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Matrix metalloproteinases (MMPs) are key drivers of various diseases, including cancer. While several antibodies against MMPs are in development, our goal is to construct therapeutic anti-MMP inhibitors based on a natural broad MMP inhibitor, tissue inhibitor of metalloproteinases-2 (N-TIMP2). To confer high binding specificity toward one MMP type, we extend one of the N-TIMP2 loops, allowing it to interact with the non-conserved MMP surface. Multiple computational designs of the loop were used to design a focused library for yeast surface display, which was sorted for high binding to the target MMP-14 and low binding to off-target MMP-3. Deep sequencing of the two selected populations followed by comparative data analysis was used to identify the most promising variants, which were expressed, purified, and tested for inhibition of MMP-14 and off-target MMPs. Our best N-TIMP2 variant exhibited 29 pM binding affinity to MMP-14 and 2.4 µM affinity to MMP-3, 7500-fold more specific than WT N-TIMP2. Furthermore, the variant inhibited cell invasion with increased potency relative to WT N-TIMP2 in two breast cancer cell lines. We obtained the engineered variant high-accuracy model by including NGS data as input to AlphaFold multiple sequence alignment (MSA). Modeling results together with experimental mutagenesis demonstrate that the loop packs tightly against non-conserved residues on MMP-14 and clashes with MMP-3. This study demonstrates that introduction of loop extensions into inhibitors to stretch to the non-conserved surface of the target proteins is an attractive strategy for conferring high binding specificity in design of MMP inhibitors and other therapeutic proteins.

A major clinical challenge of ovarian cancer is the development of malignant ascites accompanied by widespread peritoneal metastasis. In ovarian clear cell carcinoma (OCCC), a challenging subtype of ovarian cancer, this problem is compounded by near-universal primary chemoresistance; patients with advanced stage OCCC thus lack effective therapies and face extremely poor survival rates. Here we show that tumor-cell-expressed serine protease inhibitor Kazal type 1 (SPINK1) is a key driver of OCCC progression and metastasis. Using cell culture models of human OCCC, we find that shRNA silencing of SPINK1 sensitizes tumor cells to anoikis and inhibits proliferation. Knockdown of SPINK1 in OCCC cells also profoundly suppresses peritoneal metastasis in mouse implantation models of human OCCC. We next identify a novel autocrine signaling axis in OCCC cells whereby tumor-cell-produced interleukin-6 (IL-6) regulates SPINK1 expression to stimulate a common protumorigenic gene expression pattern leading to anoikis resistance and proliferation of OCCC cells. We further demonstrate that this signaling pathway can be successfully interrupted with the IL-6Rα inhibitor tocilizumab, sensitizing cells to anoikis in vitro and reducing metastasis in vivo. These results suggest that clinical trials of IL-6 pathway inhibitors in OCCC may be warranted, and that SPINK1 might offer a candidate predictive biomarker in this population.

Ovarian clear cell carcinoma (OCCC) is an understudied poor prognosis subtype of ovarian cancer lacking in effective targeted therapies. Efforts to define molecular drivers of OCCC malignancy may lead to new therapeutic targets and approaches. Among potential targets are secreted proteases, enzymes which in many cancers serve as key drivers of malignant progression. Here, we found that inhibitors of trypsin-like serine proteases suppressed malignant phenotypes of OCCC cell lines. To identify the proteases responsible for malignancy in OCCC, we employed activity-based protein profiling to directly analyze enzyme activity. We developed an activity-based probe featuring an arginine diphenylphosphonate warhead to detect active serine proteases of trypsin-like specificity and a biotin handle to facilitate affinity purification of labeled proteases. Using this probe, we identified active trypsin-like serine proteases within the complex proteomes secreted by OCCC cell lines, including two proteases in common, tissue plasminogen activator and urokinase-type plasminogen activator. Further interrogation of these proteases showed that both were involved in cancer cell invasion and proliferation of OCCC cells and were also detected in in vivo models of OCCC. We conclude the detection of tissue plasminogen activator and urokinase-type plasminogen activator as catalytically active proteases and significant drivers of the malignant phenotype may point to these enzymes as targets for new therapeutic strategies in OCCC. Our activity-based probe and profiling methodology will also serve as a valuable tool for detection of active trypsin-like serine proteases in models of other cancers and other diseases.

