM AUTHOR]

Presents a thermodynamic study which probed the `two-pronged plug two-holed socket' model, for the mechanism of binding of the Src SH2 domain to phosphotyrosyl peptides. Definition of Src homology 2 (SH2) domains; Functions of SH2; Details on methods employed to conduct study; Predictions of the binding model; Discussion on study's results.

In T cells, the Tec kinases IL-2-inducible T cell kinase (ITK) and resting lymphocyte kinase (RLK) are activated by TCR stimulation and are required for optimal downstream signaling. Studies of CD4+ T cells from Itk-/- and Itk-/-Rlk-/- mice have indicated differential roles of ITK and RLK in Th1, Th2, and Th17 differentiation and cytokine production. However, these findings are confounded by the complex T cell developmental defects in these mice. In this study, we examine the consequences of ITK and RLK inhibition using a highly selective and potent small molecule covalent inhibitor PRN694. In vitro Th polarization experiments indicate that PRN694 is a potent inhibitor of Th1 and Th17 differentiation and cytokine production. Using a T cell adoptive transfer model of colitis, we find that in vivo administration of PRN694 markedly reduces disease progression, T cell infiltration into the intestinal lamina propria, and IFN-γ production by colitogenic CD4+ T cells. Consistent with these findings, Th1 and Th17 cells differentiated in the presence of PRN694 show reduced P-selectin binding and impaired migration to CXCL11 and CCL20, respectively. Taken together, these data indicate that ITK plus RLK inhibition may have therapeutic potential in Th1-mediated inflammatory diseases. [ABSTRACT FROM AUTHOR]

Reversible covalency, achieved with, for instance, highly electron-deficient olefins, offers a compelling strategy to design chemical probes and drugs that benefit from the sustained target engagement afforded by irreversible compounds, while avoiding permanent protein modification. Reversible covalency has mainly been evaluated for cysteine residues in individual kinases and the broader potential for this strategy to engage cysteines across the proteome remains unexplored. Herein, we describe a mass-spectrometry-based platform that integrates gel filtration with activity-based protein profiling to assess cysteine residues across the human proteome for both irreversible and reversible interactions with small-molecule electrophiles. Using this method, we identify numerous cysteine residues from diverse protein classes that are reversibly engaged by cyanoacrylamide fragment electrophiles, revealing the broad potential for reversible covalency as a strategy for chemical-probe discovery., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this chapter, we have described the biophysical investigations which have dissected the mechanisms of SH2 domain function. Due to nearly a decade and a half of investigation on SH2 domains, much about their binding mechanism has been characterized. SH2 domains have been found to have a positively charged binding cavity, largely conserved between different SH2 domains, which coordinates binding of the pTyr in the target. The ionic interactions between this pocket and the pTyr, in particular, between Arg beta B5 and the phosphate, provide the majority of the binding energy stabilizing SH2 domain-target interactions. The specificity in SH2 domain-target interactions emanates most often from the interactions between the residues C-terminal to the pTyr in the target and the specificity determining residues in the C-terminal half of the SH2 domain. However, the interactions in the specificity determining region of SH2 domains are weak, and hence single SH2 domains show only a modest level of specificity for tyrosine phosphorylated targets. Greater specificity in SH2 domain-containing protein-tyrosine phosphorylated target interactions can be achieved by placing SH2 domains in tandem (as is often found) or possibly through specific localization of SH2 domain-containing proteins within the cell. Although a relatively good understanding of how SH2 domains function in isolation has been obtained, the ways in which SH2 domain binding is coupled to allosteric transmission of signals in larger SH2 domain-containing proteins are still not clear. Hence, the future should bring further investigations of the mechanisms by which SH2 domain ligation alters the enzymatic activity and cellular localization of SH2 domain-containing proteins.