Recent advances in the design and development of small-molecule MMP-2 inhibitors.

MMP-2 is crucial for ECM remodeling and embryonic development. MMP-2 is a key biomolecular target for its strong association with cancer progression, metastasis, and angiogenesis. Again, the implication of MMP-2 in other diseases is well-established. Though several MMPIs failed after extensive clinical studies due to a lack of selectivity, poor pharmacokinetics, and dose-related toxicities, there is still a huge opportunity to develop specific MMP-2 inhibitors to battle against such life-threatening diseases as cardiovascular diseases, diabetes, renal diseases, and inflammatory diseases. Here, the development of small-molecule MMP-2 inhibitors for the last five years, comprising various ZBGs and diverse scaffolds, as well as their structural information along with their in-depth biological implications in cancers and other diseases, has been discussed in detail. This study may reinforce the importance of potential and selective MMP-2 inhibition as a therapeutic approach, paving the way for future research into optimizing small-molecule MMP-2 inhibitors for clinical applications. As the development of these MMP-2 inhibitors advances, further in vivo studies and structure–activity relationship optimizations will be essential to translate these promising results into viable therapeutic options for several cancers and other life-threatening diseases.

MMP-2, cancer, clinical trials, small-molecule MMP-2 inhibitor, hydroxamate, carboxylic acid, phosphonate, ZBG 1. Introduction The extracellular matrix (ECM) is an intricate arrangement of proteins that facilitates the structural framework of cells. The ECM operates as a storage site for proteins like chemokines, cytokines, growth factors, and various signaling molecules that collectively influence cellular functions in health and disease conditions [1]. Before the 1950s, no enzyme was known to degrade collagen. Matrix metalloproteinases (MMPs) are those enzymes known to perform this task [2]. The term MMP was coined in the 1980s by Okada and coworkers [3]. The ECM, being dynamic, is continuously remodeled by MMPs that act as the foundation for its gaining recognition [4]. MMPs are zinc-dependent enzymes responsible for ECM remodeling. MMPs belong to the metzincin protease superfamily, characterized by the conserved HExxHxxGxxH domain, which binds the catalytic zinc for enzymatic activity [5]. Despite structural similarities and overlapping substrate specificities, 23 different human MMPs have been recognized to date, with versatile roles in physiological processes such as tumor progression, angiogenesis, and cell invasion. MMPs are linked to several illnesses, including various cancers [6], periodontal disease [7], atherosclerosis [8], arthritis [9], and inflammation [10]. The growing catalog of diseases associated with MMPs has made them an attractive target for both diagnostic and therapeutic approaches [11]. MMP-2, commonly referred to as gelatinase A, is one of the crucial enzymes of the MMP family that plays pivotal roles in ECM remodeling and embryonic development [6]. Along with MMP-9 (also known as gelatinase B), MMP-2 participates in degrading type IV and V collagen, fibronectin, laminin, and elastin [12]. MMP-2 behaves like an interstitial collagenase, cleaving triple-helix collagens at a ¾N-terminal cleavage site [2,13]. It digests denatured collagen with help from a fibronectin-like domain [2,13]. Gelatinases regulate non-matrix proteins, like fibroblast growth factor receptor 1 (FGFR1), insulin-like growth factor-binding protein-3 and −5 (IGF-BP-3, IGF-BP-5), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and transforming growth factor-β (TGF-β) [13]. Nevertheless, MMP-2 interacts with different functional proteins and simultaneously influences several molecular pathways. MMP-2 also breaks down tau (tubulin-associated unit) protein, promoting the neurodegenerative processes [14]. Deficiency of MMP-2 can impair TGF-β signaling, affecting diseases like thoracic aortic aneurysm [15]. MMP-2 enhances tumor invasion and metastasis by activating vascular endothelial growth factor (VEGF), IGF, and TGF-β signaling pathways [16]. MMP-2 also plays a role in modulating immune responses and inflammation by breaking down TNF-α [17]. It facilitates epidermal growth factor receptor (EGFR) activation, which leads to cell proliferation [18]. Considering its versatile roles, MMP-2 has been implicated in numerous diseases like various cancers, cardiovascular diseases, neurodegenerative diseases, diabetes, COPD, fibrosis, and inflammatory diseases [6,11,19]. In this context, thousands of matrix metalloproteinase inhibitors (MMPIs) were synthesized over more than three decades, of which Periostat (also known as doxycycline hydrate) is the only FDA-approved MMPI for periodontal disease [20]. A number of anticancer MMPIs have undergone clinical trials but failed due to lack of selectivity and off-target effects, metabolic instability, poor pharmacokinetics, and various dose-limiting toxicities, like musculoskeletal syndrome (MSS) [11,19,21]. Selective gelatinase inhibitors may avoid such problems, such as MSS, making them potential therapeutic targets [19,21–23]. Not only that, researchers are also trying to inculcate MMP-2 as nano-probes that help to detect real-time and long-term tumor tracking activity because MMP-2 serves in cancer imaging by acting as a biomarker for cancerous cell detection [24]. MMP-2 is used in targeted drug therapy due to its overexpression in tumors, which allows for enzyme-specific drug carrier activation [24]. It also breaks down the physiological barrier in tumor cells, thus helping to enhance the penetration of the photosensitizer in photodynamic therapy [25]. MMP-2 responsive systems help normalize the tumor microenvironment, making tumors more susceptible to the treatment [24]. For example, they assist in deep tumor penetration by improving oxygen supply for photodynamic therapy [24]. Small-molecule MMP-2 inhibitors and monoclonal antibodies are being developed to reduce off-target effects and toxicity associated with broad-spectrum MMPIs [26]. The most common traditional strategy for designing MMPIs involves targeting the active site, the catalytic Zn2+ [6,27]. MMPIs were designed comprising a backbone structure and a zinc-binding group (ZBG) to bind the catalytic Zn2+ ion to inactivate the enzyme [6,28,29]. Therefore, several ZBGs have been employed, including the most common carboxylates and hydroxamates [6,30,31]. Recent proposals aim for the development of small-molecule inhibitors that can be targeted for selective suppression for the improvement of the selectivity and efficiency of the inhibitors [32,33]. Small-molecule inhibitors (SMIs) are low molecular weight compounds, designed to interact with specific and most probably single molecular targets, potentially disrupting their function [34]. SMIs are often suitable for oral administration and are widely utilized in diverse therapeutic applications, ranging from enzyme inhibition to receptor modulation [34]. The first-generation MMPIs comprising commonly hydroxamate ZBG block the Zn2+ ion, which is crucial for inhibitory efficacy [6,22,35,36]. However, these small-molecule MMPIs faced challenges like instability as they broke down into less useful chemicals, and they lacked selectivity, as they blocked several other important enzymes besides MMPs, resulting in side effects [6,19,35,36]. Advanced strategies for selective MMP inhibition target the S1‘ pocket [11,19,31,35–38] (a deep structural cleft near the Zn2+ ion in MMPs) at the catalytic site or allosteric inhibition (binds to regions outside the active site) disrupting enzyme shape and function [39,40]. Research continues to refine MMP-2 inhibitors for better efficacy, bioavailability, and pharmacological stability. These advancements are shaping the next generation of MMPIs for therapeutic use. The search to refine these small-molecule MMPIs continues owing to their pharmacokinetic stability and simplicity of administration. Therefore, in this article, the latest advancement strategies to design and develop potent MMP-2 inhibitors are explored elaborately. 