Serine protease inhibitors of the Kunitz-bovine pancreatic trypsin inhibitor (BPTI) family are ubiquitous biological regulators of proteolysis. These small proteins are resistant to proteolysis, but can be slowly cleaved within the protease-binding loop by target proteases, thereby compromising their activity. For the human protease mesotrypsin, this cleavage is especially rapid. Here, we aimed to stabilize the Kunitz domain structure against proteolysis through disulfide engineering. Substitution within the Kunitz inhibitor domain of the amyloid precursor protein (APPI) that incorporated a new disulfide bond between residues 17 and 34 reduced proteolysis by mesotrypsin 74-fold. Similar disulfide engineering of tissue factor pathway inhibitor-1 Kunitz domain 1 ((KD1)TFPI1) and bikunin Kunitz domain 2 ((KD2)bikunin) likewise stabilized these inhibitors against mesotrypsin proteolysis 17- and 6.6-fold, respectively. Crystal structures of disulfide-engineered APPI and (KD1)TFPI1 variants in a complex with mesotrypsin at 1.5 and 2.0 Å resolution, respectively, confirmed the formation of well-ordered disulfide bonds positioned to stabilize the binding loop. Long all-atom molecular dynamics simulations of disulfide-engineered Kunitz domains and their complexes with mesotrypsin revealed conformational stabilization of the primed side of the inhibitor-binding loop by the engineered disulfide, along with global suppression of conformational dynamics in the Kunitz domain. Our findings suggest that the Cys-17–Cys-34 disulfide slows proteolysis by dampening conformational fluctuations in the binding loop and minimizing motion at the enzyme–inhibitor interface. The generalizable approach developed here for the stabilization against proteolysis of Kunitz domains, which can serve as important scaffolds for therapeutics, may thus find applications in drug development.

Large cell carcinoma (LCC) is a rare and aggressive lung cancer subtype with poor prognosis and no targeted therapies. Tumor-associated fibroblasts (TAFs) derived from LCC tumors exhibit premature senescence, and coculture of pulmonary fibroblasts with LCC cell lines selectively induces fibroblast senescence, which in turn drives LCC cell growth and invasion. Here we identify MMP1 as overexpressed specifically in LCC cell lines, and we show that expression of MMP1 by LCC cells is necessary for induction of fibroblast senescence and consequent tumor promotion in both cell culture and mouse models. We also show that MMP1, in combination with TGF-β1, is sufficient to induce fibroblast senescence and consequent LCC promotion. Furthermore, we implicate PAR-1 and oxidative stress in MMP1/TGF-β1-induced TAF senescence. Our results establish an entirely new role for MMP1 in cancer, and support a novel therapeutic strategy in LCC based on targeting senescent TAFs., This work was further supported by grants from the Agencia Estatal de Investigaci´on (AEI/FEDER) (PI13/02368, SAF2016-79527-R and PID2019-110944RB-I00 (AEI/ 10.13039/501100011033) to JA, PI16/ 00890 to NR), Fundaci´o Privada Cellex (to JA), National Institutes of Health (grant R01 GM132100 to ER), Generalitat de Catalunya (AGAUR SGR661 and CERCA Programme to JA), Junta Provincial de Barcelona de l’Associaci´o Espanyola Contra el C`ancer (AECC-B16-917 to JA), Italian Ministry of Health (RF-2016-02362946 to LR), Italian Association for Cancer Research (AIRC-IG21431 to LR), Sociedad Espa˜nola de Neumología y Cirugía Tor´acica – SEPAR (SEPAR 437 to NR), COST Action (PROTEOSTASIS BM1307), and by fellowships from Ciˆencia sem Fronteiras CNPq (to RI), and Universitat de Barcelona/beca APIF (to PD).

