Protease-activated receptors in health and disease.

Proteases are signaling molecules that specifically control cellular functions by cleaving protease-activated receptors (PARs). The four known PARs are members of the large family of G protein-coupled receptors. These transmembrane receptors control most physiological and pathological processes and are the target of a large proportion of therapeutic drugs. Signaling proteases include enzymes from the circulation; from immune, inflammatory epithelial, and cancer cells; as well as from commensal and pathogenic bacteria. Advances in our understanding of the structure and function of PARs provide insights into how diverse proteases activate these receptors to regulate physiological and pathological processes in most tissues and organ systems. The realization that proteases and PARs are key mediators of disease, coupled with advances in understanding the atomic level structure of PARs and their mechanisms of signaling in subcellular microdomains, has spurred the development of antagonists, some of which have advanced to the clinic. Herein we review the discovery, structure, and function of this receptor system, highlight the contribution of PARs to homeostatic control, and discuss the potential of PAR antagonists for the treatment of major diseases.

Inflammation and thrombosis in COVID-19 pathophysiology: proteinase-activated and purinergic receptors as drivers and candidate therapeutic targets.

Evolving information has identified disease mechanisms and dysregulation of host biology that might be targeted therapeutically in coronavirus disease 2019 (COVID-19). Thrombosis and coagulopathy, associated with pulmonary injury and inflammation, are emerging clinical features of COVID-19. We present a framework for mechanisms of thrombosis in COVID-19 that initially derive from interaction of SARS-CoV-2 with ACE2, resulting in dysregulation of angiotensin signaling and subsequent inflammation and tissue injury. These responses result in increased signaling by thrombin (proteinase-activated) and purinergic receptors, which promote platelet activation and exert pathological effects on other cell types (e.g., endothelial cells, epithelial cells, and fibroblasts), further enhancing inflammation and injury. Inhibitors of thrombin and purinergic receptors may, thus, have therapeutic effects by blunting platelet-mediated thromboinflammation and dysfunction in other cell types. Such inhibitors include agents (e.g., anti-platelet drugs) approved for other indications, and that could be repurposed to treat, and potentially improve the outcome of, COVID-19 patients. COVID-19, caused by the SARS-CoV-2 virus, drives dysregulation of angiotensin signaling, which, in turn, increases thrombin-mediated and purinergic-mediated activation of platelets and increase in inflammation. This thromboinflammation impacts the lungs and can also have systemic effects. Inhibitors of receptors that drive platelet activation or inhibitors of the coagulation cascade provide opportunities to treat COVID-19 thromboinflammation.

New insight into the agonism of protease-activated receptors as an immunotherapeutic strategy.

The activation and mobilization of immune cells play a crucial role in immunotherapy. Existing therapeutic interventions, such as cytokines administration, aim to enhance immune cell activity. However, these approaches usually result in modest effectiveness and toxic side effects, thereby restricting their clinical application. Protease-activated receptors (PARs), a subfamily of G protein-coupled receptors, actively participate in the immune system by directly activating immune cells. The activation of PARs by proteases or synthetic ligands can modulate immune cell behavior, signaling, and responses to treat immune-related diseases, suggesting the significance of PARs agonism in immunotherapy. However, the agonism of PARs in therapeutical applications remains rarely discussed, since it has been traditionally considered that PARs activation facilitates disease progressions. This review aims to comprehensively summarize the activation, rather than inhibition, of PARs in immune-related physiological responses and diseases. Additionally, we will discuss the emerging immunotherapeutic potential of PARs agonism, providing a new strategic direction for PARs-mediated immunotherapy.

Beyond Anticoagulation: A Comprehensive Review of Non-Vitamin K Oral Anticoagulants (NOACs) in Inflammation and Protease-Activated Receptor Signaling.

