The urokinase-type plasminogen activator receptor (uPAR) is currently gaining momentum as a promising molecular target for treatment of various solid cancers. For patient stratification, we developed a high-affinity uPAR-targeting peptide (AE105) detecting primary cancer lesions as well as occult metastasis by positron emission tomography (PET) imaging. uPAR-targeting by AE105 is also used for optical imaging in fluorescence-guided surgery of, for example, head-and-neck cancers. Recently, we showed that a monoclonal anti-uPAR antibody (FL1), in the form of an antibody-drug conjugate (FL1-ADC), efficiently eradicate pancreatic ductal carcinomas in surrogate mouse models leading to long-term remissions. In the current study, we solved high-resolution cryo-EM structures of FL1 in complex with two different conformational states of uPAR. Combined with comprehensive kinetic data from surface plasmon resonance studies, our cryo-EM structures provide essential insights into how FL1 binding impacts the interdomain flexibility of uPAR by restricting the movement of its N-terminal LU domain. This constraint from the bound FL1 drives uPAR into its open conformation, which leads to a pronounced reduction in the binding affinity for both its natural protease ligand (300-fold) and the PET imaging probe AE105 (25-fold). Collectively, these consequences of FL1-binding on uPAR conformation are considered beneficial for both targeted cancer treatment with FL1-ADCs and for the accompanying evaluation of treatment efficacy by longitudinal AE105-based PET imaging.

Hereditary angioedema (HAE) is associated with episodic kinin-induced swelling of the skin and mucosal membranes. Most patients with HAE have low plasma C1-inhibitor activity, leading to increased generation of the protease plasma kallikrein (PKa) and excessive release of the nanopeptide bradykinin from high-molecular-weight kininogen (HK). However, disease-causing mutations in at least 10% of patients with HAE appear to involve genes for proteins other than C1-inhibitor. A point mutation in the Kng1 gene encoding HK and low-molecular weight kininogen (LK) was identified recently in a family with HAE. The mutation changes a methionine (Met379) to lysine (Lys379) in both proteins. Met379 is adjacent to the Lys380-Arg381 cleavage site at the N-terminus of the bradykinin peptide. Recombinant wild-type (Met379) and variant (Lys379) versions of HK and LK were expressed in HEK293 cells. PKa-catalyzed kinin release from HK and LK was not affected by the Lys379 substitutions. However, kinin release from HK-Lys379 and LK-Lys379 catalyzed by the fibrinolytic protease plasmin was substantially greater than from wild-type HK-Met379 and LK-Met379. Increased kinin release was evident when fibrinolysis was induced in plasma containing HK-Lys379 or LK-Lys379 compared with plasma containing wild-type HK or LK. Mass spectrometry revealed that the kinin released from wild-type and variant kininogens by PKa is bradykinin. Plasmin also released bradykinin from wild-type kininogens but cleaved HK-Lys379 and LK-Lys379 after Lys379 rather than Lys380, releasing the decapeptide Lys-bradykinin (kallidin). The Met379Lys substitutions make HK and LK better plasmin substrates, reinforcing the relationship between fibrinolysis and kinin generation.

Two series of macrocyclic inhibitors addressing the S1 pocket and the prime site of the fibrinolytic serine protease plasmin have been developed. In the first series the P1 tranexamoyl residue was coupled to 4‐aminophenylalanine in P1’ position, which provided moderately potent inhibitors with inhibition constants around 1 µM. In the second series, a substituted biphenylalanine was incorporated as P1’ residue leading to approximately 1000‐fold stronger plasmin inhibitors, the best compounds possess subnanomolar inhibition constants. The most effective compounds already exhibit a certain selectivity as plasmin inhibitors compared to other trypsin‐like serine proteases such as trypsin, plasma kallikrein, thrombin, activated protein Ca, as well as factors XIa and Xa. For inhibitor 28 of the second series, the co‐crystal structure in complex with a Ser195Ala microplasmin mutant revealed the P2’ residue adopts multiple conformations. Most polar contacts to plasmin and surrounding water molecules are mediated through the P1 tranexamoyl residue, whereas the bound conformation of the macrocycle is mainly stabilized by two intramolecular hydrogen bonds.