2. Implication of MMP-2 in several diseases MMP-2 is a multi-functional enzyme integral to various physiological and pathological processes, including several disease conditions [6,10,19] as outlined in Figure 1. Here, in this article, some of these major disease conditions where MMP-2 plays a crucial role have been discussed. 2.1. MMP-2 in cancer MMP-2 has been implicated in several cancers through versatile mechanisms [19,21]. MMP-2 accelerates tumor metastasis by degrading key components of the ECM and basement membranes, enabling cancer cells to invade the neighboring tissues, which further enter the bloodstream and lead to extravasation into distant organs [35,41–44]. It specifically adds to capillary basal lamina degradation and facilitates tumor cell migration past the blood vessels. It also promotes angiogenesis by releasing pro-angiogenic factors like VEGF from the ECM, supporting tumor vascularization and growth [45,46]. In melanoma, MMP-2 increases VEGF-A secretion, enhancing vascular endothelial activation and promoting tumor cell adhesion and extravasation [45,46]. Additionally, MMP-2 aids immune evasion by modulating immune responses, such as restraining interleukin (IL) receptors on T-cells, thereby suppressing anti-tumor immunity [47]. Employing these mechanisms, MMP-2 makes a substantial impact on cancer progression, making it a critical target for anti-metastatic therapies. MMP-2 enhances cancer cell migration and promotes tumor cell survival by resisting apoptosis and supporting angiogenesis [48,49]. MMP-2 plays a pivotal role in the metastasis of cancerous cells by degrading type IV collagen [50]. Particularly, the tissue inhibitor of metalloproteinase-1 (TIMP1) and the tissue inhibitor of metalloproteinase-2 (TIMP2) suppress the activity of MMP, which prevents cancer cells from invasion and proliferation [51]. In the case of MMP-2, TIMP2 acts as an inhibitor that inhibits cell invasion and suppresses different types of cancer. However, the balance of MMP-2 and TIMP2 plays a very crucial role in patients’ survival [52]. Notably, patients exhibiting both MMP-2 (+) and TIMP2 (+) expression had better survival rate than patients with MMP-2 (+) and TIMP2 (-) expression. Interestingly, expression with MMP-2 (-) and TIMP2 (+) showed the most promising outcome [52]. MMP-2 inhibition is regulated by the Jak2/STAT3 pathway, S1P2 receptor, Rho-ROCK pathway, NF-κB-mediated epithelial–mesenchymal transition (EMT), TGF-β1 inhibition, and ERK1/2/JNK1/2 pathways [50]. MMP-2 overexpression can be triggered by ERK1/2 & Wnt-β-catenin pathways, CXCL-1 Chemokine, PI3K/AKT/mTOR pathway, AKT, and NF-κB pathways mediated by histone deacetylase 6 (HDAC6) [50]. Therefore, MMP-2 acts as both a biomarker and an end-stage player in cancer metastasis. MMP-2 is critical in epithelial–mesenchymal transition [53], which is regulated through genetic, epigenetic [54], and signaling pathways [50]. Therefore, it may increase cell plasticity and invasion [55]. MMP-2 influences both dormancy and reactivation of cancer cells. MMP-2 supports dormancy by releasing ECM fragments [56] (such as endostatin [57], arrestin [58], restin [59], and collagen IV chains [60]) that inhibit angiogenesis. Again, MMP-2 can activate the TGF-β isoforms, ending dormancy and promoting cancer cell proliferation [50]. Tumors directly produce MMP-2 that can trigger proteolysis of key immune receptors (e.g., TNF-R, IL-6 R, IL-2 R) and weaken immune responses [61,62]. 2.2. MMP-2 in cardiovascular diseases MMP-2 has been linked to various cardiovascular diseases as it exacerbates oxidative stress [63] and inflammation by the release of various pro-inflammatory cytokines [64]. Malfunctioning of MMP-2 contributes to excessive ECM degradation, leading to fibrosis, ventricular dilation, and impaired cardiac function [65]. The degradation subjected by MMP-2 on the elastin, collagen, and several other ECM components results in the weakening of the aortic wall, leading to the formation of aneurysms [66]. Several studies suggested that the TIMP-2 deficiency exacerbates the formation of aortic aneurysms [67]. ECM remodeling induced by MMP-2 can also lead to smooth muscle cell proliferation, endothelial cell detachment, and vascular remodeling that contributes to chronic thromboembolic pulmonary hypertension (CTEPH) [68,69]. MMP-2 contributes to destabilizing the atherosclerotic plaques by degrading arterial ECM [70], facilitating inflammatory cell infiltration, and reducing nitric oxide bioavailability [71], which may lead to endothelial dysfunction, plaque rupture, and vascular events like heart attacks or strokes [67,72]. Elevated MMP-2 levels disrupt vascular integrity, contribute to the breakdown of the blood–brain barrier (BBB), and promote leukocyte transmigration in ischemic stroke [73–75]. 2.3. MMP-2 in renal impairment Fibrosis, tubular cell necrosis, and amplification of the inflammatory state, especially in ischemia-reperfusion injury, are all a result of MMP-2 activity [70]. MMP-2 upregulation plays prime roles as an early marker of acute kidney injury [76–79]. It works by inducing recovery through tubular regeneration, but when inhibited, the kidney gets damaged persistently. Impaired MMP-2 accelerates ECM degradation, tubule-interstitial fibrosis, and glomerulosclerosis, which will entail a threat to renal function [80,81]. In the early stage of chronic kidney disease (CKD), MMP-2 has been found to assist in tissue repair, but its amplified activity exacerbates renal damage in the later stages, which results in glomerular basement membrane degradation. Regulation of MMP-2 activity could balance its protective and pathological effects, aiding in CKD management and prevention of disease progression [82–86]. 2.4. MMP-2 in diabetes mellitus Diabetes mellitus is a chronic autoimmune disorder caused by the demise of the insulin-producing pancreatic β cells, leading to absolute insulin deficiency and hyperglycemia [87]. MMP-2 aids in the development of Type I diabetes by involving several pathways, including JNK signaling, and the p53 pathway [88]. Again, MMP-2 plays a crucial role in platelet activation, which can aggravate diabetic vasculopathy [89]. Not only that, MMP-2 promotes cell migration and epidermal–mesenchymal transition, accentuating the diabetic liver disease [90]. Moreover, high glucose induces the expression of both full-length and truncated isoforms of MMP-2, which are involved in diabetic cardiomyopathy [91]. MMP-2 contributes to diabetic renal fibrosis followed by CKD. In Type I diabetes patients, MMP-2 is a novel marker for neurovascular complications and microangiopathies, but it is not reliable for early-stage fibrosis prediction in children [92,93]. Type II diabetes mellitus is distinguished by insulin resistance and a continuous decline in insulin production, accounting for 90% of diabetes cases [94,95]. In Type II diabetes mellitus, MMP-2 plays a pivotal role in inflammation, vascular damage, and genetic susceptibility. Elevated MMP-2 levels are associated with pro-inflammatory markers like ILs, which worsen the diabetic symptoms [96,97]. 2.5. MMP-2 in other diseases Regarding the nervous system, MMP-2 regulates synaptic plasticity, central nervous system development, and regeneration in post-injury scenarios [98–100]. It influences dendritic spine development, cell adhesion, and plays a role in neuro-inflammatory pathways. Regarding musculoskeletal health, MMP-2 supports bone remodeling, bone formation, and ECM degradation [101,102]. Mutations either in the MMP-2 gene or due to dysregulation lead to very severe bone abnormalities and pathological conditions [103]. MMP-2 assists in maintaining homeostasis in the reproductive system [104]. In females, it helps the cyclic endometrial changes that occur during the menstrual cycle, implantation, and so on [105]. For males, it assists in sperm count and sperm motility [105]. High levels of MMP-2 are associated with endometriosis [104]. MMP-2 participates in ECM remodeling in folliculogenesis and tissue repair during ovulation and parturition in the ovary [106]. 