Non-vitamin K oral anticoagulants (NOACs) have revolutionized anticoagulant therapy, offering improved safety and efficacy over traditional agents like warfarin. This review comprehensively examines the dual roles of NOACs-apixaban, rivaroxaban, edoxaban, and dabigatran-not only as anticoagulants, but also as modulators of inflammation via protease-activated receptor (PAR) signaling. We highlight the unique pharmacotherapeutic properties of each NOAC, supported by key clinical trials demonstrating their effectiveness in preventing thromboembolic events. Beyond their established anticoagulant roles, emerging research suggests that NOACs influence inflammation through PAR signaling pathways, implicating factors such as factor Xa (FXa) and thrombin in the modulation of inflammatory responses. This review synthesizes current evidence on the anti-inflammatory potential of NOACs, exploring their impact on inflammatory markers and conditions like atherosclerosis and diabetes. By delineating the mechanisms by which NOACs mediate anti-inflammatory effects, this work aims to expand their therapeutic utility, offering new perspectives for managing inflammatory diseases. Our findings underscore the broader clinical implications of NOACs, advocating for their consideration in therapeutic strategies aimed at addressing inflammation-related pathologies. This comprehensive synthesis not only enhances understanding of NOACs' multifaceted roles, but also paves the way for future research and clinical applications in inflammation and cardiovascular health.

Vascular Endothelial Cells Produce Coagulation Factors That Control Their Growth via Joint Protease-Activated Receptor and C5a Receptor 1 (CD88) Signaling.

As per the classical view of the coagulation system, it functions solely in plasma to maintain hemostasis. An experimental approach modeling vascular reconstitution was used to show that vascular endothelial cells (ECs) endogenously synthesize coagulation factors during angiogenesis. Intracellular thrombin generated from this synthesis promotes the mitotic function of vascular endothelial cell growth factor A (VEGF-A). The thrombin concurrently cleaves C5a from EC-synthesized complement component C5 and unmasks the tethered ligand for EC-expressed protease-activated receptor 4 (PAR4). The two ligands jointly trigger EC C5a receptor-1 (C5ar1) and PAR4 signaling, which together promote VEGF receptor 2 growth signaling. C5ar1 is functionally associated with PAR4, enabling C5a or thrombin to elicit Gαi and/or Gαq signaling. EC coagulation factor and EC complement component synthesis concurrently down-regulate with contact inhibition. The connection of these processes with VEGF receptor 2 signaling provides new insights into mechanisms underlying angiogenesis. Knowledge of endogenous coagulation factor/complement component synthesis and joint PAR4/C5ar1 signaling could be applied to other cell types.

Factor VII activating protease (FSAP) inhibits the outcome of ischemic stroke in mouse models.

The outcome of ischemic stroke can be improved by further refinements of thrombolysis and reperfusion strategies. Factor VII activating protease (FSAP) is a circulating serine protease that could be important in this context. Its levels are raised in patients as well as mice after stroke and a single nucleotide polymorphism (SNP) in the coding sequence, which results in an inactive enzyme, is linked to an increased risk of stroke. In vitro, FSAP cleaves fibrinogen to promote fibrinolysis, activates protease-activated receptors, and decreases the cellular cytotoxicity of histones. Based on these facts, we hypothesized that FSAP can be used as a treatment for ischemic stroke. A combination of tissue plasminogen activator (tPA), a thrombolytic drug, and recombinant serine protease domain of FSAP (FSAP-SPD) improved regional cerebral perfusion and neurological outcome and reduced infarct size in a mouse model of thromboembolic stroke. FSAP-SPD also improved stroke outcomes and diminished the negative consequences of co-treatment with tPA in the transient middle cerebral artery occlusion model of stroke without altering cerebral perfusion. The inactive MI-isoform of FSAP had no impact in either model. FSAP enhanced the lysis of blood clots in vitro, but in the tail transection model of hemostasis, FSAP-SPD treatment provoked a faster clotting time indicating that it also has pro-coagulant actions. Thus, apart from enhancing thrombolysis, FSAP has multiple effects on stroke progression and represents a promising novel therapeutic strategy in the treatment of ischemic stroke.

Species Differences in Platelet Protease-Activated Receptors.

Protease-activated receptors (PARs) are a class of integral membrane proteins that are cleaved by a variety of proteases, most notably thrombin, to reveal a tethered ligand and promote activation. PARs are critical mediators of platelet function in hemostasis and thrombosis, and therefore are attractive targets for anti-platelet therapies. Animal models studying platelet PAR physiology have relied heavily on genetically modified mouse strains, which have provided ample insight but have some inherent limitations. The current review aims to summarize the notable PAR expression and functional differences between the mouse and human, in addition to highlighting some recently developed tools to further study human physiology in mouse models.