Pathogens often manipulate the host plasminogen activation system to facilitate escape from the initial site of infection, promote systemic invasion, and evade immune protection. Plasminogen, which is known for its involvement in fibrinolysis, immune modulation, and wound healing, has recently been linked to Clostridioides difficile spore germination. By identifying the mechanisms that drive spore germination and their contribution to C. difficile pathogenesis, in animal model studies, we gain insights into their role in disease severity and mortality. Moreover, inhibiting plasminogen activation using a specific single-chain variable fragment (scFv) has shown the potential to reduce infection severity and mortality. These findings suggest that targeting plasminogen-mediated pathways can be a promising therapeutic approach for managing C. difficile colitis.

The Front Cover shows new macrocyclic plasmin inhibitors containing a C-terminal P1 benzylamine group. Their N-terminal substitution provided analogues with sub-nanomolar Ki values. Additional inhibitors containing an asymmetric linker possess Ki values close to 2 nM. For the first time, crystal structures of these macrocyclic inhibitors in complex with a Ser195Ala microplasmin mutant were determined, which explain their excellent potency and selectivity. More information can be found in the Research Article by Simon J. A. Wiedemeyer, Guojie Wu, Ruby H. P. Law, Torsten Steinmetzer et al.

Hereditary angioedema (HAE) is an inherited disorder characterized by recurrent episodes of severe soft tissue swelling involving skin and mucous membranes. In most cases, edema is caused by excessive formation of the vasoactive nanopeptide bradykinin (BK) due to dysregulation of the plasma kallikrein-kinin system (KKS). The KKS consists of the zymogens prekallikrein (PK) and factor XII (FXII) and the cofactor/substrate high-molecular-weight kininogen (HK). PK and FXII reciprocally convert each other to the proteases plasma kallikrein (PKa) and FXIIa. PKa cleaves HK after Lys362 and Arg371 to release bradykinin. In most cases of HAE, increased KKS activation is due to low plasma activity of C1-Inhibitor (C1-INH), the main regulator of PKa and FXIIa. However, ~10% of HAE patients have normal C1-INH activity (HAEnC1). A relationship between fibrinolysis and kinin formation was first described in 1971. The fibrinolytic protease plasmin activates FXII and cleaves HK, although it is unclear that this results in significant bradykinin generation. Recently mutations in the PLG gene encoding plasminogen (Lys311Glu) and the KNG1 gene encoding HK and the related protein low-molecular-weight kininogen (LK, Met361Lys) were identified in patients with HAEnC1. Here we describe how the fibrinolytic system contributes to angioedema in patients with these mutations. Human Glu-plasminogen (Plg), HK and LK were expressed in HEK293 cells. Lys311 in plasminogen was changed to glutamic acid, and Met361 in HK and LK were changed to lysine, by site directed mutagenesis. Wild type (Plg-Lys311) and variant (Plg-Glu311) plasminogen were converted to plasmin (Plm-Lys311 and Plm-Glu311) with urokinase. The capacity of plasmin to cleave HK and LK were studied with SDS-PAGE, western blots and kinin ELISA. Cleavage of wild type HK-Met361 and LK-Met361 and variant HK-Lys361 and LK-Lys361 by PKa and plasmin were assessed in a similar manner. The types of kinin released during reactions were determined by mass spectroscopy. Normal plasma was supplemented with Plg-Lys311 or Plg-Glu311, and tPA was added to generate plasmin. Bradykinin release was substantially greater with Plm-Glu311 than Plm-Lys311. This held when reactions were run in plasma lacking FXII or PK, indicating activation of the KKS was not responsible for kinin production. Using plasma-derived proteins, PKa released bradykinin 100-fold faster from HK than LK, and released bradykinin from HK ~50-fold faster than did plasmin. PKa and plasmin released bradykinin at comparable relatively slow rates from LK. Plm-Glu311 released bradykinin from HK 10-fold faster, and from LK 3-fold faster, than in reactions with Plm-Lys311. In experiments in which kininogen deficient plasma was supplemented with either HK or LK to a physiologic concentration, the majority of bradykinin released by Plm-Glu311 was from LK. PKa-catalyzed release of bradykinin from wild type HK-Met361 and variant HK-Lys361 comparably. The same held for LK-Met361 and LK-Lys361. However, plasmin released kinin from HK-Lys361 and LK-Lys361 3.3-fold and 13-fold faster than from HK-Met361 and LK-Met361. While the kinin released from HK-Met361 and LK-Met361 was bradykinin, plasmin released the decapeptide Lys-bradykinin (kallidin) from HK-Lys361 and LK-Lys36. Our studies indicate that angioedema in some patients with HAEnC1 may be due to fibrinolytic activity, and not to KKS activity. Both HK and LK may be substrates in such patients. As the plasma LK concentration is ~4-fold higher than HK, it may be the main pathologic kinin source. The plasmin Lys311Glu substitution converts the protease into a more efficient kininogenase. Fibrinolytic activity is normally brisk in the mouth, perhaps explaining why patients with the Lys311Glu mutation primarily suffer from oral-lingual angioedema. The findings raise the possibility that oral-lingual edema after tPA infusion or ACE inhibitor administration may also be due to plasmin-mediated cleavage of LK and HK. The Met361Lys substitution in HK and LK introduces a novel protease cleavage site immediately upstream from a normal PKa cleavage site. The novel site is preferred over the normal site by plasmin, but not PKa. Several treatments for HAE directed at neutralizing PKa or FXIIa activity have been developed. Our results suggest that such drugs may be less effective in patients with HAEnC1 in which disease mechanisms operate independently of the KKS.

