Conformation-Specific Inhibitory Anti-MMP-7 Monoclonal Antibody Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Chemotherapeutic Cell Kill.

Matrix metalloproteases (MMPs) undergo post-translational modifications including pro-domain shedding. The activated forms of these enzymes are effective drug targets, but generating potent biological inhibitors against them remains challenging. We report the generation of anti-MMP-7 inhibitory monoclonal antibody (GSM-192), using an alternating immunization strategy with an active site mimicry antigen and the activated enzyme. Our protocol yielded highly selective anti-MMP-7 monoclonal antibody, which specifically inhibits MMP-7's enzyme activity with high affinity (IC50 = 132 ± 10 nM). The atomic model of the MMP-7-GSM-192 Fab complex exhibited antibody binding to unique epitopes at the rim of the enzyme active site, sterically preventing entry of substrates into the catalytic cleft. In human PDAC biopsies, tissue staining with GSM-192 showed characteristic spatial distribution of activated MMP-7. Treatment with GSM-192 in vitro induced apoptosis via stabilization of cell surface Fas ligand and retarded cell migration. Co-treatment with GSM-192 and chemotherapeutics, gemcitabine and oxaliplatin elicited a synergistic effect. Our data illustrate the advantage of precisely targeting catalytic MMP-7 mediated disease specific activity.

Expanding small-molecule target space to mRNA translation regulation.

Multiple layers of regulation are in place on mRNA translation to ensure that cells respond in a fast manner to environmental cues in a tissue-specific and mRNA-selective manner. Here, we discuss mRNA translation regulatory mechanisms and potential drug-intervention targets. Taking on a new scientific rational of translation regulation and consequently a new target space, we have developed a unique discovery platform that is able to identify selective small molecule drugs that affect translation of specific proteins. This approach has enabled targeting of proteins that have been considered undruggable. Our discovery platform was repeatedly utilized to identify compounds in multiple therapeutic programs, including fibrosis, oncology, anti-virals and Huntington's disease. In fibrosis, the lead compound ANI-21 has demonstrated a tissue-specific effect in lowering the translation of Collagen-I and superior efficacy over best standard of care, in both cell and animal models, mediated by a novel mechanism of action. This program is expected to enter clinical studies within 12-18 months.

LOXL2 Inhibition Paves the Way for Macrophage-Mediated Collagen Degradation in Liver Fibrosis.

Liver fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) proteins and enzymes, especially fibrillary collagens, and represents a major cause of morbidity and mortality worldwide. Lysyl oxidases (LOXs) drive covalent crosslinking of collagen fibers, thereby promoting stabilization and accumulation of liver fibrosis while limiting its resolution. Here we show in a carbon tetrachloride (CCl4)-induced liver fibrosis murine model that treatment with a novel anti-lysyl oxidase like 2 (LOXL2) neutralizing antibody, which targets extracellular LOXL2, significantly improves fibrosis resolution. LOXL2 inhibition following the onset of fibrosis accelerated and augmented collagen degradation. This was accompanied by increased localization of reparative monocyte-derived macrophages (MoMFs) in the proximity of fibrotic fibers and their representation in the liver. These cells secreted collagenolytic matrix metalloproteinases (MMPs) and, in particular, the membrane-bound MT1-MMP (MMP-14) collagenase. Inducible and selective ablation of infiltrating MoMFs negated the increased "on-fiber" accumulation of MMP-14-expressing MoMFs and the accelerated collagenolytic activity observed in the anti-LOXL2-treated mice. Many studies of liver fibrosis focus on preventing the progression of the fibrotic process. In contrast, the therapeutic mechanism of LOXL2 inhibition presented herein aims at reversing existing fibrosis and facilitating endogenous liver regeneration by paving the way for collagenolytic macrophages.

The apparent competitive action of ECM proteases and cross-linking enzymes during fibrosis: Applications to drug discovery.

Progressive loss of organ function in most organs is associated with fibrosis, a tissue state associated with abnormal matrix buildup. If highly progressive, the fibrotic process eventually leads to organ failure and death. Fibrosis is a basic connective tissue lesion defined by the increase in the amount of fibrillar extracellular matrix (ECM) components in a tissue or organ. In addition, intrinsic changes in important structural cells can induce the fibrotic response by regulating the differentiation, recruitment, proliferation and activation of extracellular matrix-producing myofibroblasts. ECM enzymes belonging to the family of matrix metalloproteinases (MMPs) and lysyl oxidases (LOXs) play a crucial role in ECM remodeling and regeneration. MMPs have a catalytic role in degradation of ECM, whereas LOX/LOXLs mediate ECM, especially collagen, cross-linking and stiffening. Importantly, enzymes from both families are elevated during the fibrotic response to tissue injury and its resolution. Yet, the apparent molecular competition or antagonistic activities of these enzyme families during the various stages of fibrosis is often overlooked. In this review, we discuss the diverse roles of MMPs and LOX/LOXL2 in chronic organ fibrosis. Finally, we review contemporary therapeutic strategies for fibrosis treatment, based on neutralization of MMP and LOX activity, as well as the development of novel drug delivery approaches.

Tumor macrophages are pivotal constructors of tumor collagenous matrix.

Tumor-associated macrophages (TAMs) promote tumor development, invasion, and dissemination by various mechanisms. In this study, using an orthotopic colorectal cancer (CRC) model, we found that monocyte-derived TAMs advance tumor development by the remodeling of its extracellular matrix (ECM) composition and structure. Unbiased transcriptomic and proteomic analyses of (a) TAM-abundant and -deficient tumor tissues and (b) sorted tumor-associated and -resident colonic macrophage subpopulations defined a distinct TAM-induced ECM molecular signature composed of an ensemble of matricellular proteins and remodeling enzymes they provide to the tumor microenvironment. Remarkably, many of these ECM proteins are specifically increased in human CRC versus healthy colon. Specifically, we demonstrate that although differentiating into TAMs, monocytes up-regulate matrix-remodeling programs associated with the synthesis and assembly of collagenous ECM, specifically collagen types I, VI, and XIV. This finding was further established by advanced imaging showing that TAMs instruct the deposition, cross-linking, and linearization of collagen fibers during tumor development, especially at areas of tumor invasiveness. Finally, we show that cancer-associated fibroblasts are significantly outnumbered by TAMs in this model and that their expression of collagen XIV and I is reduced by TAM deficiency. Here, we outline a novel TAM protumoral function associated with building of the collagenous ECM niche.

Extracellular Matrix Proteolysis by MT1-MMP Contributes to Influenza-Related Tissue Damage and Mortality.

Mounting an effective immune response, while also protecting tissue integrity, is critical for host survival. We used a combined genomic and proteomic approach to investigate the role of extracellular matrix (ECM) proteolysis in achieving this balance in the lung during influenza virus infection. We identified the membrane-tethered matrix metalloprotease MT1-MMP as a prominent host-ECM-remodeling collagenase in influenza infection. Selective inhibition of MT1-MMP protected the tissue from infection-related structural and compositional tissue damage. MT1-MMP inhibition did not significantly alter the immune response or cytokine expression. The available flu therapeutic Oseltamivir did not prevent lung ECM damage and was less effective than anti-MT1-MMP in influenza virus Streptococcus pneumoniae coinfection paradigms. Combination therapy of Oseltamivir with anti-MT1-MMP showed a strong synergistic effect and resulted in complete recovery of infected mice. This study highlights the importance of tissue resilience in surviving infection and the potential of such host-pathogen therapy combinations for respiratory infections.