Protease-activated receptor-2 dependent and independent responses of bone cells to prostate cancer cell secretory products.

BACKGROUND: Metastatic prostate cancer lesions in the skeleton are frequently characterized by excessive formation of bone. Prostate cancer cells secrete factors, including serine proteases, that are capable of influencing the behavior of surrounding cells. Some of these proteases activate protease-activated receptor-2 (PAR2 ), which is expressed by osteoblasts (bone-forming cells) and precursors of osteoclasts (bone-resorbing cells). The aim of the current study was to investigate a possible role for PAR2 in regulating the behavior of bone cells exposed to metastatic prostate cancer cells. METHODS: The effect of medium conditioned by the PC3, DU145, and MDA-PCa-2b prostate cancer cell lines was investigated in assays of bone cell function using cells isolated from wildtype and PAR2 -null mice. Osteoclast differentiation was assessed by counting tartrate-resistant acid phosphatase-positive multinucleate cells in bone marrow cultured in osteoclastogenic medium. Osteoblasts were isolated from calvariae of neonatal mice, and BrdU incorporation was used to assess their proliferation. Assays of alkaline phosphatase activity and quantitative PCR analysis of osteoblastic gene expression were used to assess osteoblast differentiation. Responses of osteoblasts to medium conditioned by MDA-PCa-2b cells were analyzed by RNAseq. RESULTS: Conditioned medium (CM) from all three cell lines inhibited osteoclast differentiation independently of PAR2 . Media from PC3 and DU145 cells had no effect on assays of osteoblast function. Medium conditioned by MDA-PCa-2b cells stimulated BrdU incorporation in both wildtype and PAR2 -null osteoblasts but increased alkaline phosphatase activity and Runx2 and Col1a1 expression in wildtype but not PAR2 -null cells. Functional enrichment analysis of RNAseq data identified enrichment of multiple gene ontology terms associated with lysosomal function in both wildtype and PAR2 -null cells in response to MDA-PCa-2b-CM. Analysis of individual genes identified osteogenesis-associated genes that were either upregulated by MDA-PCa-2b-CM selectively in wildtype cells or downregulated selectively in PAR2 -null cells. CONCLUSIONS: Factors secreted by prostate cancer cells influence bone cell behavior through both PAR2 -dependent and -independent mechanisms. Both PAR2 -independent suppression of osteoclast differentiation and PAR2 -dependent stimulation of osteogenesis are likely to determine the nature of prostate cancer metastases in bone.

Protease-activated receptor-mediated platelet aggregation in patients with type 2 diabetes on potent P2Y12 inhibitors.

BACKGROUND: Antiplatelet therapy is a cornerstone in the secondary prevention of ischemic events following percutaneous coronary intervention (PCI). The new P2Y12 receptor inhibitors prasugrel and ticagrelor have been shown to improve patients' outcomes. Whether or not these drugs have equal efficacy in individuals with or without diabetes is disputed. Furthermore, platelets can be activated by thrombin, which is, at least in part, independent of P2Y12 -mediated platelet activation. Protease-activated receptor (PAR)-1 and -4 are thrombin receptors on human platelets. We sought to compare the in vitro efficacy of prasugrel (n = 121) and ticagrelor (n = 99) to inhibit PAR-mediated platelet aggregation in individuals with type 2 diabetes (prasugrel n = 26, ticagrelor n = 29). MATERIALS AND METHODS: We compared P2Y12 -, PAR-1- and PAR-4-mediated platelet aggregation as assessed by multiple electrode platelet aggregometry between prasugrel- and ticagrelor-treated patients without and with type 2 diabetes who underwent acute PCI. RESULTS: Overall, there were no differences of P2Y12 -, PAR-1- and PAR-4-mediated platelet aggregation between prasugrel- and ticagrelor-treated patients. However, both drugs inhibited P2Y12 -mediated platelet aggregation stronger, and thereby to a similar extent in patients with type 2 diabetes than in those without diabetes. There was no correlation between either P2Y12 -, or PAR-1- or PAR-4-mediated platelet aggregation and levels of HbA1c or the body mass index (BMI). However, we observed patients with high residual platelet reactivity in response to PAR-1 and PAR-4 stimulation in all cohorts. CONCLUSION: Prasugrel and ticagrelor inhibit P2Y12 - and PAR-mediated platelet aggregation in individuals with diabetes to a similar extent, irrespective of HbA1c levels and BMI.

