Raeeszadeh‐Sarmazdeh, Maryam

Matrix metalloproteinases (MMPs) are a family of zinc‐dependent endopeptidases that function to mediate tissue remodeling; however, they are also implicated in pathological conditions such as cancer, inflammatory and degenerative diseases. There are 23 human MMPs and significant sequence and structural homology exists among them and with other metalloproteinases. This represents a substantial challenge in developing therapeutically viable selective inhibitors due to off‐target effects. Tissue inhibitors of metalloproteinases (TIMPs) are a family of four endogenous protein protease inhibitors that regulate MMP activity. Our lab and others have utilized TIMPs as scaffolds, engineering them to selectively target specific MMPs for potential therapeutic applications. We previously employed a yeast surface display platform to identify TIMP‐1 variants possessing enhanced MMP‐3 affinity. In the current study, we used structures of MMP‐3 co‐crystallized with either WT TIMP‐1 or an engineered TIMP‐1 variant as seeds for molecular dynamics simulations. Simulations were also conducted on unbound WT and engineered TIMP‐1 to examine MMP‐independent dynamics that distinguish the engineered variant. The simulation data were analyzed with root‐mean‐square deviation/fluctuation, time‐dependent secondary structure, temporally resolved hydrogen bonding, conformational clustering, and dynamical network algorithms to discern structural and dynamical determinants of the increased affinity. Key observations that demonstrate the increased effectiveness of engineered TIMP‐1 as an MMP‐3 inhibitor were secondary structural changes in the MMP‐binding loops in proximity to mutations, attenuated global and mutation‐adjacent dynamics, decreased conformational dynamics, and increased persistence of MMP‐3‐compatible conformations. There were also indications of greater interdomain cooperativity within the engineered TIMP‐1 variant: increased interdomain hydrogen bonding, coordinated motions of the N‐terminal region with the C‐terminal domain, and increased intercommunity information flow/motion transfer. From these observations, we hypothesize the increased interdomain interactions represent a mechanism of dynamical stabilization such that mainly cooperative motion between domains presides, rather than random independent motions. Our findings suggest propagated motions between domains may influence the MMP affinity of TIMPs. Furthermore, these data suggest future strategies to design next‐generation mutant TIMP libraries by targeting regions dynamically coupled to the MMP binding loops for variation, thus exploiting natural protein dynamics to achieve desired selectivity.

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.

ABSTRACTMatrix metalloproteinases (MMPs) are a family of zinc endopeptidases that cleave nearly all components of the extracellular matrix as well as many other soluble and cell‐associated proteins. MMPs have been implicated in normal physiological processes, including development, and in the acquisition and progression of the malignant phenotype. Disappointing results from a series of clinical trials testing small molecule, broad spectrum MMP inhibitors as cancer therapeutics led to a re‐evaluation of how MMPs function in the tumor microenvironment, and ongoing research continues to reveal that these proteins play complex roles in cancer development and progression. It is now clear that effective targeting of MMPs for therapeutic benefit will require selective inhibition of specific MMPs. Here, we provide an overview of the MMP family and its biological regulators, the tissue inhibitors of metalloproteinases (TIMPs). We then summarize recent research from model systems that elucidate how specific MMPs drive the malignant phenotype of breast cancer cells, including acquisition of cancer stem cell features and induction of the epithelial–mesenchymal transition, and we also outline clinical studies that implicate specific MMPs in breast cancer outcomes. We conclude by discussing ongoing strategies for development of inhibitors with therapeutic potential that are capable of selectively targeting the MMPs most responsible for tumor promotion, with special consideration of the potential of biologics including antibodies and engineered proteins based on the TIMP scaffold. J. Cell. Biochem. 118: 3531–3548, 2017. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.