"Reagentless" immunosensors are emerging to address the challenge of practical and sensitive detection of important biomarkers in real biological samples without the need for multistep assays and user intervention, with applications ranging from research tools to point-of-care diagnostics. Selective target binding to an affinity reagent is detected and reported in one step without the need for washing or additional reporters. In this study, we used a structure-guided approach to identify a mutation site in an antibody fragment for the polarity-dependent fluorophore, Anap, such that upon binding of the protein target cardiac troponin I, the Anap-labeled antibody would produce a detectable and dose-dependent shift in emission wavelength. We observed a significant emission wavelength shift of the Anap-labeled anti-cTnI mutant, with a blue shift of up to 37 nm, upon binding to the cTnI protein. Key differences in the resulting emission spectra between target peptides in comparison to whole proteins were also found; however, the affinity and binding characteristics remained unaffected when compared to the wild-type antibody. We also highlighted the potential flexibility of the approach by incorporating a near-infrared dye, IRDye800CW, into the same mutation site, which also resulted in a dose-dependent wavelength shift upon target incubation. These reagents can be used in experiments and devices to create simpler and more efficient biosensors across a range of research, medical laboratory, and point-of-care platforms.

Plasminogen (Plg) is the inactive form of plasmin (Plm) that exists in two major glycoforms, referred to as glycoforms I and II (GI and GII). In the circulation, Plg assumes an activation-resistant 'closed' conformation via interdomain interactions and is mediated by the lysine binding site (LBS) on the kringle (KR) domains. These inter-domain interactions can be readily disrupted when Plg binds to lysine/arginine residues on protein targets or free L-lysine and analogues. This causes Plg to convert into an 'open' form which is crucial for activation by host activators. In this study, we investigated how various ligands affect the kinetics of Plg conformational change using small-angle X-ray scattering (SAXS). We began by examining the open and closed conformations of Plg using size-exclusion chromatography (SEC) coupled with SAXS. Next, we developed a high throughput (HTP) 96-well SAXS assay setup. This method enables us to determine the Kopen value, which is used to compare ligands’ effect on Plg conformation directly. Based on our analysis using Plg GII, we have found that the Kopen for ε-aminocaproic acid (EACA) is approximately three times greater than that of Tranexamic acid (TXA), which is widely recognized as a highly effective ligand. We demonstrated further that Plg undergoes a conformational change when it binds to the C-terminal peptides of the inhibitor α2-antiplasmin and receptor Plg‑RKT. Our findings suggest that, besides the C-terminal lysine, internal lysine(s) are also necessary for the formation of open Plg. Finally, we compared the conformational changes of Plg GI and GII directly and found that the closed form of GI, which has an N-linked glycosylation, is less stable. To summarize, we have successfully determined the response of Plg to various ligand/receptor peptides by directly measuring the kinetics of its conformational changes.