3. Structural overview of MMP-2 MMP isoforms contain these basic structures, like the Zn2+, signaling peptide, pro-peptide domain, and catalytic domain, but mainly differ based on the subgroups. However, the key differences of each MMP isoform have been represented in our earlier studies [11,107,108]. One major point of variation lies in the size of the S1‘ pocket, which influences substrate specificity and inhibitor binding. For instance, MMP-1 and MMP-7 possess a small pocket, MMP-2, MMP-8, MMP-9, MMP-12, and MMP-14 have a medium-sized pocket, and MMP-3, MMP-10, and MMP-13 have a large S1‘ pocket [19]. MMP-2 belongs to the gelatinase family, which also comprises MMP-9. In the 1970s, MMP-2 was identified as a crucial turning point in the research of MMPs [109]. MMP-2 was first sequenced in 1988 and named 72 kDa gelatinase A, which helps degrade both collagen and gelatin [110]. Later, Guy Salvesen and Hideaki Nagase successfully purified MMP-2 from human rheumatoid synovial fibroblasts [111]. MMP-2 helps break down various ECM components, including gelatine, laminin, fibronectin, elastin, and collagen IV [112], making it a key target in tissue repair and cancer treatment [113]. Besides its participation in key physiological functions like embryonic development, in vitro wound healing, and tissue regeneration. In the structure of MMP-2, there exists a pre-domain that facilitates proper enzyme secretion, followed by a pro-domain that helps in maintaining the latency by interacting with the catalytic Zn2+ ion [114]. It comprises a conserved cysteine residue that interrelates the catalytic Zn2+ ion, hindering unwarranted substrate cleavage. Activation of MMP-2 involves proteolytic removal or disruption of this domain, a mechanism often referred to as the “cysteine switch.” MMP-2 has a unique catalytic domain that houses the active site with a zinc ion coordinated by three histidine residues within the conserved sequence motif HExxHxxGxxH which is responsible for the proteolytic activity, along with a collagen-binding domain that includes three fibronectin type II modules (FN-II motifs) which bind to an extra 175 amino acids, aiding ECM degradation and remodeling [113,115]. It contains two hydrophobic S1‘ pockets and S1 pockets [113]. Alongside the catalytic domain, MMP-2 consists of the Zn2+-binding motifs with a conserved methionine residue near the motif that is essential for enzymatic activity [114,116]. Coupled with the Zn2+ ion that is essential for catalysis [113], MMPs also include a Ca2+ ion that aids the structural stability of the protein. Two Ca2+ ions are found to stabilize the catalytic domain, and one Ca2+ ion stabilizes the C-terminal domain as seen in the pro-MMP-2 structure [113]. MMP-2 primarily exists as an inactive pro-enzyme (proMMP-2) that requires activation, typically by other MMPs or enzymes like MMP-14 [13,117,118]. Its activity is strongly regulated by TIMPs, particularly TIMP-2, ensuring ECM homeostasis [119,120]. Its structure incorporates two zinc ions, each serving distinct roles. The presence of both zinc ions is crucial for the functions of MMP-2; the catalytic zinc directly participates in substrate cleavage, while the structural zinc maintains the enzyme’s architecture, enabling effective catalysis [29,121]. The catalytic Zn2+ ion is situated in the MMP active site that is coordinated by three histidine residues from the conserved HExxHxxGxxH motif. The Zn2+ ion is essential for the enzyme’s proteolytic activity, facilitating the hydrolysis of peptide bonds in substrate proteins [29,121]. However, the structural zinc ion is located in a site distinct from the catalytic center that is coordinated by four cysteine residues. This Zn2+ ion facilitates the structural integrity and stability of the enzyme, maintaining its conformation and ensuring proper domain interactions [29,121]. MMPs also contain a hemopexin/vitronectin-like domain, which is responsible for substrate binding and activation. This domain is connected by a hinge region, which provides structural flexibility and connectivity. However, they are absent in some MMPs like MMP-7, MMP-23, and MMP-26 [122]. This domain facilitates interactions with TIMPs and supports dimerization and substrate binding [123]. The crystallographic insights state that the structures of pro-MMP-2 with TIMP-2 provide insights into inhibitor binding and serve as templates for designing novel inhibitors [115–124]. Various domains of the MMP-2 enzyme, as well as the surface contour of the MMP-2 catalytic domain, including the alignment of several MMPs as well as the active site of MMP-2 with co-crystallized inhibitor obtained from the RCSB Protein Data Bank (RCSB PDB) [https://www.rcsb.org/], including the key amino acid residues, are represented in Figure 2. From the Figure 2(C), it is visible that the catalytic site of these MMP isoforms including MMP-2 is limited by the S1 loop, the β-IV sheet, and the α-II helix while three histidine residues (i.e., His120, His124, and His130) holding the catalytic Zn2+ ion in a proximity of the catalytic Glu121 residue and the tunnel-like hydrophobic S1‘ pocket (Figure 2(C,D)). Additionally, the presence of the Ω-loop at the opening of the S1‘ pocket also acts as a gate for the S1’ pocket (Figure 2(C,D)) [19]. Regarding the S1‘ pocket of these MMP isoforms, it is observed that the sidechain of the S1 loop-forming residues contributes significantly to the binding of the ligands inside the S1‘ pocket, therefore providing selective ligand binding for isoform selectivity [19]. Interestingly, depending upon the sidechain of S1 loop residues, these MMP isoforms can be clustered into (a) small/shallow S1‘ pocket containing MMPs (MMP-1 and MMP-7), (b) medium/intermediated S1‘ pocket containing MMPs (MMP-2, MMP-8, MMP-9, and MMP-12) as well as (c) the deep/large S1‘ pocket containing MMPs (MMP-3, MMP-10, and MMP-13) [19,21]. The comparison of the NMR solution structure of MMP-2 (PDB ID 1HOV) with crystallographic data of MMP-2 (PDB IDs 1CK7 and 1QIB) has underscored the significance of Arg250 and Phe249 with n-pentyl side chain that are essential for active site binding [19]. Also, the changing orientation of the Ω-loop and residues at the Loop 5 (L5)-βIV junction can limit or broaden the entrance of the S1‘ pocket, affecting ligand binding and complex formation stability due to the presence of residues with variable sidechains. Presence of such variable residues (Ile141 for MMP-2, Asn218 for MMP-8, Met422 for MMP-9, Thr239 for MMP-12, Ser239 for MMP-1, and Ile218 for MMP-13, Figure 3) can affect the orientation of the Ω-loop, particularly the conformation of proline residues in these MMP isoforms, thereby affecting the isoform selectivity for the ligands. It has also been suggested that the replacement of specific amino acids, i.e., Phe431-Arg432 from MMP-2‘s Ω-loop with other amino acids such as Pro429-Pro430 in MMP-9, can decrease its Ω-loop flexibility, significantly affecting the ligand selectivity toward both MMP-2 and MMP-9 [19]. Furthermore, interaction between the variable Ω-loop amino acids (Phe431−Arg432 for MMP-2, Pro429−Pro430 for MMP-9) and the ligands/molecules can also lead to specific inhibition of MMP-2 or MMP-9 [19]. Apart from the S1‘ pocket, the solvent-exposed S2‘ pocket can also contribute to isoform selectivity of molecules/ligands against MMP-2, where the His85 and Tyr74 can play crucial roles for stable ligand binding for MMP-2 selectivity [19,21]. Nevertheless, from the juxtapositional analysis of the MMP-2 active site with other MMP isoforms, it is visible that not only the active site amino acid residues but also the size of their side chain residues, their interaction with the other adjacent amino acid residues and the functional groups of the ligands/molecules, van der Waals and inductive interactions of the ligand with the active site and terminal S1‘ pocket residues during dynamic conditions can affect the orientation, size, volume, and flexibility of the active site, therefore governing the binding of that particular molecule to pertain MMP-2 selectivity. 