Detection of Endogenous Matrix Metalloprotease-12 Active Form with a Novel Broad Spectrum Activity-based Probe

Matrix metalloproteases (MMPs) have attracted considerable attention as critical mediators of pathological tissue remodeling processes. However it remains an unresolved challenge to detect their active forms in biological samples. To prove the efficacy of a recently developed MMP activity-based probe, we examined the content in MMP active forms of bronchoalveolar lavage fluids (BALf) from male C57BL/6 mice exposed to ultrafine carbon black nanoparticles, a model of chronic obstructive pulmonary disease. This probe was shown to label proteins, mostly expressed in BALf of mice exposed to nanoparticles. Using competition assays with a selective MMP-12 inhibitor as well as MMP-12 knock-out mice, one of these proteins was identified as the active form of the catalytic domain of MMP-12. This new probe can detect the active form of MMP-12 down to a threshold of 1 fmol. Radioactive counting showed the concentration of the active form of MMP-12 to be around 1 fmol/μl in BALf from nanoparticle-treated mice. A less sensitive probe would therefore not have detected MMP-12. As the probe can detect other MMPs in the femtomolar range, it is a potentially powerful tool for monitoring the levels of MMP active forms in various diseases.     

The intermediate S1' pocket of the endometase/matrilysin-2 active site revealed by enzyme inhibition kinetic studies, protein sequence analyses, and homology modeling

Human matrix metalloproteinase-26 (MMP-26/endometase/matrilysin-2) is a newly identified MMP and its structure has not been reported. The enzyme active site S1' pocket in MMPs is a well defined substrate P1' amino acid residue-binding site with variable depth. To explore MMP-26 active site structure-activity, a series of new potent mercaptosulfide MMP inhibitors (MMPIs) with Leu or homophenylalanine (Homophe) side chains at the P1' site were selected. The Homephe side chain is designed to probe deep S1' pocket MMPs. These inhibitors were tested against MMP-26 and several MMPs with known x-ray crystal structures to distinguish shallow, intermediate, and deep S1' pocket characteristics. MMP-26 has an inhibition profile most similar to those of MMPs with intermediate S1' pockets. Investigations with hydroxamate MMPIs, including those designed for deep pocket MMPs, also indicated the presence of an intermediate pocket. Protein sequence analysis and homology modeling further verified that MMP-26 has an intermediate S1' pocket formed by Leu-204, His-208, and Tyr-230. Moreover, residue 233 may influence the depth of an MMP S1' pocket. The residue at the equivalent position of MMP-26 residue 233 is hydrophilic in intermediate-pocket MMPs (e.g. MMP-2, -8, and -9) and hydrophobic in deep-pocket MMPs (e.g. MMP-3, -12, and -14). MMP-26 contains a His-233 that renders the S1' pocket to an intermediate size. This study suggests that MMPIs, protein sequence analyses, and molecular modeling are useful tools to understand structure-activity relationships and provides new insight for rational inhibitor design that may distinguish MMPs with deep versus intermediate S1' pockets.     

Cigarette smoke upregulates pulmonary vascular matrix metalloproteinases via TNF-alpha signaling

Cigarette smoke exposure causes vascular remodeling and pulmonary hypertension by poorly understood mechanisms. To ascertain whether cigarette smoke exposure affects production of matrix metalloproteinases (MMPs) in the pulmonary vessels, we exposed C57Bl/6 (C57) mice or mice lacking TNF-alpha receptors (TNFRKO) to smoke daily for 2 wk or 6 mo. Using laser capture microdissection and RT-PCR analysis, we examined gene expression of MMP-2, MMP-9, MMP-12, MMP-13, and tissue inhibitor of metalloproteinase (TIMP-1) and examined protein production by immunohistochemistry for MMP-2, MMP-9, and MMP-12 in small intrapulmonary arteries. At 2 wk, mRNA levels of TIMP-1 and all MMPs were increased in the C57, but not TNFRKO, mice, and immunoreactive protein for MMP-2, MMP-9, and MMP-12 was also increased in the C57 mice. Increased gelatinase activity was identified by in situ and bulk tissue zymography. At 6 mo, only MMP-12 mRNA levels remained increased in the C57 mice, but at a much lower level; however, MMP-2 mRNA levels increased in the TNFRKO mice. We conclude that smoke exposure increases MMP production in the small intrapulmonary arteries but that, with the exception of MMP-12, increased MMP production is transient. MMPs probably play a role in smoke-induced vascular remodeling, as they do in other forms of pulmonary hypertension, implying that MMP inhibitors might be beneficial. MMP production is largely TNF-alpha dependent, further supporting the importance of TNF-alpha in the pathogenesis of cigarette smoke-induced lung disease.     