Two series of macrocyclic plasmin inhibitors with a C-terminal benzylamine group were synthesized. The substitution of the N-terminal phenylsulfonyl group of a previously described inhibitor provided two analogues with sub-nanomolar inhibition constants. Both compounds possess a high selectivity against all other tested trypsin-like serine proteases. Furthermore, a new approach was used to selectively introduce asymmetric linker segments. Two of these compounds inhibit plasmin with K-i values close to 2 nM. For the first time, four crystal structures of these macrocyclic inhibitors could be determined in complex with a Ser195Ala microplasmin mutant. The macrocyclic core segment of the inhibitors binds to the open active site of plasmin without any steric hindrance. This binding mode is incompatible with other trypsin-like serine proteases containing a sterically demanding 99-hairpin loop. The crystal structures obtained experimentally explain the excellent selectivity of this inhibitor type as previously hypothesized.

Plasminogen (Plg), the zymogen of plasmin (Plm), is a glycoprotein involved in fibrinolysis and a wide variety of other physiological processes. Plg dysregulation has been implicated in a range of diseases. Classically, human Plg is categorized into two types, supposedly having different functional features, based on the presence (type I) or absence (type II) of a single N-linked glycan. Using high- resolution native mass spectrometry, we uncovered that the proteoform profiles of human Plg (and Plm) are substantially more extensive than this simple binary classification. In samples derived from human plasma, we identified up to 14 distinct proteoforms of Plg, including a novel highly stoichiometric phosphorylation site at Ser339. To elucidate the potential functional effects of these post- translational modifications, we performed proteoformresolved kinetic analyses of the Plg-to-Plm conversion using several canonical activators. This conversion is thought to involve at least two independent cleavage events: one to remove the N-terminal peptide and another to release the active catalytic site. Our analyses reveal that these processes are not independent but are instead tightly regulated and occur in a step-wise manner. Notably, N-terminal cleavage at the canonical site (Lys77) does not occur directly from intact Plg. Instead, an activation intermediate corresponding to cleavage at Arg68 is initially produced, which only then is further processed to the canonical Lys77 product. Based on our results, we propose a refined categorization for human Plg proteoforms. In addition, we reveal that the proteoform profile of human Plg is more extensive than that of rat Plg, which lacks, for instance, the here-described phosphorylation at Ser339.

Perforin is a pore-forming protein whose normal function enables cytotoxic T and natural killer (NK) cells to kill virus-infected and transformed cells. Conversely, unwanted perforin activity can also result in auto-immune attack, graft rejection and aberrant responses to pathogens. Perforin is critical for the function of the granule exocytosis cell death pathway and is therefore a target for drug development. In this study, by screening a fragment library using NMR and surface plasmon resonance, we identified 4,4-diaminodiphenyl sulfone (dapsone) as a perforin ligand. We also found that dapsone has modest (mM) inhibitory activity of perforin lytic activity in a red blood cell lysis assay in vitro. Sequential modification of this lead fragment, guided by structural knowledge of the ligand binding site and binding pose, and supported by SPR and ligand-detected 19F NMR, enabled the design of nanomolar inhibitors of the cytolytic activity of intact NK cells against various tumour cell targets. Interestingly, the ligands we developed were largely inert with respect to direct perforin-mediated red blood cell lysis but were very potent in the context of perforin's action on delivering granzymes in the immune synapse, the context in which it functions physiologically. Our work indicates that a fragment-based, structure-guided drug discovery strategy can be used to identify novel ligands that bind perforin. Moreover, these molecules have superior physicochemical properties and solubility compared to previous generations of perforin ligands.

The protein NINJ1 drives membrane rupture associated with certain types of cell death. Investigation of NINJ1 reveals mechanistic details of how it functions, raising the possibility of developing new therapeutics.