4. Evolution and current status of MMP inhibitors Various promising MMPIs have been evaluated in detail in various phases of clinical trials to fight against numerous cancers (Figure 4). However, all of them failed due to poor bioavailability, nonspecific broad-spectrum MMP inhibition, dose-related toxicities, variations among preclinical animal models and human clinical trials, along with the limitation of these MMPIs to combat advanced stages of cancers [6,11,19]. A number of such MMPIs were restricted due to dose-dependent toxicities, especially MSS characterized by joint stiffness, tendinitis, pain, and inflammation [19]. Batimastat is a synthetic hydroxamic acid-based pseudo-peptide having broad-spectrum MMP inhibition and was the first MMPI molecule to enter phase I clinical trials. Preclinical studies showed promising results in slowing tumor growth and metastasis in murine models in the 1990s [125]. In early 1991, the first clinical trial was commenced, which paved the way to 1993 phase II clinical trials that were carried out with seven malignant ascites patients receiving intraperitoneal doses [126]. However, the phase III trials were discontinued due to poor patient compliance with intraperitoneal/intrapleural administration and side effects like slow drug accumulation and peritoneal irritation [127]. Marimastat is structurally analogous to Batimastat with a similar broad-spectrum inhibitory profile. Unlike batimastat, marimastat could be administered orally, improving patient compliance. It entered into clinical trials in the mid-1990s, was tested in patients with metastatic solid tumors, and demonstrated tumor marker reduction [128]. However, it failed in phase III clinical trials due to significant MSS, causing joint stiffness and inflammation [129]. Ilomastat is a hydroxamic acid-based zinc-binding inhibitor that forms a bidentate complex at the active site, and it cannot be administered orally. As one of the potent MMP inhibitors, it had good biological activity against cancer and other diseases, but it also presented several limitations. Notably, hydroxamic acid might chelate with other subsidies containing zinc ions and some oxidative state metals like iron III due to its nonselective, broad-spectrum inhibitory activity [130]. In the early 2000s, in phase II/III clinical trials, it was investigated for treating corneal ulcers but was discontinued due to poor bioavailability [131]. Prinomastat, also known as AG-3340, is a novel class of comparatively selective and powerful MMPI . Agouron Pharmaceuticals, Inc., a subsidiary of Pfizer Company, La Jolla, California, USA, developed it. Parinomostat inhibits MMP-2/3/9/13 while sparing MMP-1 to avoid MSS-related toxicities and presents good oral pharmacokinetics, antitumor, and anti-angiogenic effects [132]. The Phase II clinical trial was terminated due to unexpected thromboembolic events and MSS toxicities in esophageal adenocarcinoma patients [133]. Combination therapy with gemcitabine and cisplatin in non-small cell lung cancer (NSCLC) was terminated due to MSS and lack of survival benefits in its phase III clinical trial [134]. Bayer designed a weaker Zn2+-binding butanoic acid analog, tanomastat, to reduce toxicity [135]. In the preclinical trials, it demonstrated anti-angiogenic activity in colon cancer, breast cancer, and melanoma models [136]. In phase I clinical trials, it was well tolerated, with no MSS-related toxicities, and was orally bioavailable [137]. Phase III clinical trials were discontinued due to faster disease progression in tanomastat-treated patients with small-cell lung cancer (SCLC). In the case of non-small cell lung cancer (NSCLC), there was an improvement in time to disease progression, but no overall survival benefit was noticed [138]. In pancreatic cancer, it was less effective compared to gemcitabine, and its failure to provide expected results triggered a shift in focus from MSS avoidance to improving the efficacy of the MMP inhibitor [139]. Bristol-Myers-Squibb (BMS) developed a broad-spectrum MMPI Rebimastat that spares MMP-mediated shedding [140]. Rebimastat has a marcaptoacyl moiety for catalytic zinc chelation. In the phase I clinical trial, a rodent model, no dose-limiting toxicities were seen at doses of 900, 1800, or 2400 mg/day, whereas the phase II clinical trials reported MSS-related toxicities, including arthralgia and myalgia, along with skin rash, fatigue, nausea, headache, and taste alterations [141,142]. Novartis developed the first sulfonamide-based broad-spectrum MMP inhibitor CGS-27023A (MMI-270) [143]. It is a hydroxamic acid molecule chelated to the Zn2+ and has a bulkier moiety that binds with the S2‘ pocket in the enzyme surface and is believed to increase the specificity [144]. CGS-27023A entered into clinical trials for osteoarthritis, solid tumors, and colorectal cancer but was immediately withdrawn because of extreme MSS and limited activity as seen in the case of Marimastat and Batimastat [145]. Doxycycline was FDA-approved for periodontitis, and it inhibits MMP-2/7/8/9 [146,147]. This is a chemically modified tetracycline (CMT) that can suppress MMP activity by blocking the production of the enzymes by inhibiting gene transcription, neutralizing reactive oxygen species, or binding metal ions such as Zn2+ [148]. Minocycline is the most potent MMPI among tetracyclines that inhibits MMP-2/9 with preclinical success in melanoma, lung, breast, and prostate cancer models [149,150]. Another CMT is COL-3 (Metastat) that entered into Phase I/II clinical trials and helped stabilize the refractory metastatic cancer, but did not improve survival [151,152]. However, reports have suggested its responsiveness in AIDS-related Kaposi’s sarcoma [142]. It is challenging to design and discover selective inhibitors of specific MMP isoforms. However, this can be a promising target for combating several diseases. Therefore, a number of clinical studies were conducted over the last five years with various MMPs. The recent outcomes of clinical trials with various MMPIs [153] are listed in Table 1. Table 1. | Clinical Trial No | MMP Inhibitor | Trial phase | Disease conditions | Status | Year | |---|---|---|---|---|---| | NCT06477185 | Doxycycline | Phase II | Tuberculosis-associated lung fibrosis and associated lung function decline | Ongoing | 2025 | | NCT03508232 | Doxycycline Hyclate | Phase II | Protection of the heart muscle after a heart attack | Ongoing | 2025 | | NCT06201793 | Minocycline and Mesalazine | Phase II | Ulcerative colitis (UC) | Ongoing | 2025 | | NCT00003010 | Marimastat | Phase III | Breast cancer | Ongoing | 2023 | | NCT04876638 | Minocycline | Phase II | Aneurysmal subarachnoid hemorrhage | Ongoing | 2023 | | NCT03194204 | Doxycycline | ___ | Rheumatoid arthritis | Completed | 2020 | | NCT01375491 | Doxycycline | Phase IV | Type II diabetes and obesity | Completed | 2020 | | NCT01469585 | Doxycycline | ___ | Irregular bleeding in women were experienced when taking continuous oral contraceptive pills | Completed | 2020 | | NCT00003011 | Marimastat | Phase III | Small cell lung cancer | Completed | 2020 | | NCT00006229 | Rebimastat Carboplatin Paclitaxel | Phase II and Phase III | Non small cell lung cancer | Completed | 2020 | 4.1. MMP-2 inhibitors bearing hydroxamic acid moiety 4.1.1. Hydroxamate derivatives of N-arylsulfonyl amino acids Lee et al. [154] designed and synthesized 40 hydroxamate derivatives by N-arylsulfonylation