Large cell carcinoma (LCC) is a rare and aggressive lung cancer subtype with poor prognosis and no targeted therapies. Tumor-associated fibroblasts (TAFs) derived from LCC tumors exhibit premature senescence, and coculture of pulmonary fibroblasts with LCC cell lines selectively induces fibroblast senescence, which in turn drives LCC cell growth and invasion. Here we identify MMP1 as overexpressed specifically in LCC cell lines, and we show that expression of MMP1 by LCC cells is necessary for induction of fibroblast senescence and consequent tumor promotion in both cell culture and mouse models. We also show that MMP1, in combination with TGF-ß1, is sufficient to induce fibroblast senescence and consequent LCC promotion. Furthermore, we implicate PAR-1 and oxidative stress in MMP1/TGF-ß1-induced TAF senescence. Our results establish an entirely new role for MMP1 in cancer, and support a novel therapeutic strategy in LCC based on targeting senescent TAFs.

Matrix metalloproteinases (MMPs) have long been known as key drivers in the development and progression of diseases, including cancer and neurodegenerative, cardiovascular, and many other inflammatory and degenerative diseases, making them attractive potential drug targets. Engineering selective inhibitors based upon tissue inhibitors of metalloproteinases (TIMPs), endogenous human proteins that tightly yet nonspecifically bind to the family of MMPs, represents a promising new avenue for therapeutic development. Here, we used a counter-selective screening strategy for directed evolution of yeast-displayed human TIMP-1 to obtain TIMP-1 variants highly selective for the inhibition of MMP-3 in preference over MMP-10. As MMP-3 and MMP-10 are the most similar MMPs in sequence, structure, and function, our results thus clearly demonstrate the capability for engineering full-length TIMP proteins to be highly selective MMP inhibitors. We show using protein crystal structures and models of MMP-3-selective TIMP-1 variants bound to MMP-3 and counter-target MMP-10 how structural alterations within the N-terminal and C-terminal TIMP-1 domains create new favorable and selective interactions with MMP-3 and disrupt unique interactions with MMP-10. While our MMP-3-selective inhibitors may be of interest for future investigation in diseases where this enzyme drives pathology, our platform and screening strategy can be employed for developing selective inhibitors of additional MMPs implicated as therapeutic targets in disease.

Human tissue kallikrein (KLK) proteases are hormone-like signaling molecules with important functions in cancer pathophysiology. KLK-related peptidase 6 (KLK6), specifically, is highly up-regulated in several types of cancer, where its increased activity promotes cancer invasion and metastasis. This characteristic suggests KLK6 as an attractive target for therapeutic interventions. However, inhibitors that specifically target KLK6 have not yet been reported, possibly because KLK6 shares a high sequence homology and structural similarity with other serine proteases and resists inhibition by many polypeptide inhibitors. Here, we present an innovative combinatorial approach to engineering KLK6 inhibitors via flow cytometry–based screening of a yeast-displayed mutant library of the human amyloid precursor protein Kunitz protease inhibitor domain (APPI), an inhibitor of other serine proteases, such as anionic and cationic trypsins. On the basis of this screening, we generated APPI(M17L,I18F,S19F,F34V) (APPI-4M), an APPI variant with a KLK6 inhibition constant (K(i)) of 160 pm and a turnover time of 10 days. To the best of our knowledge, APPI-4M is the most potent KLK6 inhibitor reported to date, displaying 146-fold improved affinity and 13-fold improved proteolytic stability compared with WT APPI (APPI(WT)). We further demonstrate that APPI-4M acts as a functional inhibitor in a cell-based model of KLK6-dependent breast cancer invasion. Finally, the crystal structures of the APPI(WT)/KLK6 and APPI-4M/KLK6 complexes revealed the structural and mechanistic bases for the improved KLK6 binding and proteolytic resistance of APPI-4M. We anticipate that APPI-4M will have substantial translational potential as both imaging agent and therapeutic.