Patients with hereditary angioedema (HAE) experience episodes of bradykinin (BK)-induced swelling of skin and mucosal membranes. The most common cause is reduced plasma activity of C1 inhibitor, the main regulator of the proteases plasma kallikrein (PKa) and factor XIIa (FXIIa). Recently, patients with HAE were described with a Lys311 to glutamic acid substitution in plasminogen (Plg), the zymogen of the protease plasmin (Plm). Adding tissue plasminogen activator to plasma containing Plg-Glu311 vs plasma containing wild-type Plg (Plg-Lys311) results in greater BK generation. Similar results were obtained in plasma lacking prekallikrein or FXII (the zymogens of PKa and FXIIa) and in normal plasma treated with a PKa inhibitor, indicating Plg-Glu311 induces BK generation independently of PKa and FXIIa. Plm-Glu311 cleaves high and low molecular weight kininogens (HK and LK, respectively), releasing BK more efficiently than Plm-Lys311. Based on the plasma concentrations of HK and LK, the latter may be the source of most of the BK generated by Plm-Glu311. The lysine analog ε-aminocaproic acid blocks Plm-catalyzed BK generation. The Glu311 substitution introduces a lysine-binding site into the Plg kringle 3 domain, perhaps altering binding to kininogens. Plg residue 311 is glutamic acid in most mammals. Glu311 in patients with HAE, therefore, represents reversion to the ancestral condition. Substantial BK generation occurs during Plm-Glu311 cleavage of human HK, but not mouse HK. Furthermore, mouse Plm, which has Glu311, did not liberate BK from human kininogens more rapidly than human Plg-Lys311. This indicates Glu311 is pathogenic in the context of human Plm when human kininogens are the substrates.

Class A tick evasins are natural chemokine-binding proteins that block the signaling of multiple chemokines from the CC subfamily through their cognate receptors, thus suppressing leukocyte recruitment and inflammation. Development of tick evasins as chemokine-targeted anti-inflammatory therapeutics requires an understanding of the factors controlling their chemokine recognition and selectivity. To investigate the role of the evasin N-terminal region for chemokine recognition, we prepared chimeric evasins by interchanging the N-terminal regions of four class A evasins, including a newly identified evasin, EVA-RPU02. We show through chemokine binding analysis of the parental and chimeric evasins that the N-terminal region is critical for chemokine binding affinity and selectivity. Notably, we found some chimeras were unable to bind certain cognate chemokine ligands of both parental evasins. Moreover, unlike any natural evasins characterized to date, some chimeras exhibited specific binding to a single chemokine. These results indicate that the evasin N terminus interacts cooperatively with the "body" of the evasin to enable optimum chemokine recognition. Furthermore, the altered chemokine selectivity of the chimeras validates the approach of engineering the N termini of evasins to yield unique chemokine recognition profiles.

Nine days after medial septal lesion a 20% increase in the number of muscarinic antagonist binding sites in rat hippocampus was observed without any change in the affinity for agonist or antagonist. Chronic atropine treatment (s.c. 5 mg/kg, twice a day for 14 days, 20 mg/kg once a day for 14 days or 100 mg/kg for 4 days and 20 mg/kg for 10 days, once daily) led to an increase in the number of muscarinic antagonist binding sites in rat hippocampus with 35, 80 and 80% respectively and also lowered the affinity for3H-antagonists in a dose dependent manner. Agonist binding studies also indicated an increase in receptor number and a decrease in affinity. The latter change can possibly be explained by the presence of residual atropine 24 h after the last injection. If this is taken into account we may conclude that muscarinic supersensitivity evoked either by severing the input or by chronic pharmacologic blockade both produced 'new receptors' with ligand binding properties similar to the original receptors.