Synthesis and evaluation of a novel fluorescent photoprobe for imaging matrix metalloproteinases

The measurement of matrix metalloproteinase (MMP) activity in diseases like inflammation, oncogenesis, or atherosclerosis in vivo is highly desirable. Fine-tuned pyrimidine-2,4,6-triones (barbiturates) offer nonpeptidyl lead structures for developing imaging agents for specifically visualization of activated MMPs in vivo. The aim of this study was to modify a C-5-disubstituted barbiturate and thus design a highly affine, nonpeptidic, optical MMP inhibitor (MMPI)-ligand for imaging of activated MMPs in vivo. A convergent 10 step synthesis was developed, starting with a malonic ester and (4-bromophenoxy)benzene to generate 5-bromo-pyrimidine-2,4,6-trione as the key intermediate. To minimize the interactions between activated MMPs and the dye of the conjugate 6, a PEGylated piperazine derivative was used as a spacer and an azide as a protected amino function. After linking both building blocks, reducing the azide ( Staudinger reaction) and labeling with Cy 5.5, we obtained the nonhydroxamate MMP inhibitor 6 with high affinity (IC 50-value: 48 nM for MMP-2) measured in a fluorogenic assay using commercially available MMP-substrates and the purified enzyme. Zymography revealed an efficient blocking of enzyme activity of purified MMP-2 and MMP-9 and of MMP-containing cell supernatants (HT-1080), (A-673) using the PEGylated barbiturate 5. Fluorescence microscopy studies using a highly (A-673) and a moderate (HT-1080) MMP-2 secreting cell line showed efficient binding of the Cy 5.5 labeled tracer 6 to the MMP-2 positive cells while MMP-2 negative cells (MCF-7) did not bind. Therefore, this new barbiturate-based MMP-probe has a high affinity and specificity toward MMP-2 and -9 and is thus a promising candidate for sensitive MMP detection in vivo.     

E. coli expression of TIMP-4 and comparative kinetic studies with TIMP-1 and TIMP-2: insights into the interactions of TIMPs and matrix metalloproteinase 2 (gelatinase A)

The inhibitory properties of TIMP-4 for matrix metalloproteinases (MMPs) were compared to those of TIMP-1 and TIMP-2. Full-length human TIMP-4 was expressed in E. coli, folded from inclusion bodies, and the active component was purified by MMP-1 affinity chromatography. Progress curve analysis of MMP inhibition by TIMP-4 indicated that association rate constants (k(on)) and inhibition constants (K(i)) were similar to those for other TIMPs ( approximately 10(5) M(-)(1) s(-)(1) and 10(-)(9)-10(-)(12) M, respectively). Dissociation rate constants (k(off)) for MMP-1 and MMP-3 determined using alpha(2)-macroglobulin to capture MMP dissociating from MMP-TIMP complexes were in good agreement with values deduced from progress curves ( approximately 10(-)(4) s(-)(1)). K(i) and k(on) for the interactions of TIMP-1, -2, and -4 with MMP-1 and -3 were shown to be pH dependent. TIMP-4 retained higher reactivity with MMPs at more acidic conditions than either TIMP-1 or TIMP-2. Molecular interactions of TIMPs and MMPs investigated by IAsys biosensor analysis highlighted different modes of interaction between proMMP-2-TIMP-2 (or TIMP-4) and active MMP-2-TIMP-2 (or TIMP-4) complexes. The observation that both active MMP-2 and inactive MMP-2 (with the active site blocked either by the propeptide or a hydroxamate inhibitor) have essentially identical affinities for TIMP-2 suggests that there are two TIMP binding sites on the hemopexin domain of MMP-2: one with high affinity that is involved in proMMP-2 or hydroxamate-inhibited MMP-2; and the other with low affinity involved in formation of the complex of active MMP-2 and TIMP-2. Similar models of interaction may apply to TIMP-4. The latter low-affinity site functions in conjunction with the active site of MMP-2 to generate a tight enzyme-inhibitor complex.     