High structural and sequence similarity within protein families can pose significant challenges to the development of selective inhibitors, especially toward proteolytic enzymes. Such enzymes usually belong to large families of closely similar proteases and may also hydrolyze, with different rates, protein- or peptide-based inhibitors. To address this challenge, we employed a combinatorial yeast surface display library approach complemented with a novel pre-equilibrium, competitive screening strategy for facile assessment of the effects of multiple mutations on inhibitor association rates and binding specificity. As a proof of principle for this combined approach, we utilized this strategy to alter inhibitor/protease association rates and to tailor the selectivity of the amyloid β-protein precursor Kunitz protease inhibitor domain (APPI) for inhibition of the oncogenic protease mesotrypsin, in the presence of three competing serine proteases, anionic trypsin, cationic trypsin and kallikrein-6. We generated a variant, designated APPIP13W/M17G/I18F/F34V, with up to 30-fold greater specificity relative to the parental APPIM17G/I18F/F34V protein, and 6500- to 230 000-fold improved specificity relative to the wild-type APPI protein in the presence of the other proteases tested. A series of molecular docking simulations suggested a mechanism of interaction that supported the biochemical results. These simulations predicted that the selectivity and specificity are affected by the interaction of the mutated APPI residues with nonconserved enzyme residues located in or near the binding site. Our strategy will facilitate a better understanding of the binding landscape of multispecific proteins and will pave the way for design of new drugs and diagnostic tools targeting proteases and other proteins.

Large cell carcinoma (LCC) is an aggressive lung cancer subtype with poor prognosis and no targeted therapies. We previously reported that tumor-associated fibroblasts (TAFs) derived from LCC tumors exhibit premature senescence, and coculture of pulmonary fibroblasts with LCC cell lines selectively induces fibroblast senescence, which in turn drives LCC cell growth and invasion. Here we report that MMP1 is overexpressed specifically in LCC cell lines. Notably, silencing MMP1 expression in LCC cancer cell lines using shRNA revealed that MMP1 expression by LCC cells is necessary for induction of fibroblast senescence in coculture experiments with normal pulmonary fibroblasts, as revealed by the analysis of a panel of standard senescence markers, including β-galactosidase (SA-βgal) staining, permanent growth arrest and expression of senescence-associated secretory factors. Injecting control (shScr) or shMMP1 LCC cells into immunodeficient nude mice revealed that tumor growth, tumor take and cancer cell dissemination to the lung were reduced in shMMP1 H460 tumors compared to control tumors. We also observed fewer senescent fibroblasts in tumors from shMMP1 H460 cells using Sentragor staining, which allows identification of senescent cells in paraffin embedded tissues. Moreover, we found that recombinant active MMP1 in combination with TGF-β1 were sufficient to induce normal fibroblast senescence. In terms of the potential underlying mechanisms, treatment with the antioxidant n-acetyl cysteine (NAC) significantly attenuated the increase in SA-βgal+ fibroblasts elicited by co-stimulation with rMMP1 and TGF-β1, and its corresponding conditioned medium elicited a significantly lower growth and invasion in LCC cancer cells, revealing the oxidative stress implication in fibroblast senescence induction and associated pro-tumorigenic secretome. In summary, our results establish a new role for MMP1 in cancer and support that LCC cells elicit a tumor-supporting niche through the aberrant secretion of MMP1 and TGF-β1 to induce senescence in adjacent fibroblasts. Furthermore, we implicate oxidative stress in MMP1/TGF-β1-induced TAF senescence and support a novel therapeutic strategy in LCC based on targeting senescent TAFs. Citation Format: Marta Gabasa, Evette S. Radisky, Rafael Ikemori, Giulia Bertolini, Marselina Arshakyan, Alexandra Hockla, Paula Duch, Ornella Rondinone, Alejandro Llorente, Maria Maqueda, Alexandre Perera, Noemí Reguart, Luca Roz, Derek C. Radisky, Jordi Alcaraz. MMP1 and TGF-β1 cooperate to drive tumor progression in large cell carcinoma of the lung through fibroblast senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2515.