Background: CUB domain-containing protein 1 (CDCP1) is a cell surface receptor regulating key signalling pathways in malignant cells. CDCP1 has been proposed as a molecular target to abrogate oncogenic signalling pathways and specifically deliver anti-cancer agents to tumors. However, the development of CDCP1-targeting agents has been questioned by its frequent proteolytic processing which was thought to result in shedding of the CDCP1 extracellular domain limiting its targetability. In this study, we investigated the relevance of targeting CDCP1 in the context of pancreatic ductal adenocarcinoma (PDAC) and assess the impact of CDCP1 proteolysis on the effectiveness of CDCP1 targeting agents. Methods: The involvement of CDCP1 in PDAC progression was assessed by association analysis in several PDAC cohorts and the proteolytic processing of CDCP1 was evaluated in PDAC cell lines and patient-derived cells. The consequences of CDCP1 proteolysis on its targetability in PDAC cells was assessed using immunoprecipitation, immunostaining and biochemical assays. The involvement of CDCP1 in PDAC progression was examined by loss-of-function in vitro and in vivo experiments employing PDAC cells expressing intact or cleaved CDCP1. Finally, we generated antibody-based imaging and therapeutic agents targeting CDCP1 to demonstrate the feasibility of targeting this receptor for detection and treatment of PDAC tumors. Results: High CDCP1 expression in PDAC is significantly associated with poorer patient survival. In PDAC cells proteolysis of CDCP1 does not always result in the shedding of CDCP1-extracellular domain which can interact with membrane-bound CDCP1 allowing signal transduction between the different CDCP1-fragments. Targeting CDCP1 impairs PDAC cell functions and PDAC tumor growth independently of CDCP1 cleavage status. A CDCP1-targeting antibody is highly effective at delivering imaging radionuclides and cytotoxins to PDAC cells allowing specific detection of tumors by PET/CT imaging and superior anti-tumor effects compared to gemcitabine in in vivo models. Conclusion: Independent of its cleavage status, CDCP1 exerts oncogenic functions in PDAC and has significant potential to be targeted for improved radiological staging and treatment of this cancer. Its elevated expression by most PDAC tumors and lack of expression by normal pancreas and other major organs, suggest that targeting CDCP1 could benefit a significant proportion of PDAC patients. These data support the further development of CDCP1-targeting agents as personalizable tools for effective imaging and treatment of PDAC.

CUB-domain containing protein 1 (CDCP1) is a cancer associated cell surface protein that amplifies pro-tumorigenic signalling by other receptors including EGFR and HER2. Its potential as a cancer target is supported by studies showing that anti-CDCP1 antibodies inhibit cell migration and survival in vitro, and tumor growth and metastasis in vivo. Here we characterize two anti-CDCP1 antibodies, focusing on immuno-conjugates of one of these as a tool to detect and inhibit ovarian cancer. Methods: A panel of ovarian cancer cell lines was examined for cell surface expression of CDCP1 and loss of expression induced by anti-CDCP1 antibodies 10D7 and 41-2 using flow cytometry and Western blot analysis. Surface plasmon resonance analysis and examination of truncation mutants was used to analyse the binding properties of the antibodies for CDCP1. Live-cell spinning-disk confocal microscopy of GFP-tagged CDCP1 was used to track internalization and intracellular trafficking of CDCP1/antibody complexes. In vivo, zirconium 89-labelled 10D7 was detected by positron-emission tomography imaging, of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. The efficacy of cytotoxin-conjugated 10D7 was examined against ovarian cancer cells in vitro and in vivo. Results: Our data indicate that each antibody binds with high affinity to the extracellular domain of CDCP1 causing rapid internalization of the receptor/antibody complex and degradation of CDCP1 via processes mediated by the kinase Src. Highlighting the potential clinical utility of CDCP1, positron-emission tomography imaging, using zirconium 89-labelled 10D7, was able to detect subcutaneous and intraperitoneal xenograft ovarian cancers in mice, including small (diameter <3 mm) tumor deposits of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. Furthermore, cytotoxin-conjugated 10D7 was effective at inhibiting growth of CDCP1-expressing ovarian cancer cells in vitro and in vivo. Conclusions: These data demonstrate that CDCP1 internalizing antibodies have potential for killing and detection of CDCP1 expressing ovarian cancer cells.

Group A Streptococcus pyogenes (GAS) is a human pathogen that commonly causes superficial infections such as pharyngitis, but can also lead to systemic and fatal diseases. GAS infection remains to be a major threat in regions with insufficient medical infrastructures, leading to half a million deaths annually worldwide. The pathogenesis of GAS is mediated by a number of virulence factors, which function to facilitate bacterial colonization, immune evasion, and deep tissue invasion. In this review, we will discuss the mechanism of molecular interaction between the host protein and virulence factors that target the fibrinolytic system, including streptokinase (SK), plasminogen-binding group A streptococcal M-like protein (PAM), and streptococcal inhibitor of complement (SIC). We will discuss our current understanding, through structural studies, on how these proteins manipulate the fibrinolytic system during infection.