of amino acids with valine, leucine, and phenylalanine, as well as cyclic amino acids like proline and pipecolic acid. In vitro MMP inhibition assay showed stronger and more selective MMP-2 inhibition (IC50 500 nM). Several compounds displayed selective MMP-2 inhibition over MMP-1. The molecular docking study revealed that compound 1exhibited a higher binding affinity for MMP-2 (−9.86 kcal/mol) compared to MMP-1 (−8.37 kcal/mol), suggesting that longer, bulky, and hydrophobic groups such as biphenyl are preferable for fitting into the S1‘ pocket of MMP-2, responsible for MMP-2 inhibitory potency and selectivity. The SAR analysis pointed out that steric and hydrophobic features were crucial for imparting MMP-2 inhibition. Excessive bulky structures associated with the sulfonamide moiety (such as 4-tert-butylphenyl, 2,4,6-trimethylphenyl, and 4-adamantylphenyl) exerted less effective MMP-2 inhibition due to improper fitting into the narrow S1’ pocket, whereas longer and more flexible structures like N-4-butoxyphenyl explored strong selectivity toward MMP-2 inhibition, presumably due to proper fitting into the MMP-2 S1’ pocket. On the other hand, bulky amino acids (namely phenylalanine and valine) improved MMP-2 selectivity. Molecules like compounds 1–3 (Supplementary Figure S1) resulted in a significant decrease in gelatinase inhibition comparable to the reference MMP-2 inhibitor ARP-101, as evidenced by zymogen assay and gelatine degradation assay. However, the in vitro wound healing assay suggested that compounds 2 and 3 had weaker cell migration effects compared to ARP-101, but compound 1 did not show any effect at a 5 µM dose. It further suggested that these compounds can be suitable for cosmetic preparations targeting anti-photoaging through preservation of skin repair. Nevertheless, the RT-qPCR analysis demonstrated that these compounds had no significant effect on the alteration of MMP-2 mRNA levels, revealing that these inhibitors directly target enzymatic activity without altering gene expression. This post-transcriptional mechanism may offer the advantage of maintaining skin homeostasis through MMP-2 inhibition without interfering with cellular regulatory mechanisms. 4.1.2. P-tosyl glutamine derivatives with hydroxamate moiety Das and coworkers [155] recently reported a set of p-tosyl glutamine derivatives bearing either a carboxylic acid or hydroxamate moiety. All these compounds exhibited comparatively better cytotoxic efficacy against the chronic myeloid leukemia (CML) cell line K562 over other solid tumor cell lines such as A549, U87-MG, and HT1080, but were nontoxic toward the normal cell line HEK-293. The most cytotoxic molecules (compounds 4–6, Supplementary Figure S1) were evaluated against MMPs comprising medium-sized S1’ pockets (i.e., MMP-2, MMP-9, MMP-8, and MMP-12). All of them exerted potent MMP-2 inhibition and showed some selectivity over MMP-8 and MMP-12. The cell cycle analysis revealed that compounds 4 and 6 yielded 9.5% and 6.9% cells, respectively, indicating the cell cycle arrest in the sub-G1 phase in the K562 cell line. Nevertheless, the apoptosis assay revealed that compounds 4 and 6 induced 34.7% and 23.4% apoptosis in the K562 cell line. Not only that, compound 5produced a time-dependent apoptosis in the K562 cell line as evidenced by Acridine Orange/Ethidium bromide (AO/EB) staining. Both these molecules (compounds 4 and 6) exerted significant antiangiogenic activity in the ACHN cell line. Both compounds 4 and 6 reduced 25.20% and 28.2% MMP-2 expression, respectively, in the K562 cell line. The MD simulation study revealed stable binding interactions between compound 4 and MMP-2 (PDB: 1HOV) through strong bidentate chelation between the hydroxamate moiety and catalytic Zn2+ ion, as well as several hydrogen bonding and water-mediated hydrogen bonding interactions involving amino acid residues, namely Gly81, Glu121, Asp80, Ala139, and Pro140. 4.1.3. Biphenylsulfonyl glutamine derivatives with hydroxamate moiety Recently, Baidya and coworkers [156] reported some biphenylsulfonyl glutamine derivatives comprising a hydroxamate moiety. All these compounds resulted in potent antileukemic efficacy in the K562 cell line with some selectivity over solid tumor cell lines A549, U87-MG, and HT1080, as well as nontoxic behavior toward normal cell line HEK-293. Among these compounds, the most promising antileukemic agents (compounds 7–10, Supplementary Figure S3) exerted potent MMP-2 inhibition with some selectivity over MMP-9 and MMP-8. Importantly, compounds 8 and 9 revealed a reduction of 21.3% and 17.8% MMP-2 expression, respectively, in the K562 cell line. In the K562 cell line, compounds 8 and 9 resulted in an induction of 45.4% and 39.8% apoptosis, respectively. Both these compounds were also found to arrest the cell cycle and exhibited significant antiangiogenic potential. The MD simulation study revealed that compound 8 exhibited strong bidentate chelation with the catalytic Zn2+ ion with its hydroxamate moiety at the MMP-2 active site (PDB: 1HOV). Nevertheless, the terminal phenyl moiety of the biphenyl scaffold produced π-π interactions with both Tyr142 and His120. Nevertheless, water-mediated hydrogen bonding with amino acids Pro140 and Glu121 also played a significant role in the stable binding interaction. A further study by Baidya and coworkers [157] revealed some biphenylsulfonyl derivatives bearing D(-)-glutamic acid moiety. All these compounds exerted potent antileukemic efficacy while tested in the K562 cell line, with cytotoxicity conducted in other solid tumor cell lines. The most potent molecules (compounds 11–14, Supplementary Figure S4) were subjected to the MMP inhibitory assay, and it was noticed that all of them exhibited potent and selective MMP-2 inhibition over MMP-9 and MMP-8. Compounds 11 and 12 reduced 15.8% and 18.4% MMP-2 expression, respectively, in the K562 cell line. Both of them also significantly induced apoptosis (35.1% by compound 11 and 41.6% by compound 12) with significant cell cycle arrest in the K562 cell line, as well as promising antiangiogenic efficacy in the ACHN cell line. Both these compounds also resulted in a significant decrease in cell viability as evidenced by the Live/dead cell assay. The MD simulation study explored that compound 12 produced a stable binding interaction through strong bidentate Zn2+ chelation with the hydroxamate moiety as well as π-π interactions with Tyr142 and His120 at the MMP-2 active site (PDB: 1HOV). Not only that, direct hydrogen bonding and water-mediated hydrogen bonding with amino acids, namely Leu83, Ala84, Pro140, His85, and Ala86, also played effective roles in stronger and stable binding between compound 12 and MMP-2 (PDB: 1HOV). The density functional theory (DFT) analysis further validated the observations of the MD simulation study. 4.2. MMP-2 inhibitors bearing carboxylic acid moiety 4.2.1. N-arylsulfonamido D-valine derivatives The study conducted by Konken et al. [158] analyzed 18 N-substituted N-arylsulfonamido D-valine derivatives and evaluated them for MMP inhibition. All these compounds were found to be more effective gelatinase (MMP-2 and MMP-9) inhibitors than other MMPs evaluated. Not only that, this study further suggested that the incorporation of a fluoroalkyltriazole group at the terminal thiophene’s methyl group did not drastically reduce the inhibitor potency, but enhanced the selectivity toward MMP-2. Therefore, this may be suitable for F-18 labeling using a prosthetic group approach and subsequent application for molecular imaging of MMP-2 activity by PET. The presence of a mono-fluorinated substituent led to additional interaction with the S2‘ pocket and showed higher potency. Long alkyl chains on the valine nitrogen presented a decrease in inhibitory effect, while the dodecyl or TEG groups with azide helped restore the activity. Compound 15 (fluorescein-labeled) showed strong MMP-2 inhibition, making it a promising fluorescence imaging tool (Supplementary Figure S5). Compound 15 selectively binds to gelatinases in inflammatory cuffs in a neuroinflammation model and pancreatic β-cells with a reduction in MMP-2 and MMP-9 activity. 