Sankaran, Banumathi

Selective inhibition of the more than 100 S1 family serine proteases is a long-standing challenge due to their active site similarity. Mesotrypsin, implicated in cancer progression, exemplifies these difficulties; no current inhibitors achieve selectivity over other human trypsins. We found an unexpected autoinhibited conformation of mesotrypsin via x-ray crystallography, revealing a cryptic pocket adjacent to the active site. Using high-throughput virtual screening targeting this cryptic pocket, we identified a conformationally selective small-molecule inhibitor that stabilizes the inactive state of mesotrypsin. This inhibitor demonstrates selectivity for mesotrypsin over other trypsins. Our findings challenge the accepted view of digestive trypsins as constitutively active enzymes lacking potential for allosteric regulation. Furthermore, analyses of other structures suggest that dynamic sampling of closed states with analogous allosteric cryptic pockets appears widespread among S1 serine proteases. These observations point to a potentially generalizable strategy to achieve selective inhibition, offering broad implications for drug development targeting serine proteases in cancer and other diseases.

Serine protease inhibitors of the Kunitz‐bovine pancreatic trypsin inhibitor (BPTI) family, such as tissue factor pathway inhibitor‐1 Kunitz domain 1 ( KD1 TFPI1) and amyloid precursor protein inhibitor (APPI), are biological regulators of proteolysis and serve as important scaffolds for developing therapeutics. Mesotrypsin is a unique human protease with cancer‐promoting properties which rapidly cleaves and inactivates serine protease inhibitors; because of this, it is a challenging therapeutic target and an excellent model for developing therapeutic inhibitors with improved proteolytic resistance. We hypothesize that stabilization of the Kunitz inhibitor binding loop by disulfide bond anchoring to the scaffold can reduce proteolytic susceptibility and improve binding efficacy. Here we have engineered a new disulfide bond into both KD1 TFPI1 and an APPI variant previously engineered for mesotrypsin affinity. The new disulfide bond anchors the binding loop to the scaffold, resulting in reduced mesotrypsin proteolytic cleavage by 17‐fold and 3‐fold, respectively. To gain insight into the structural basis for improved proteolytic stability, we crystallized mutant KD1 TFPI1 and APPI with mesotrypsin and solved these novel structures. We then performed all‐atom molecular dynamics simulations using these mesotrypsin‐mutant inhibitor complexes compared to models lacking the engineered disulfide bond. Our simulation results support our hypothesis that the cause of decreased proteolysis rates is dampened conformational fluctuations in the binding loop and minimized motion at the enzyme‐inhibitor interface. We also performed simulations using unbound inhibitors to show that stabilization of the binding loop to states similar to the bound configuration correlates with stronger binding affinity. Our disulfide engineering approach is potentially a useful tool in biopharmaceutical development for mesotrypsin, other serine proteases, and possibly for other enzyme targets. Support or Funding Information NIH grant R01CA154387 to E.S.R., European Research Council grant 336041 to N.P., US‐Israel Binational Science Foundation grant BSF2015134 to E.S.R. and N.P. Xray diffraction data were measured at the Berkeley Center for Structural Biology at the Advanced Light Source and at the European Synchrotron Radiation Facility. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Matrix metalloproteinases (MMPs) play a pivotal role in progression of several diseases including cancer and fibrosis, which makes these enzymes promising targets for developing protein therapeutics. The challenge in using MMP inhibitors as drugs in MMP‐associated diseases is lack of high selectivity toward specific target MMPs. Therefore, development of selective MMP inhibitors is a critical goal. Tissue inhibitors of metalloproteinases (TIMPs), as natural MMP inhibitors, offer ideal scaffolds for engineering highly selective MMP‐targeted therapeutics. We have previously shown a key role for MMP‐3 in the pathogenesis of pulmonary fibrosis. TIMP‐1 is a natural MMP‐3 inhibitor with picomolar affinity; it also binds to other MMPs with a spectrum of affinities. Engineering