Diversification of PAR signaling through receptor crosstalk.

Protease activated receptors (PARs) are among the first receptors shown to transactivate other receptors: noticeably, these interactions are not limited to members of the same family, but involve receptors as diverse as receptor kinases, prostanoid receptors, purinergic receptors and ionic channels among others. In this review, we will focus on the evidence for PAR interactions with members of their own family, as well as with other types of receptors. We will discuss recent evidence as well as what we consider as emerging areas to explore; from the signalling pathways triggered, to the physiological and pathological relevance of these interactions, since this additional level of molecular cross-talk between receptors and signaling pathways is only beginning to be explored and represents a novel mechanism providing diversity to receptor function and play important roles in physiology and disease.

Protease Activated Receptors: A Pathway to Boosting Mesenchymal Stromal Cell Therapeutic Efficacy in Acute Respiratory Distress Syndrome?

Acute Respiratory Distress Syndrome is the most common cause of respiratory failure among critically ill patients, and its importance has been heightened during the COVID-19 pandemic. Even with the best supportive care, the mortality rate in the most severe cases is 40-50%, and the only pharmacological agent shown to be of possible benefit has been steroids. Mesenchymal stromal cells (MSCs) have been tested in several pre-clinical models of lung injury and been found to have significant therapeutic benefit related to: (a) potent immunomodulation; (b) secretion of epithelial and endothelial growth factors; and (c) augmentation of host defense to infection. Initial translational efforts have shown signs of promise, but the results have not yielded the anticipated outcomes. One potential reason is the relatively low survival of MSCs in inflammatory conditions as shown in several studies. Therefore, strategies to boost the survival of MSCs are needed to enhance their therapeutic effect. Protease-activated receptors (PARs) may represent one such possibility as they are G-protein coupled receptors expressed by MSCs and control several facets of cell behavior. This review summarizes some of the existing literature about PARs and MSCs and presents possible future areas of investigation in order to develop potential, PAR-modified MSCs with enhanced therapeutic efficiency.

Coagulation, Protease-Activated Receptors, and Diabetic Kidney Disease: Lessons from eNOS-Deficient Mice.

Endothelial nitric oxide synthase (eNOS) dysfunction is known to exacerbate the progression and prognosis of diabetic kidney disease (DKD). One of the mechanisms through which this is achieved is that low eNOS levels are associated with hypercoagulability, which promotes kidney injury. In the extrinsic coagulation cascade, the tissue factor (factor III) and downstream coagulation factors, such as active factor X (FXa), exacerbate inflammation through activation of the protease-activated receptors (PARs). Recently, it has been shown that the lack of or reduced eNOS expression in diabetic mice, as a model of advanced DKD, increases renal tissue factor levels and PAR1 and 2 expression in their kidneys. Furthermore, pharmaceutical inhibition or genetic deletion of coagulation factors or PARs ameliorated inflammation in DKD in mice lacking eNOS. In this review, we summarize the relationship between eNOS, coagulation, and PARs and propose a novel therapeutic option for the management of patients with DKD.

Protease Activated Receptors and Arthritis.

The catabolic and destructive activity of serine proteases in arthritic joints is well known; however, these enzymes can also signal pain and inflammation in joints. For example, thrombin, trypsin, tryptase, and neutrophil elastase cleave the extracellular N-terminus of a family of G protein-coupled receptors and the remaining tethered ligand sequence then binds to the same receptor to initiate a series of molecular signalling processes. These protease activated receptors (PARs) pervade multiple tissues and cells throughout joints where they have the potential to regulate joint homeostasis. Overall, joint PARs contribute to pain, inflammation, and structural integrity by altering vascular reactivity, nociceptor sensitivity, and tissue remodelling. This review highlights the therapeutic potential of targeting PARs to alleviate the pain and destructive nature of elevated proteases in various arthritic conditions.

Emerging Roles of Protease-Activated Receptors (PARs) in the Modulation of Synaptic Transmission and Plasticity.