Phage display of tissue inhibitor of metalloproteinases-2 (TIMP-2): identification of selective inhibitors of collagenase-1 (metalloproteinase 1 (MMP-1))

Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a broad spectrum inhibitor of the matrix metalloproteinases (MMPs), which function in extracellular matrix catabolism. Here, phage display was used to identify variants of human TIMP-2 that are selective inhibitors of human MMP-1, a collagenase whose unregulated action is linked to cancer, arthritis, and fibrosis. Using hard randomization of residues 2, 4, 5, and 6 (L1) and soft randomization of residues 34-40 (L2) and 67-70 (L3), a library was generated containing 2 × 10(10) variants of TIMP-2. Five clones were isolated after five rounds of selection with MMP-1, using MMP-3 as a competitor. The enriched phages selectively bound MMP-1 relative to MMP-3 and contained mutations only in L1. The most selective variant (TM8) was used to generate a second library in which residues Cys(1)-Gln(9) were soft-randomized. Four additional clones, selected from this library, showed a similar affinity for MMP-1 as wild-type TIMP-2 but reduced affinity for MMP-3. Variants of the N-terminal domain of TIMP-2 (N-TIMP-2) with the sequences of the most selective clones were expressed and characterized for inhibitory activity against eight MMPs. All were effective inhibitors of MMP-1 with nanomolar K(i) values, but TM8, containing Ser(2) to Asp and Ser(4) to Ala substitutions, was the most selective having a nanomolar K(i) value for MMP-1 but no detectable inhibitory activity toward MMP-3 and MMP-14 up to 10 μM. This study suggests that phage display and selection with other MMPs may be an effective method for discovering tissue inhibitor of metalloproteinase variants that discriminate between specified MMPs as targets.     

Constraining specificity in the N-domain of tissue inhibitor of metalloproteinases-1; gelatinase-selective inhibitors

The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs). Since unregulated MMP activities are linked to arthritis, cancer, and atherosclerosis, TIMP variants that are selective inhibitors of disease-related MMPs have potential therapeutic value. The structures of TIMP/MMP complexes reveal that most interactions with the MMP involve the N-terminal pentapeptide of TIMP and the C-D beta-strand connector which occupy the primed and unprimed regions of the active site. The loop between beta-strands A and B forms a secondary interaction site for some MMPs, ranging from multiple contacts in the TIMP-2/membrane type-1 (MT1)-MMP complex to none in the TIMP-1/MMP-1 complex. TIMP-1 and its inhibitory domain, N-TIMP-1, are weak inhibitors of MT1-MMP; inhibition is not improved by grafting the longer AB loop from TIMP-2 into N-TIMP-1, but this change impairs binding to MMP-3 and MMP-7. Mutational studies with N-TIMP-1 suggest that its weak inhibition of MT1-MMP, as compared to other N-TIMPs, arises from multiple (>3) sequence differences in the interaction site. Substitutions for Thr2 of N-TIMP-1 strongly influence MMP selectivity; Arg and Gly, that generally reduce MMP affinity, have less effect on binding to MMP-9. When the Arg mutation is added to the N-TIMP-1(AB2) mutant, it produces a gelatinase-specific inhibitor with Ki values of 2.8 and 0.4 nM for MMP-2 and -9, respectively. Interestingly, the Gly mutant has a Ki of 2.1 nM for MMP-9 and >40 muM for MMP-2, indicating that engineered TIMPs can discriminate between MMPs in the same subfamily.     