of TIMP‐1 for more selective inhibition of MMP‐3 may provide protein therapeutics with applications in treating lung fibrosis. I have used directed evolution and yeast surface display toward development of selective MMP‐3‐targeted biologic agents through protein engineering of TIMP‐1. I have screened a library of TIMP‐1 mutants generated by random mutagenesis of residues comprising the MMP‐interacting loops of TIMP‐1 to select for enhanced MMP‐3 binding on the yeast surface. Diversified residues have been chosen based on structural studies of TIMP‐1/MMP‐3 interactions. TIMP‐1 mutants isolated after rounds of Fluorescent‐Activated Cell Sorting (FACS) showed substantial improvement in MMP‐3 binding compared to the wild‐type TIMP‐1, both in the surface display context and in enzyme inhibition studies using soluble purified proteins. I have also developed a counter‐selection strategy to screen TIMP‐1 mutants that bind selectively to MMP‐3 in the presence of MMP‐10, an MMP with the greatest sequence and structural similarity to MMP‐3. The isolated TIMP‐1 mutants after five rounds of competitive screening showed up to 20‐fold improvement in binding selectivity, highlighting the significant potential of this approach for development of inhibitors with single‐MMP selectivity. In ongoing studies, I have identified some of the key TIMP‐1 mutations responsible for improvements in MMP‐3 affinity and selectivity. I have co‐crystallized one of the top affinity mutants with the catalytic domain of MMP‐3, and have obtained synchrotron diffraction data at a resolution of 2.2 Å; efforts to solve this crystal structure are underway. I anticipate that this structure will offer insights into the structural basis for improvements in selectivity, and will help to guide future library design for refinement of my directed evolution methodology for generating selective engineered TIMPs. This work provides a promising and innovative strategy to develop protein therapeutics based on natural enzyme inhibitors, critically improving selectivity to eliminate offtarget effects. Support or Funding Information This project is funded through two grants from Department of Defense (DoD) (ID # W81XWH‐16‐2‐003015), and National Institute of Health (NIH) (R21CA205471). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of matrix metalloproteinases (MMPs), enzymes known for contributions to diseases including cancer and fibrosis. The TIMP N‐terminal domain, regarded as the “inhibitory domain”, has been the center of attention both for understanding natural regulation of MMPs by TIMPs, and for developing protein‐based MMP inhibitors for therapeutic applications. The role of the TIMP C‐terminal domain in MMP inhibition has not been well studied. I have used directed evolution via yeast surface display for protein engineering of full‐length human TIMP‐1 toward development of MMP‐3‐targeted ultra‐binders. TIMP‐1 variants with up to 10‐fold improvement in MMP‐3 binding (compared to WT‐TIMP‐1) were isolated after six rounds of library screening using fluorescence‐activated cell sorting (FACS). Analysis of individual and paired mutations from the selected TIMP‐1 variants revealed a cooperative effect between distant mutations on the N‐ and C‐terminal TIMP domains, positioned on opposite sides of the interaction interface with MMP‐3. I have co‐crystallized two TIMP‐1 variants containing single and composite mutations in complex with the catalytic domain of MMP‐3 and solved the X‐ray crystal structures of these complexes. The protein structures reveal substantial conformational changes in the TIMP‐1 binding interfaces near the two cooperative mutations. Affinity appears to be strengthened by tighter cinching of a reciprocal tyrosine clasp formed between the N‐terminal loop of TIMP‐1 and the proximal MMP3 interface, and adoption of an alpha‐helical conformation by a segment of the TIMP‐1 C‐terminal domain, resulting in stabilized interactions between the two TIMP‐1 domains and improved hydrophobic interactions between TIMP‐1 and MMP‐3. Our protein engineering and structural studies provide novel insight into the cooperative function of N‐ and C‐terminal TIMP domains and the significance of peripheral TIMP epitopes in MMP recognition. Our results further point to new approaches for design of TIMP‐based protein therapeutics with improved affinity and selectivity. Support or Funding Information This work was supported by grants DOD W81XWH‐16‐2‐0030, and NIH R21 CA205471. Diffraction data were measured at the Berkeley National Synchrotron Light Source. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Enzymes of the matrix metalloproteinase (MMP) family have been shown to play critical roles in cancer progression and metastasis, making them ideal targets for developing cancer therapeutics. MMP‐9 plays a significant role in tumor progression and metastasis of triple negative breast cancer (TNBC), while MMP‐3, the biological activator of MMP‐9, can also promote mammary carcinoma through inducing epithelial‐mesenchymal‐transition (EMT) in tumor cells. These enzymes are attractive as therapeutic targets; however, broad spectrum MMP inhibitors have proven ineffective, so inhibitors with greater selectivity are desired. Tissue inhibitor of metalloproteinases‐1 (TIMP‐1) is a natural inhibitor of MMP‐3 and MMP‐9 with picomolar affinity, and offers a scaffold for engineering of selective MMP‐targeted therapeutics. To overcome the challenges of wide multispecificity of TIMP‐1 for different classes of MMPs, we used state‐of‐the‐art directed evolution and yeast surface display techniques recruiting high‐throughput library screening technology to fully evolve novel protein‐based drugs, based on the TIMP‐1 scaffold, with high selectivity for MMP‐3 or MMP‐9. We have developed a counter‐selection strategy to screen TIMP‐1 mutants that bind selectively to MMP‐3 in the presence of MMP‐10, an MMP with the greatest sequence and structural similarity to MMP‐3. The isolated TIMP‐1 mutants after five rounds of competitive screening showed up to 20‐fold improvement in binding selectivity, highlighting the significant potential of this approach for development of inhibitors with single‐MMP selectivity. In ongoing studies, I have identified some of the key TIMP‐1 mutations responsible for improvements in MMP‐3 affinity and selectivity. I have co‐crystallized the top affinity and selectivity mutants with the catalytic domain of MMP‐3, and have solved the structure. These structures provide insights into the structural basis for improvements in binding affinity and selectivity. These studies and methodology developed in this project, will lay the foundation for developing novel therapeutic strategies for MMP‐related diseases and understanding protein‐protein interaction between MMPs and their inhibitors.

Engineered protein therapeutics offer advantages, including strong target affinity, selectivity and low toxicity, but like natural proteins can be susceptible to proteolytic degradation, thereby limiting their effectiveness. A compelling therapeutic target is mesotrypsin, a protease up-regulated with tumour progression, associated with poor prognosis, and implicated in tumour growth and progression of many cancers. However, with its unique capability for cleavage and inactivation of proteinaceous inhibitors, mesotrypsin presents a formidable challenge to the development of biological inhibitors. We used a powerful yeast display platform for directed evolution, employing a novel multi-modal library screening strategy, to engineer the human amyloid precursor protein Kunitz protease inhibitor domain (APPI) simultaneously for increased proteolytic stability, stronger binding affinity and improved selectivity for mesotrypsin inhibition. We identified a triple mutant APPIM17G/I18F/F34V, with a mesotrypsin inhibition constant (Ki) of 89 pM, as the strongest mesotrypsin inhibitor yet reported; this variant displays 1459-fold improved affinity, up to 350 000-fold greater specificity and 83-fold improved proteolytic stability compared with wild-type APPI. We demonstrated that APPIM17G/I18F/F34V acts as a functional inhibitor in cell-based models of mesotrypsin-dependent prostate cancer cellular invasiveness. Additionally, by solving the crystal structure of the APPIM17G/I18F/F34V–mesotrypsin complex, we obtained new insights into the structural and mechanistic basis for improved binding and proteolytic resistance. Our study identifies a promising mesotrypsin inhibitor as a starting point for development of anticancer protein therapeutics and establishes proof-of-principle for a novel library screening approach that will be widely applicable for simultaneously evolving proteolytic stability in tandem with desired functionality for diverse protein scaffolds.