4.2.2. Glutamine-based MMP-2 inhibitors Das et al. [159] reported some p-tosyl glutamic acid derivatives as effective antiproliferative agents (compounds 16–20, Supplementary Figure S6) evaluated against chronic myeloid leukemia cell line K562, NSCLC cell line A549, glioblastoma cell line U-87 MG, and fibrosarcoma cell line HT1080. Importantly, all these compounds were found to be effective against the K562 cell line over other cell lines tested. Not only that, all these molecules exerted ineffectiveness against normal human embryonic kidney cells HEK-293, indicating nontoxicity toward HEK-293 and selectivity toward CML. The most effective antileukemic agents were evaluated against MMPs comprising a medium-sized S1‘ pocket (i.e., MMP-2, MMP-9, MMP-8, and MMP-12). All these molecules exhibited better efficacy toward MMP-2 inhibition compared to other MMPs tested. Among these compounds, the most effective compounds 17 and 20 resulted in sub-G1 cell cycle arrest, respectively, in the K562 cell line. The apoptosis assay revealed that compounds 17 and 20 induced 24.1% and 37.1% apoptosis, respectively, in the K562 cell line. Both of these molecules responded to the antiangiogenic activity performed in the ACHN cell line. Nevertheless, compounds 17 and 20 reduced 19.6% and 21.1% MMP-2 expression levels, respectively, in the K562 cell line. The molecular dynamics (MD) simulation study revealed that compound 20 formed a stable binding interaction at the MMP-2 active site (PDB: 1HOV) through bidentate chelation with the carboxylic acid moiety and Zn2+ ion. Apart from that, the phenyl ring formed a π-π stacking interaction with Tyr142 and His120. 4.3. Triazine dendrimers with hydroxamate and carboxylic acid ZBG Haiba et al. [160] developed some S-triazine-based dendrimers. All the dendrimers (compounds 21–24, Supplementary Figure S7) resulted in good MMP-2 inhibitory efficacy with good antiproliferation against the cancer cell lines MDA-MB-231 and Caco-2. The flow cytometric analysis revealed that compounds 21 and 23 yielded more than 60% apoptotic induction in both these cell lines, better than the reference drug molecule, doxorubicin. The wound healing assay revealed that both these compounds exerted highly effective antimetastatic efficacy through inhibition of Caco-2 cell migration (83.9%). In another study, Ramadan et al. [161] synthesized some S-tryazine-based dendrimers to incorporate potential ZBG as well as carboxy terminal groups to facilitate direct conjugation with quercetin through biodegradable ester bonds. Both the dendrimers (i.e., 25 and 26, Supplementary Figure S8) resulted in promising MMP-2 and MMP-9 inhibition comparable to the reference drug NNGH. The PEGylated lactobionic acid targeted dendrimer quercetin conjugates (i.e., (TPhyG3-OH)-QUR-LA-PEG and (TPhyG4-OH)-QUR-LA-PEG) resulted in effective cytotoxicity against MCF-7 (IC50 = 18.809 µM and 12.690 µM, respectively) and HepG-2 (IC50 = 5.306 µM and 4.137 µM, respectively), better than the free drug quercetin. The flow cytometry analysis revealed that both the PEGylated dendrimers resulted in more than 74% apoptosis in the MCF-7 cell line and more than 55% apoptosis in the HepG-2 cell line. The SAR analysis revealed that dendrimer 26 was more promising compared to the respective dendrimer 25 and quercetin regarding anticancer efficacy, apoptotic induction, and VEGF gene expression, since dendrimer 26 causes excess quercetin and lactobionic acid molecules. 4.4. MMP-2 inhibitors bearing phosphonate moiety 4.4.1. Amino-phosphonate derivatives Huang et al. [162] reported the design and synthesis of 16 novel Combretastatin A-4 derivatives conjugated with aminophosphonates as dual inhibitors of tubulin and MMPs. All these compounds exhibited effective antiproliferative activity against various cancer cell lines, namely HepG-2, HT29, A549, MGC-803, and LO2. Among these compounds, compound 27 (Supplementary Figure S9) displayed the most cytotoxicity against all these cell lines. However, other compounds (such as compounds 28–30, Supplementary Figure S9) also provided excellent cytotoxicity against cell lines HepG-2, HT29, A549, and MGC-803, comparable to combretastatin. The SAR analysis revealed that substituents like methoxy, methyl, and fluoro at the meta and para positions of the phenyl ring enhanced antiproliferative activity. Moreover, increasing the carbon chain length adjacent to the ester function may decrease the antiproliferative efficacy. All these compounds (compounds 27–30) also produced moderate and nonselective MMP-2 inhibition with respect to MMP-3 and MMP-9. Compound 27 resulted in significant efficacy against cisplatin-resistant ovarian cancer cell line SK-OV-3/CDDP and doxorubicin-resistant breast cancer cell line MCF-7/DOX with an IC50 of 0.39 µM and 0.25 µM, respectively. Better than combretastatin, cisplatin, and doxorubicin. Furthermore, compound 27 induced effective apoptosis in the A549 cell line (25.9%) better than combretastatin (12.85%) as suggested by the Annexin V-FITC/PI double staining assay. Not only that, compound 27 significantly arrested the G2/M phase of the cell cycle (86.97% at 5 µM) in a dose-dependent manner in the A549 cell line. Moreover, a significant reduction of cell migration was noticed (about 12.1%) after the treatment of compound 27 in a dose-dependent manner, better than combretastatin (about 26.6%) while tested in the A549 cell line. Again, compound 27 produced a significant increase in intracellular ROS level (41.80% at 5 µM), better than combretastatin in the A549 cell line. Nevertheless, compound 27 also enhanced the accumulation of CHOP, p-eIF2a, and p-PERK in the A549 cell line, suggesting the induction of endoplasmic reticulum (ER) stress as evidenced by Western blot analysis. Moreover, compound 27 upregulated the expression of Bax and downregulated the expression of Bcl-2, as well as an increase in caspase-3 and p53 levels. Regarding the in vivo studies, compound 27 significantly inhibited tumor growth (73.2% at 30 mg/kg) in a dose-dependent manner as seen in the A549 xenograft model in BALB/c mice without any significant change in loss of weight and histopathological changes. 4.4.2. Chalcone-based phosphonate derivatives A number of Chalcone derivatives conjugated with aminophosphonates were designed and synthesized by Li et al. [163]. Among these compounds, compound 31 (Supplementary Figure S10) displayed the most effective antiproliferation against cancer cell lines, namely A549, NCI-H460, MCH-7, and MDA-MB-231. The SAR analysis revealed that incorporation of substituted groups, methoxy, methyl, fluoro, and nitro at the meta position of the phenyl ring may increase the antiproliferative efficacy. Compound 31 also produced moderate MMP-2 inhibition, nonselective over MMP-3 and MMP-9. Furthermore, the Western blot analysis revealed that compound 31 also yielded a significant decrease in MMP-2 inhibition, indicating excellent antimetastatic effects. Nevertheless, it also inhibited cell migration (about 45.1% at 0.5 µM) in the A549 cell line. Additionally, compound 31 yielded a significant dose-dependent inhibition in A549 cell migration as evidenced by the wound healing assay. Moreover, administration of compound 31 suppressed the expression of NF-κB and p-IKKβ, playing a pivotal role in pathogenesis in lung cancer in A549 cell line. Again, the Annexin V-FITC/PI assay disclosed that compound 31 produced a significant apoptosis in the A549 cell line (41.2% at a 0.5 µM dose). Further, compound 31 blocked G2/M cell cycle significantly in the A549 cell line (81.28% at 0.5 µM). Not only that, compound 31 significantly decreased expression levels of cyclin B1, Cdc2, and Cdc25c, the important cell cycle regulatory proteins, in the A549 cell line. Moreover, treatment with compound 31 also produced an increase in intracellular ROS levels and induced DNA damage as well as elevated levels of caspase-3 in the A549 cell line. In A549 xenograft in BALB/c nude mice, administration of compound 31 resulted in a significant inhibition of tumor growth (74.7% at 20 mg/kg) without any significant toxic effects on the major organs of mice. The molecular docking study revealed that compound 31 produced hydrogen bonding with Thr143, and the carbonyl oxygen atom was found closer to the catalytic Zn2+ ion (PDB ID: 1HOV). 