Protease-activated receptors (PARs) are a class of G protein-coupled receptors (GPCRs) with a unique mechanism of activation, prompted by a proteolytic cleavage in their N-terminal domain that uncovers a tethered ligand, which binds and stimulates the same receptor. PARs subtypes (PAR1-4) have well-documented roles in coagulation, hemostasis, and inflammation, and have been deeply investigated for their function in cellular survival/degeneration, while their roles in the brain in physiological conditions remain less appreciated. Here, we describe PARs' effects in the modulation of neurotransmission and synaptic plasticity. Available evidence, mainly concerning PAR1-mediated and PAR2-mediated regulation of glutamatergic and GABAergic transmission, supports that PARs are important modulators of synaptic efficacy and plasticity in normal conditions.

GPCR Partners as Cancer Driver Genes: Association with PH-Signal Proteins in a Distinctive Signaling Network.

The essential role of G-protein coupled receptors (GPCRs) in tumor growth is recognized, yet a GPCR based drug in cancer is rare. Understanding the molecular path of a tumor driver gene may lead to the design and development of an effective drug. For example, in members of protease-activated receptor (PAR) family (e.g., PAR1 and PAR2), a novel PH-binding motif is allocated as critical for tumor growth. Animal models have indicated the generation of large tumors in the presence of PAR1 or PAR2 oncogenes. These tumors showed effective inhibition when the PH-binding motif was either modified or were inhibited by a specific inhibitor targeted to the PH-binding motif. In the second part of the review we discuss several aspects of some cardinal GPCRs in tumor angiogenesis.

Astragalin Inhibits Cigarette Smoke-Induced Pulmonary Thrombosis and Alveolar Inflammation and Disrupts PAR Activation and Oxidative Stress-Responsive MAPK-Signaling.

Epidemiological evidence shows that smoking causes a thrombophilic milieu that may play a role in the pathophysiology of chronic obstructive pulmonary disease (COPD) as well as pulmonary thromboembolism. The increased nicotine level induces a prothrombotic status and abnormal blood coagulation in smokers. Since several anticoagulants increase bleeding risk, alternative therapies need to be identified to protect against thrombosis without affecting hemostasis. Astragalin is a flavonoid present in persimmon leaves and green tea seeds and exhibits diverse activities of antioxidant and anti-inflammation. The current study investigated that astragalin attenuated smoking-induced pulmonary thrombosis and alveolar inflammation. In addition, it was explored that molecular links between thrombosis and inflammation entailed protease-activated receptor (PAR) activation and oxidative stress-responsive mitogen-activated protein kinase (MAPK)-signaling. BALB/c mice were orally administrated with 10-20 mg/kg astragalin and exposed to cigarette smoke for 8 weeks. For the in vitro study, 10 U/mL thrombin was added to alveolar epithelial A549 cells in the presence of 1-20 µM astragalin. The cigarette smoking-induced the expression of PAR-1 and PAR-2 in lung tissues, which was attenuated by the administration of ≥10 mg/kg astragalin. The oral supplementation of ≥10 mg/kg astragalin to cigarette smoke-challenged mice attenuated the protein induction of urokinase plasminogen activator, plasminogen activator inhibitor-1and tissue factor, and instead enhanced the induction of tissue plasminogen activator in lung tissues. The astragalin treatment alleviated cigarette smoke-induced lung emphysema and pulmonary thrombosis. Astragalin caused lymphocytosis and neutrophilia in bronchoalveolar lavage fluid due to cigarette smoke but curtailed infiltration of neutrophils and macrophages in airways. Furthermore, this compound retarded thrombin-induced activation of PAR proteins and expression of inflammatory mediators in alveolar cells. Treating astragalin interrupted PAR proteins-activated reactive oxygen species production and MAPK signaling leading to alveolar inflammation. Accordingly, astragalin may interrupt the smoking-induced oxidative stress-MAPK signaling-inflammation axis via disconnection between alveolar PAR activation and pulmonary thromboembolism.

Proteolytic Cleavage of Bioactive Peptides and Protease-Activated Receptors in Acute and Post-Colitis.