The roles of substrate thermal stability and P2 and P1' subsite identity on matrix metalloproteinase triple-helical peptidase activity and collagen specificity

The hydrolysis of collagen (collagenolysis) is one of the committed steps in extracellular matrix turnover. Within the matrix metalloproteinase (MMP) family distinct preferences for collagen types are seen. The substrate determinants that may guide these specificities are unknown. In this study, we have utilized 12 triple-helical substrates in combination with 10 MMPs to better define the contributions of substrate sequence and thermal stability toward triple helicase activity and collagen specificity. In general, MMP-13 was found to be distinct from MMP-8 and MT1-MMP(Delta279-523), in that enhanced substrate thermal stability has only a modest effect on activity, regardless of sequence. This result correlates to the unique collagen specificity of MMP-13 compared with MMP-8 and MT1-MMP, in that MMP-13 hydrolyzes type II collagen efficiently, whereas MMP-8 and MT1-MMP are similar in their preference for type I collagen. In turn, MMP-1 was the least efficient of the collagenolytic MMPs at processing increasingly thermal stable triple helices and thus favors type III collagen, which has a relatively flexible cleavage site. Gelatinases (MMP-2 and MMP-9(Delta444-707)) appear incapable of processing more stable helices and are thus mechanistically distinct from collagenolytic MMPs. The collagen specificity of MMPs appears to be based on a combination of substrate sequence and thermal stability. Analysis of the hydrolysis of triple-helical peptides by an MMP mutant indicated that Tyr(210) functions in triple helix binding and hydrolysis, but not in processing triple helices of increasing thermal stabilities. Further exploration of MMP active sites and exosites, in combination with substrate conformation, may prove valuable for additional dissection of collagenolysis and yield information useful in the design of more selective MMP inhibitors.     

Substrate specificity determinants of human macrophage elastase (MMP-12) based on the 1.1 A crystal structure

The macrophage elastase enzyme (MMP-12) expressed mainly in alveolar macrophages has been identified in the mouse lung as the main destructive agent associated with cigarette smoking, which gives rise to emphysema, both directly via elastin degradation and indirectly by disturbing the proteinase/antiproteinase balance via inactivation of the alpha1-proteinase inhibitor (alpha1-PI), the antagonist of the leukocyte elastase. The catalytic domain of human recombinant MMP-12 has been crystallized in complex with the broad-specificity inhibitor batimastat (BB-94). The crystal structure analysis of this complex, determined using X-ray data to 1.1 A and refined to an R-value of 0.165, reveals an overall fold similar to that of other MMPs. However, the S-shaped double loop connecting strands III and IV is fixed closer to the beta-sheet and projects its His172 side-chain further into the rather hydrophobic active-site cleft, defining the S3 and the S1-pockets and separating them from each other to a larger extent than is observed in other MMPs. The S2-site is planar, while the characteristic S1'-subsite is a continuous tube rather than a pocket, in which the MMP-12-specific Thr215 replaces a Val residue otherwise highly conserved in almost all other MMPs. This alteration might allow MMP-12 to accept P1' Arg residues, making it unique among MMPs. The active-site cleft of MMP-12 is well equipped to bind and efficiently cleave the AlaMetPhe-LeuGluAla sequence in the reactive-site loop of alpha1-PI, as occurs experimentally. Similarities in contouring and particularly a common surface hydrophobicity both inside and distant from the active-site cleft explain why MMP-12 shares many substrates with matrilysin (MMP-7). The MMP-12 structure is an excellent template for the structure-based design of specific inhibitors for emphysema therapy and for the construction of mutants to clarify the role of this MMP.     

Classical Macrophage Activation Up-Regulates Several Matrix Metalloproteinases through Mitogen Activated Protein Kinases and Nuclear Factor-κB