Serine proteases have been implicated as key drivers and facilitators of lung cancer malignancy, and while these proteins represent straightforward targets for therapeutic inhibitors, identification of optimal points for intervention has been complicated by the complex networks in which these enzymes function. Here we implicate a signaling pathway consisting of PRSS3/mesotrypsin and kallikrein-related peptidase 5 (KLK5) in lung adenocarcinoma malignancy. We show that elevated PRSS3/mesotrypsin expression is prognostic for poor outcome for patients with lung adenocarcinoma, and that genetic or pharmacologic targeting of PRSS3/mesotrypsin reduces lung adenocarcinoma cell invasiveness and proliferation. We further show that genetic targeting of KLK5, a known target of PRSS3/mesotrypsin, phenocopies the effect of PRSS3/mesotrypsin knockdown, and also that elevated expression of KLK5 is similarly prognostic for outcome in lung adenocarcinoma. Finally, we use transcriptional profiling experiments to show that PRSS3/mesotrypsin and KLK5 control a common malignancy-promoting pathway. These experiments implicate a potential PRSS3/mesotrypsin-KLK5 signaling module in lung adenocarcinoma and reveal the potential therapeutic benefit of selectively targeting these pathways.

Characterizing the binding selectivity landscape of interacting proteins is crucial both for elucidating the underlying mechanisms of their interaction and for developing selective inhibitors. However, current mapping methods are laborious and cannot provide a sufficiently comprehensive description of the landscape. Here, we introduce a novel and efficient strategy for comprehensively mapping the binding landscape of proteins using a combination of experimental multi-target selective library screening and in silico next-generation sequencing analysis. We map the binding landscape of a non-selective trypsin inhibitor, the amyloid protein precursor inhibitor (APPI), to each of the four human serine proteases (kallikrein-6, mesotrypsin, and anionic and cationic trypsins). We then use this map to dissect and improve the affinity and selectivity of APPI variants toward each of the four proteases. Our strategy can be used as a platform for the development of a new generation of target-selective probes and therapeutic agents based on selective protein–protein interactions., Characterizing the binding selectivity landscape of interacting proteins is crucial in protein engineering. Here the authors use multi-target selective library screening and in silico next-generation sequencing to map the binding landscape of proteins and produce improved proteases inhibitors.

Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. These findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.

Mesotrypsin is an isoform of trypsin that is uniquely resistant to polypeptide trypsin inhibitors and can cleave some inhibitors rapidly. Previous studies have shown that the amyloid precursor protein Kunitz protease inhibitor domain (APPI) is a specific substrate of mesotrypsin and that stabilization of the APPI cleavage site in a canonical conformation contributes to recognition by mesotrypsin. We hypothesized that other proteins possessing potential cleavage sites stabilized in a similar conformation might also be mesotrypsin substrates. Here we evaluated a series of candidate substrates, including human Kunitz protease inhibitor domains from amyloid precursor-like protein 2 (APLP2), bikunin, hepatocyte growth factor activator inhibitor type 2 (HAI2), tissue factor pathway inhibitor-1 (TFPI1), and tissue factor pathway inhibitor-2 (TFPI2), as well as E-selectin, an unrelated protein possessing a potential cleavage site displaying canonical conformation. We find that Kunitz domains within APLP2, bikunin, and HAI2 are cleaved by mesotrypsin with kinetic profiles of specific substrates. TFPI1 and TFPI2 Kunitz domains are cleaved less efficiently by mesotrypsin, and E-selectin is not cleaved at the anticipated site. Cocrystal structures of mesotrypsin with HAI2 and bikunin Kunitz domains reveal the mode of mesotrypsin interaction with its canonical substrates. Our data suggest that major determinants of mesotrypsin substrate specificity include sequence preferences at the P1 and P′2 positions along with conformational stabilization of the cleavage site in the canonical conformation. Mesotrypsin up-regulation has been implicated previously in cancer progression, and proteolytic clearance of Kunitz protease inhibitors offers potential mechanisms by which mesotrypsin may mediate pathological effects in cancer.