The protease activity in inflammatory bowel disease (IBD) and irritable bowel syndrome has been studied extensively using synthetic fluorogenic substrates targeting specific sets of proteases. We explored activities in colonic tissue from a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat model by investigating the cleavage of bioactive peptides. Pure trypsin- and elastase-like proteases on the one hand and colonic tissue from rats with TNBS-induced colitis in the acute or post-inflammatory phase on the other, were incubated with relevant peptides to identify their cleavage pattern by mass spectrometry. An increased cleavage of several peptides was observed in the colon from acute colitis rats. The tethered ligand (TL) sequences of peptides mimicking the N-terminus of protease-activated receptors (PAR) 1 and 4 were significantly unmasked by acute colitis samples and these cleavages were positively correlated with thrombin activity. Increased cleavage of ß-endorphin and disarming of the TL-sequence of the PAR3-based peptide were observed in acute colitis and linked to chymotrypsin-like activity. Increased processing of the enkephalins points to the involvement of proteases with specificities different from trypsin- or chymotrypsin-like enzymes. In conclusion, our results suggest thrombin, chymotrypsin-like proteases and a set of proteases with different specificities as potential therapeutic targets in IBD.

Age-Dependent Control of Collagen-Dependent Platelet Responses by Thrombospondin-1-Comparative Analysis of Platelets from Neonates, Children, Adolescents, and Adults.

Platelet function is developmentally regulated. Healthy neonates do not spontaneously bleed, but their platelets are hypo-reactive to several agonists. The mechanisms underlying immature platelet function in neonates are incompletely understood. This critical issue remains challenging for the establishment of age-specific reference ranges. In this study, we evaluated platelet reactivity of five pediatric age categories, ranging from healthy full-term neonates up to adolescents (11-18 years) in comparison to healthy adults (>18 years) by flow cytometry. We confirmed that platelet hypo-reactivity detected by fibrinogen binding, P-selectin, and CD63 surface expression was most pronounced in neonates compared to other pediatric age groups. However, maturation of platelet responsiveness varied with age, agonist, and activation marker. In contrast to TRAP and ADP, collagen-induced platelet activation was nearly absent in neonates. Granule secretion markedly remained impaired at least up to 10 years of age compared to adults. We show for the first time that neonatal platelets are deficient in thrombospondin-1, and exogenous platelet-derived thrombospondin-1 allows platelet responsiveness to collagen. Platelets from all pediatric age groups normally responded to the C-terminal thrombospondin-1 peptide RFYVVMWK. Thus, thrombospondin-1 deficiency of neonatal platelets might contribute to the relatively impaired response to collagen, and platelet-derived thrombospondin-1 may control distinct collagen-induced platelet responses.

Temporal Roles of Platelet and Coagulation Pathways in Collagen- and Tissue Factor-Induced Thrombus Formation.

In hemostasis and thrombosis, the complex process of thrombus formation involves different molecular pathways of platelet and coagulation activation. These pathways are considered as operating together at the same time, but this has not been investigated. The objective of our study was to elucidate the time-dependency of key pathways of thrombus and clot formation, initiated by collagen and tissue factor surfaces, where coagulation is triggered via the extrinsic route. Therefore, we adapted a microfluidics whole-blood assay with the Maastricht flow chamber to acutely block molecular pathways by pharmacological intervention at desired time points. Application of the technique revealed crucial roles of glycoprotein VI (GPVI)-induced platelet signaling via Syk kinase as well as factor VIIa-induced thrombin generation, which were confined to the first minutes of thrombus buildup. A novel anti-GPVI Fab EMF-1 was used for this purpose. In addition, platelet activation with the protease-activating receptors 1/4 (PAR1/4) and integrin αIIbß3 appeared to be prolongedly active and extended to later stages of thrombus and clot formation. This work thereby revealed a more persistent contribution of thrombin receptor-induced platelet activation than of collagen receptor-induced platelet activation to the thrombotic process.

Challenges and Opportunities in Protease-Activated Receptor Drug Development.

Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors (GPCRs) that transduce cellular responses to extracellular proteases. PARs have important functions in the vasculature, inflammation, and cancer and are important drug targets. A unique feature of PARs is their irreversible proteolytic mechanism of activation that results in the generation of a tethered ligand that cannot diffuse away. Despite the fact that GPCRs have proved to be the most successful class of druggable targets, the development of agents that target PARs specifically has been challenging. As a consequence, researchers have taken a remarkable diversity of approaches to develop pharmacological entities that modulate PAR function. Here, we present an overview of the diversity of therapeutic agents that have been developed against PARs. We further discuss PAR biased signaling and the influence of receptor compartmentalization, posttranslational modifications, and dimerization, which are important considerations for drug development.