4.5. Atypical MMP-2 inhibitors 4.5.1. Heterocycles bearing both silicon and phosphorus Some silicon and phosphorus-containing heterocyclic molecules (compounds 32–43, Supplementary Figure S11) as MMP-2 inhibitors were designed and synthesized by El-Hussieny et al. [164]. The thiolactam analog (compound 35) and the azaphosphetidin-4-one analog (compound 37) were the most effective MMP-2 inhibitors in this series, with greater efficacy compared to quercetin. Again, compound containing the oxazaphosphinan-2-one ring (compound 39) and azaphosphetidin-4-thione ring (compound 41) exerted comparatively better efficacy than quercetin. Again, the compound containing azaphosphetidin-4-one (compound 37) with a silicon group was found to be better than the respective compound 40 with no silicon. However, for the thiaphosphetanimine analog (compound 50) without any silicon was better compared to the respective analog with silicon (compound 54). Compounds 35 and 37 were docked into the active site of MMP-2 (PDB: 1HOV). The thioketo group of the thiolactam ring of compound 35 was found to coordinate the catalytic Zn2+ ion, whereas the phenyl rings interacted with Leu82 and His124 through hydrophobic interactions. On the other hand, the carbonyl group of the azaphosphetidin-4-one moiety of compound 37 coordinated to the Zn2+ ion. Moreover, the phenyl rings of compound 37 interacted with Leu42 and His130 through hydrophobic interactions. Not only that, the trimethylsilyl group of compound 37 was found to interact with the imidazole moiety of His120. 4.5.2. Isatin-thiazole conjugates Veeranna et al. [165] synthesized some isatin-linked thiazole hydrazonederivatives. The gelatin zymography assay depicted that three bromoindolinone derivatives with chloro (compound 44, Supplementary Figure S12), fluoro (compound 45, Supplementary Figure S12) and bromo (compound 46, Supplementary Figure S12) substitutions at the para position of terminal phenyl ring possess good MMP-2 inhibitory activity (>60% MMP-2 inhibition) comparatively better than MMP-9 inhibition (>10% MMP-9 inhibition). 4.5.3. Brominated-Ormeloxifene Sikander et al. [166] reported the synthesis and antiproliferative efficacy of Brominated-Ormeloxifene (Br-ORM) against cervical cancer. Br-ORM (compound 47, Supplementary Figure S13) was found to downregulate the expression of both MMP-2 and MMP-9, whereas it upregulated the expression of E-cadherin in CaSki and SiHA cervical cancer cell lines, as evidenced by Western blot analysis. 4.5.4. Resveratrol and related analogs It has been established that FABP5 (fatty acid binding protein 5) enables fatty acid transfer to the nucleus and subsequently activates the expression of downstream genes like MMP-2 and MMP-9 related to cell migration [167]. Chen et al. [168] showed that FABP5 expression significantly reduced the prognosis of cervical cancer patients with high MMP-2 expression through TCGA database analysis. Again, RT-PCR and Western blot analysis revealed that resveratrol (compound 48, Supplementary Figure S13) inhibited both MMP-2 and MMP-9 expression in SiHA and HeLa cells in a concentration-dependent manner. Furthermore, it was found that resveratrol (compound 48) interacted with FABP5 to block fatty acid transfer into the nucleus and subsequently inhibited the expression of both MMP-2 and MMP-9 to inhibit SiHa cell migration. 4.6. Natural inhibitors of MMP-2 4.6.1. Theaflavins Guo et al. [169] performed the MMP-2 inhibitory activity of some theaflavins (49–52, Supplementary Figure S14); all of them exhibited moderate MMP-2 inhibition and binding affinity in a competitive manner. Among them, compound 49 was the most effective MMP-2 inhibitor (IC50 = 6.50 µM), comparable to epigallocatechin gallate (IC50 = 6 µM) but better than caffeic acid (IC50 = 26 µM). Other theaflavins, i.e., compounds 50–52, exerted less efficacy compared to compound 49. The enzyme kinetics study also yielded that compound 49 had the best MMP-2 binding affinity (Ki = 9.20 µM) compared to others. The molecular docking study revealed that these theaflavins had binding energies of −11.45 kcal/mol, −10.39 kcal/mol, −10.52 kcal/mol, and −8.09 kcal/mol, respectively, while binding at the active site of MMP-2 (PDB: 3AYU). The interacting region was found to comprise 18 amino acid residues, namely Pro74, Gly80, Leu81, Leu82, Ala83, His84, Ala85, Tyr112, Leu116, Val117, His120, His130, Ala136, Leu137, Ala139, Pro140, Ile141, and Tyr142. Compound 49 was found to form 11 hydrophobic interactions with amino acid residues (namely Leu82, Tyr112, Val117, His120, Leu137, Ile141, and Tyr142) and 10 hydrogen bonds with amino acid residues Gly80, Leu82, Ala83, and Leu116. Again, Compound 50 formed four hydrophobic interactions with Leu81, Leu82, His120, and Tyr142, along with eight hydrogen bonds at the active site. Nevertheless, Compound 50 also formed π-staking and salt-bridge interactions with His130 and His120, respectively. On the other hand, Compound 51 formed hydrophobic interactions with Leu81, Leu82, His84, and His120 as well as hydrogen bonding with Gly80, Leu82, Ala85, and Ala139, along with π-staking interaction with His120. Similarly, Compound 52 produced hydrophobic interactions with Leu81, Leu82, and hydrogen bonding with Pro74, Leu82, Ala83, His84, as well as π-staking and salt-bridge interaction with His120. The MD simulation study revealed the formation of a stable complex between these theaflavins and MMP-2. 4.6.2. Curcumin In a study conducted by Zareh et al. [170], it was noticed that the expression of both MMP-2 and MMP-9 genes enhanced 5-fold and 3.3-fold, respectively, after the 2-chloroethyl ethyl sulfide (CEES) exposure. Importantly, it was noticed that treatment with curcumin and doxycycline reduced MMP-2 expression markedly. Curcumin/compound 53 (Supplementary Figure S15) treatment reduced MMP-2 activity by about 32% whereas MMP-9 activity was reduced by almost 56%. However, doxycycline reduced MMP-2 activity by 12% and MMP-9 by 40%. Curcumin showed a much better ability to reduce MMP-2 and MMP-9 expression compared to doxycycline, but the difference was statistically significant only in the case of MMP-9. Giliberti et al. [171] investigated the synergistic effects of curcumin and methotrexate on osteosarcoma cells. The combination of a 1 µM dose of Curcumin and a 2.5 µM dose of methotrexate produced a good reduction of MMP-2 expression in MG-63 osteosarcoma cells. However, the reduction of MMP-9 expression was found to be better at the same dose. 4.6.3. Eriocitrin Baek et al. [172] reported that the expression levels of both MMP-2 and MMP-9 were reduced at various concentrations of eriocitrin/compound 54 (25–100 µM) (Supplementary Figure S15), and this further suppressed the activity of vascular endothelial cells and impaired their motility in the HUVEC cell line. 4.6.4. Amentoflavone Jo and coworkers [173] screened a library of 64 flavonoids to identify effective MMP-2 inhibitors. Among these compounds, amentoflavone (compound 55, Supplementary Figure S15) was the most effective MMP-2 inhibitor with an IC50 of 6.989 µM. The induced-fit docking study of amentoflavone and MMP-2 produced a good docking score of −9.323 kcal/mol. Amentoflavone was surrounded by hydrophobic amino acids namely Leu163, Leu164, Tyr193, Val198, Leu197, Tyr223, Ile222, Tyr228, Leu218, and Phe232, two hydrogen bonds were found between 4‘-OH group and Leu164 and