LBPO.TB01 · 肿瘤生物学 · Late-Breaking
选择性MMP-9抑制剂的蛋白工程揭示三阴性乳腺癌的治疗策略
Protein engineering of selective MMP-9 inhibitors reveals a therapeutic strategy for triple-negative breast cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
基质金属蛋白酶(MMPs)是肿瘤进展、侵袭和转移的关键调控因子；然而，抑制MMPs的临床努力因缺乏选择性和对保护性家族成员的意外抑制而大多失败。在三阴性乳腺癌(TNBC)中，MMP-9(明胶酶B)与肿瘤侵袭性、细胞外基质重塑和转移播散密切相关。相比之下，MMP-8已被证明通过限制炎症和抑制转移在乳腺癌中发挥抗肿瘤作用。这种功能上的分歧凸显了在TNBC中选择性靶向MMP-9同时保留MMP-8活性作为一种合理治疗策略的重要性。为应对这一挑战，我们对内源性MMP抑制剂——金属蛋白酶组织抑制剂-1(TIMP-1)——进行工程改造，使其对MMP-9相较MMP-8具有增强的选择性。构建了人TIMP-1的酵母表面展示文库，并使用荧光激活细胞分选进行筛选，通过对重组MMP-9的多轮迭代阳性选择，随后对MMP-8进行竞争性反向选择以消除脱靶结合物。该策略实现了对MMP-9特异性优于野生型TIMP-1的TIMP-1变体的富集。多个TIMP-1变体表现出对MMP-9增强的结合和抑制选择性，其中TIMP-1-C6.2成为最有前景的候选物。TIMP-1-C6.2优先抑制MMP-9，Ki为0.3 ± 0.04 nM，而对MMP-8的抑制显著较弱(Ki = 6.8 ± 0.27 nM)，对应于对MMP-9约23倍的选择性。重要的是，使用MDA-MB-231 TNBC细胞进行的Matrigel侵袭试验中的功能评估显示，TIMP-1-C6.2以剂量依赖方式显著增强了对肿瘤细胞侵袭的抑制，优于野生型TIMP-1(p < 0.0001)。这种改善的抗侵袭活性反映了精细化的靶点选择性，而非抑制效力的整体增加。总之，这些发现表明，对基于TIMP的抑制剂进行选择性工程改造，可通过抑制促肿瘤的MMP-9同时保留抗肿瘤的MMP-8，克服MMP靶向治疗的一个核心局限。这项工作为开发具有更佳安全性和治疗潜力的选择性MMP-9抑制剂用于TNBC及其他MMP驱动的癌症奠定了坚实基础。
查看英文原文 English abstract
Matrix metalloproteinases (MMPs) are key regulators of tumor progression, invasion, and metastasis; however, clinical efforts to inhibit MMPs have largely failed due to a lack of selectivity and unintended suppression of protective family members. In triple-negative breast cancer (TNBC), MMP-9 (gelatinase B) is strongly associated with tumor aggressiveness, extracellular matrix remodeling, and metastatic dissemination. In contrast, MMP-8 has been shown to exert anti-tumorigenic effects in breast cancer by limiting inflammation and suppressing metastasis. This functional divergence underscores the importance of selectively targeting MMP-9 while preserving MMP-8 activity as a rational therapeutic strategy in TNBC. To address this challenge, we engineered variants of the endogenous MMP inhibitor tissue inhibitor of metalloproteinases-1 (TIMP-1) with enhanced selectivity for MMP-9 over MMP-8. A yeast surface display library of human TIMP-1 was constructed and screened using fluorescence-activated cell sorting through iterative rounds of positive selection for recombinant MMP-9, followed by competitive counter-selection against MMP-8 to eliminate off-target binders. This strategy enabled enrichment of TIMP-1 variants with improved specificity for MMP-9 compared with wild-type TIMP-1. Several TIMP-1 variants exhibited enhanced binding and inhibitory selectivity toward MMP-9, with TIMP-1-C6.2 emerging as the most promising candidate. TIMP-1-C6.2 preferentially inhibited MMP-9 with a Ki of 0.3 ± 0.04 nM, while demonstrating substantially weaker inhibition of MMP-8 (Ki = 6.8 ± 0.27 nM), corresponding to an approximately 23-fold selectivity for MMP-9. Importantly, functional evaluation in Matrigel invasion assays using MDA-MB-231 TNBC cells revealed that TIMP-1-C6.2 significantly enhanced inhibition of tumor cell invasion compared with wild-type TIMP-1 in a dose-dependent manner (p < 0.0001). This improved anti-invasive activity reflects refined target selectivity rather than a generalized increase in inhibitory potency. Collectively, these findings demonstrate that selective engineering of TIMP-based inhibitors can overcome a central limitation of MMP-targeted therapies by suppressing pro-tumorigenic MMP-9 while sparing anti-tumorigenic MMP-8. This work provides a strong foundation for the development of selective MMP-9 inhibitors with improved safety and therapeutic potential for TNBC and other MMP-driven cancers.
利益披露 Disclosure
A. Shoari, None..
A. Hockla, None..
M. A. Coban, None..
E. E. Aitchison, None..
A. M. Dimesa, None..
E. S. Radisky, None.