Remodelling of the extracellular matrix (ECM) and cell surface by matrix metalloproteinases (MMPs) is an important function of monocytes and macrophages. Recent work has emphasised the diverse roles of classically and alternatively activated macrophages but the consequent regulation of MMPs and their inhibitors has not been studied comprehensively. Classical activation of macrophages derived in vitro from un-fractionated CD16(+/-) or negatively-selected CD16(-) macrophages up-regulated MMP-1, -3, -7, -10, -12, -14 and -25 and decreased TIMP-3 steady-state mRNA levels. Bacterial lipopolysaccharide, IL-1 and TNFα were more effective than interferonγ except for the effects on MMP-25, and TIMP-3. By contrast, alternative activation decreased MMP-2, -8 and -19 but increased MMP -11, -12, -25 and TIMP-3 steady-state mRNA levels. Up-regulation of MMPs during classical activation depended on mitogen activated protein kinases, phosphoinositide-3-kinase and inhibitor of κB kinase-2. Effects of interferonγ depended on janus kinase-2. Where investigated, similar effects were seen on protein concentrations and collagenase activity. Moreover, activity of MMP-1 and -10 co-localised with markers of classical activation in human atherosclerotic plaques in vivo. In conclusion, classical macrophage activation selectively up-regulates several MMPs in vitro and in vivo and down-regulates TIMP-3, whereas alternative activation up-regulates a distinct group of MMPs and TIMP-3. The signalling pathways defined here suggest targets for selective modulation of MMP activity.     

Designed Loop Extension Followed by Combinatorial Screening Confers High Specificity to a Broad Matrix Metalloproteinase Inhibitor

Matrix metalloproteinases (MMPs) are key drivers of various diseases, including cancer. Development of probes and drugs capable of selectively inhibiting the individual members of the large MMP family remains a persistent challenge. The inhibitory N-terminal domain of tissue inhibitor of metalloproteinases-2 (N-TIMP2), a natural broad MMP inhibitor, can provide a scaffold for protein engineering to create more selective MMP inhibitors. Here, we pursued a unique approach harnessing both computational design and combinatorial screening to confer high binding specificity toward a target MMP in preference to an anti-target MMP. We designed a loop extension of N-TIMP2 to allow new interactions with the non-conserved MMP surface and generated an efficient focused library for yeast surface display, which was then screened for high binding to the target MMP-14 and low binding to anti-target MMP-3. Deep sequencing analysis identified the most promising variants, which were expressed, purified, and tested for selectivity of inhibition. Our best N-TIMP2 variant exhibited 29 pM binding affinity to MMP-14 and 2.4 µM affinity to MMP-3, revealing 7500-fold greater specificity than WT N-TIMP2. High-confidence structural models were obtained by including NGS data in the AlphaFold multiple sequence alignment. The modeling together with experimental mutagenesis validated our design predictions, demonstrating that the loop extension packs tightly against non-conserved residues on MMP-14 and clashes with MMP-3. This study demonstrates how introduction of loop extensions in a manner guided by target protein conservation data and loop design can offer an attractive strategy to achieve specificity in design of protein ligands.     

Hydrogen sulfide protects against vascular remodeling from endothelial damage

Remodeling by its very nature implied synthesis and degradation of extracellular matrix (ECM) proteins. Although oxidative stress, matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) have been implicated in vascular remodeling, the differential role of MMPs versus TIMPs and oxidative stress in vascular remodeling was unclear. TIMP-3 induced vascular cell apoptosis, therefore, we hypothesized that during vascular injury TIMP-3, MMP-9 and -12 (elastin-degrading MMP) were increased, whereas MMP-2 (constitutive MMP) and TIMP-4 (cardioprotective TIMP) decreased. Because of the potent anti-oxidant, vasorelaxing, anti-hypertensive agent, hydrogen sulfide (H₂S) was used to mitigate the vascular remodeling due to the differential expression of MMP and TIMP. Carotid artery injury was created by inserting a PE-10 catheter and rotating several times before pulling out. The insertion hole was sealed. Mice were grouped: wild type (WT), wild-type damaged artery (WTD), WT + NaHS (sodium hydrogen sulfide, precursor of H₂S) treatment (30 μmol/L in drinking water/6 weeks) and WTD + NaHS treatment. Carotid arteries were analyzed for oxidative stress and remodeling, by measuring super oxide dismutase-1 (SOD1), p47 (NADPH oxidase subunit), nitrotyrosine, MMPs and TIMPs by in situ immunolabeling and by Western blot analyses. The results suggested robust increase in p47, nitrotyrosine, MMP-9, MMP-12, TIMP-3 and decrease in SOD1 and MMP-2 levels in the injured arteries. The treatment with H₂S ameliorated these effects. We concluded that p47, TIMP-3, MMP-9 and -12 were increased where as SOD-1, MMP-2 and TIMP-4 were decreased in the injured arteries. The treatment with H₂S mitigated the vascular remodeling by normalizing the levels of redox stress, MMPs and TIMPs.     

Protein engineering of the tissue inhibitor of metalloproteinase 1 (TIMP-1) inhibitory domain. In search of selective matrix metalloproteinase inhibitors

Studies of the structural basis of the interactions of tissue inhibitors of metalloproteinases (TIMPs) and matrix metalloproteinases (MMPs) may provide clues for designing MMP-specific inhibitors. In this paper we report combinations of mutations in the major MMP-binding region that enhance the specificity of N-TIMP-1. Mutants with substitutions for residues 4 and 68 were characterized and combined with previously studied Thr(2) mutations to generate mutants with improved selectivity or binding affinity to specific MMPs. Some combinations of mutations had non-additive effects on DeltaG of binding to MMPs, suggesting interactions between subsites in the reactive site. The T2L/V4S mutation generates an inhibitor that binds to MMP-2 20-fold more tightly than to MMP-3(DeltaC) and over 400-fold more tightly than to MMP-1. The T2S/V4A/S68Y mutant is the strongest inhibitor for stromelysin-1 among all mutants characterized to date, with an apparent K(i) for MMP-3(DeltaC) in the picomolar range. A third mutant, T2R/V4I, has no detectable inhibitory activity for MMP-1 but is an effective inhibitor of MMP-2 and -3. These selective TIMP variants may provide useful tools for investigation of biological roles of specific MMPs and for possible therapy of MMP-related diseases.     

Mechanical stretch induces MMP-2 release and activation in lung endothelium: role of EMMPRIN

High-volume mechanical ventilation leads to ventilator-induced lung injury. This type of lung injury is accompanied by an increased release and activation of matrix metalloproteinases (MMPs). To investigate the mechanism leading to the increased MMP release, we systematically studied the effect of mechanical stretch on human microvascular endothelial cells isolated from the lung. We exposed cells grown on collagen 1 BioFlex plates to sinusoidal cyclic stretch at 0.5 Hz using the Flexercell system with 17-18% elongation of cells. After 4 days of cell stretching, conditioned media and cell lysate were collected and analyzed by gelatin, casein, and reverse zymograms as well as Western blotting. RT-PCR of mRNA extracted from stretched cells was performed. Our results show that 1) cyclic stretch led to increased release and activation of MMP-2 and MMP-1; 2) the activation of MMP-2 was accompanied by an increase in membrane type-1 MMP (MT1-MMP) and inhibited by a hydroxamic acid-derived inhibitor of MMPs (Prinomastat, AG3340); and 3) the MMP-2 release and activation were preceded by an increase in production of extracellular MMP inducer (EMMPRIN). These results suggest that cyclic mechanical stretch leads to MMP-2 activation through an MT1-MMP mechanism. EMMPRIN may play an important role in the release and activation of MMPs during lung injury.     

Design, synthesis, and characterization of potent, slow-binding inhibitors that are selective for gelatinases.

Gelatinases have been shown to play a key role in angiogenesis and tumor metastasis. Small molecular weight synthetic inhibitors for these enzymes are highly sought for potential use as anti-metastatic agents. Virtually all of the known inhibitors of matrix metalloproteinases (MMPs) are broad spectrum. We report herein the synthesis and kinetic characterization of two compounds, 4-(4-phenoxyphenylsulfonyl)butane-1,2-dithiol (compound 1) and 5-(4-phenoxyphenylsulfonyl)pentane-1,2-dithiol (compound 2), that are potent and selective gelatinase inhibitors. These compounds are slow, tight-binding inhibitors of gelatinases (MMP-2 and MMP-9) with K(i) values in the nanomolar range. In contrast, competitive inhibition of the catalytic domain of membrane-type 1 metalloproteinase (MMP-14(cat)) with comparable K(i) values (K(i) approximately 200 nm) was observed. Binding to stromelysin (MMP-3) was substantially weaker, with K(i) values in the micromolar range (K(i) approximately 10 microm). No binding to matrilysin (MMP-7) and collagenase 1 (MMP-1) was detected at inhibitor concentrations up to 60 microm. We have previously shown that synthetic MMP inhibitors work synergistically with TIMP-2 in the promotion of pro-MMP-2 activation by MT1-MMP in a process that depends on the affinity of the inhibitor toward MT1-MMP. It is shown herein that the dithiols are significantly less efficient (>100-fold) than marimastat, a broad-spectrum MMP inhibitor, in enhancing pro-MMP-2 activation in cells infected to express MT1-MMP, consistent with the lower affinity of the dithiols toward MT1-MMP. Thus, in contrast to broad-spectrum MMP inhibitors, the dithiols are less likely to promote MT1-MMP-dependent pro-MMP-2 activation in the presence of TIMP-2, while maintaining their ability to inhibit active MMP-2 effectively.     

Functional mimetic peptide discovery isolated by phage display interacts selectively to fibronectin domain and inhibits gelatinase

Matrix metalloproteinases (MMPs) play critical roles in a multiple number of autoimmunity diseases progression and metastasis of solid tumor. Gelatinases including MMP-2 and MMP-9 are extremely overexpressed in multiple pathological processes. MMP-9 and MMP-2 breakdown the extracellular matrix component gelatin very efficaciously. Therefore, designing and expansion of MMPs inhibitors can be an engrossing plan for therapeutic intermediacy. Anyway, a wide range of MMPs inhibitors face failure in several clinical trials. Due to sequence and structural conservation across the various MMPs, achieving specific and selective inhibitors is very demanding. In the current study, a phage-displayed peptide library was screened using active human recombinant MMP-9 protein and evaluated by enzyme-linked immunosorbent assay. Here, we isolate novel peptide sequence from phage display peptide libraries that can be a specific gelatinase inhibitor. Interestingly, in silico molecular docking showed strong interactions between the peptide three-dimensional models and some important residues of the MMP-9 and MMP-2 proteins at the fibronectin domain. A consensus peptide sequence was then synthesized (named as RSH-12) to evaluate its inhibitory potency by in vitro assays. Zymography assay was employed to evaluate the effect of RSH-12 on gelatinolysis activity of MMP-2 and MMP-9 secretion from the HT1080 cells using different concentrations of RSH-12 and inhibiting MMP-9- and MMP-2-driven gelatin proteolysis, measured by fluorescein isothiocyanate-gelatin degradation assay and HT1080 cell invasion assay on Matrigel (gelatinous protein mixture). The negative control peptide (CP) with the irrelevant sequence and no MMP inhibition properties and the positive control compound (GM6001) as a potent inhibitor of MMPs were used to assess the selectivity and specificity of gelatinases inhibition by RSH-12. Therefore, RSH-12 decreased the gelatin degradation by specifically preventing gelatin binding to MMP-9 and MMP-2. Selective gelatinase inhibitors may prove the usefulness of the new peptide discovered in tumor targeting and anticancer and anti-inflammation therapies.     

Insights from Selective Non-phosphinic Inhibitors of MMP-12 Tailored to Fit with an S1′ Loop Canonical Conformation

After the disappointment of clinical trials with early broad spectrum synthetic inhibitors of matrix metalloproteinases (MMPs), the field is now resurging with a new focus on the development of selective inhibitors that fully discriminate between different members of the MMP family with several therapeutic applications in perspective. Here, we report a novel class of highly selective MMP-12 inhibitors, without a phosphinic zinc-binding group, designed to plunge deeper into the S₁′ cavity of the enzyme. The best inhibitor from this series, identified through a systematic chemical exploration, displays nanomolar potency toward MMP-12 and selectivity factors that range between 2 and 4 orders of magnitude toward a large set of MMPs. Comparison of the high resolution x-ray structures of MMP-12 in free state or bound to this new MMP-12 selective inhibitor reveals that this compound fits deeply within the S₁′ specificity cavity, maximizing surface/volume ratios, without perturbing the S₁′ loop conformation. This is in contrast with highly selective MMP-13 inhibitors that were shown to select a particular S₁′ loop conformation. The search for such compounds that fit precisely to preponderant S₁′ loop conformation of a particular MMP may prove to be an alternative effective strategy for developing selective inhibitors of MMPs.