Ala165. Apart from that, 4‘-OH also made hydrogen bonding with Glu202, a key residue for MMP-2 catalytic activity. Again, 4‘‘‘-OH also formed hydrogen bonding with Tyr193, whereas the 7‘‘-OH group coordinated with the catalytic Zn2+ ion. 4.6.5. Vitamin B Vo et al. [174] reviewed that vitamin Bs have a direct and indirect impact on MMP-2 activity in different disease conditions. Riboflavin was found to inhibit the mRNA expression of MMP-2, followed by a reduction of ROS, ERK activation, and the mitigation of inflammatory signals in abdominal aortic aneurysms. Thiamine was found to reduce MMP-2 activity in diabetic hearts and prevent diabetes-induced cardiac fibrosis. Vitamin B6 was found to reduce MMP-2 concentration in animal models. Similarly, folic acid also alleviated chronic spinal cord injury-induced neuropathic pain via suppressing MMP-2 expression. Not only that, most of the vitamin Bs (such as Thiamine, Riboflavin, Niacin, Pantothenic acid, Pyridoxin, Folic acid, Cyanocobalamin) were found to exert a reduction of MMP-2 inhibition to exert anticancer efficacy against various cancers (like Breast cancer, Colorectal cancer, Skin cancer, Liver cancer, Lung cancer, etc.) 4.7. Miscellaneous 4.7.1. Peptide-based nanoparticles Zhao et al. [175] developed a truncated sequence of low-molecular-weight peptide CLP002 (TR3)-based nanoparticles (TR3-M-NPs) by modification with a PEG chain through an MMP-2 specific cleavage linker. It was also noticed that TR3-M-NPs were specifically cleaved to release the active TR3-LAG peptide inside the MMP-2-rich tumor microenvironment. When the nanoparticle reached the tumor microenvironment, the TR3 peptide was activated by MMP-2-specific cleavage and further blocked PD-L1 on the cancer cell surface. 4.7.2. MMP-responsive melanin nanoparticles Fan and coworkers [176] designed some MMP-responsive melanin nanoparticles by modifying water-insoluble ultrasmall melanin nanoparticles (MNP) with detachable PEG via an MMP-2-cleavable peptide linker. The enzyme-sensitive property of such nanoparticles was evaluated by hydrodynamic diameters and TEM analysis with active MMP-2. The DLS data also revealed the MMP-2-responsive nature of such nanoparticles. They also showed that more MMP-2-activatable probes were kept in lesions due to the MMP-2 overexpression within the atherosclerotic plaque [176]. 5. Molecular modeling studies on MMP-2 inhibitors Raja et al. [177] reported the synthesis, characterization, molecular docking, and MD simulation study of two compounds, namely, (E)-N-cinnamoyl-4-methoxybenzamide (CMB)/compound 56 and (E)-3-chloro-N-cinnamoyl-4-methoxybenzamide (CCMB)/compound 57 (Supplementary Figure S16). The molecular docking study revealed that CMB and CCMB had a binding affinity of −8.50 kcal/mol and −8.35 kcal/mol, respectively, while binding to the MMP-2 active site (PDB: 1HOV). Both these compounds formed π-π interactions with His120 and π-cationic interactions with the Zn2+ ion. Apart from that, compound CMB showed non-bonded interaction with His85, Leu116, Glu121, His124, His130, Leu137, Ala139, and Ile141, whereas compound CCMB produced non-bonded interaction with Leu83, Ala84, Leu116, Val117, Glu121, Ala136, Leu137 and Tyr142 at the MMP-2 active site. The MD simulation study revealed that both these molecules exhibited a low RMSD (<0.43 nm), low RGy (<1.566 nm), a very low RMSF (<0.164 nm) and SASA (<92.156 nm2), suggesting strong and stable binding interactions at the MMP-2 active site. Jamshidi et al. [178] identified 12 antraquinones (Supplementary Figure S17), i.e., Pulmatin (compound 58), Emodin-8-glucoside (compound 59), Obtusifolin (compound 60), Rhodoptilometrin (compound 61), Aloe-emodin (compound 62), Chrysophanol (compound 63), Emodic acid (compound 64), Rhein (compound 65), Alizarin (compound 66), Danthron (compound 67), Hypericin (compound 68), and Purpurin (compound 69) as possible MMP-2 inhibitors better than the reference molecules periostat and captropril through molecular docking analysis. Among these compounds, Pulmatin (compound 58) exhibited the highest free binding energy of −12.91 kcal/mol, whereas the other three molecules (i.e., Emodin-8-glucoside, Obtusifolin, Rhodoptilometrin) exhibited the highest free binding energy of −11.80 kcal/mol, −11.62 kcal/mol, and −11.52 kcal/mol, respectively. However, the rest of the molecules had a free binding energy range of −10.64 kcal/mol to −10.28 kcal/mol. The best molecule, pulmatin, produced hydrogen bonding interactions with Thr227, Ala220, and Gly216, whereas it formed hydrophobic interactions with Val198 and His201, and Ala220. Rafey et al. [179] reported the molecular docking study of six phytoconstituents (Supplementary Figure S18) obtained from the bark of Symplocos racemosa with MMP-2 (PDB: 1QIB). The molecular docking study reavealed that the doking score of quercetin (compound 70) is the maximum, ie. −9 kcal/mol whereas, other molecules such as sympracemoside/compound 71 (docking score = −8.8 kcal/mol), symplocoside/compound 72 (docking score = −8.7 kcal/mol), betulinic acid/compound 73 (docking score = −7.4 kcal/mol), ellagic acid/compound 74 (docking score = −7.0 kcal/mol), resulted in good binding interaction at the MMP-2 active site. On the other hand, the lowest docking score was noticed for oleanolic acid/compound 75 (docking score = −6.1 kcal/mol). The best docked compounds produced a number of hydrogen bonds at the MMP-2 active site. Quercetin and sympracemoside made hydrogen bonds with His166, His211, His205, and Ala165; Symplocoside made hydrogen bonds with Pro215, Ile222, and Arg233. However, butilinic acid produced only two hydrogen bonds with Ala128 and Asp182, and oleanolic acid made only one hydrogen bond with Pro215, whereas ellagic acid did not produce any hydrogen bonding. Again, a number of hydrophobic interactions, van der Waals, and π-π interactions were noticed among these molecules and amino acid residues like His211, His201, His205, Ala165, Glu202, Val198, Pro221, Ile222, Tyr223, Gly162, and His166. Oselusi et al. [180] identified some potential MMP-2 inhibitors from Ehretia sp through computational drug discovery approaches for the treatment of chronic wounds. From a database of 74 phytoconstituents, 46 compounds were found to be effective inhibitors of at least one type of MMP. Among these phytoconstituents, four compounds (Supplementary Figure S19), i.e., Lithospermic acid/compound 76, rosmarinic acid/compound 77, *-octadecatrienoic acid/compound 78 and danshensu/compound 79 were found to be potent inhibitors of MMP-2 as evidenced by molecular docking scores of −14.99 kcal/mol, -11.90 kcal/mol, −10.31 kcal/mol and −10.08 kcal/mol, respectively while docked into the active site of MMP-2 (PDB: 1HOV). All these molecules formed a salt-bridge interaction with the catalytic Zn2+ ion of MMP-2. Lithospermic acid yielded a hydrogen bonding interaction with Asp72, Glu121, Pro140, and π-π stacking interaction with His120, as well as π-cation interaction with the Zn2+ ion. However, rosmarinic acid formed hydrogen bonding with Asp72 and Glu121 as well as π-cation interaction with Zn2+ ion. On the other hand, *-octadecatrienoic acid and danshensu formed only one hydrogen bonding interaction with Thr140 and Glu121, respectively. The MD simulation study revealed that all these compounds exhibited a low RMSD ( < 0.54 nm), low RGy ( < 1.57 nm), and a very low RMSF ( < 0.19 nm), suggesting strong and stable binding interactions with MMP-2. Among these compounds, rosmarinic acid and danshensu exhibited good physicochemical, pharmacokinetic, and drug-like properties. Gomaa et al. [181] reported some methyl 2-(3-(2-oxo-3-phenylquinoxalin-1(2 H)-yl)propanamido) alkanoate derivatives and evaluated their anticancer activity against colorectal cancer. Among these compounds, Compounds 80 and 81 (Supplementary Figure S20) resulted in good XP docking scores of −6.5 kcal/mol and −7.6 kcal/mol, respectively. The quinoxaline ring, along with a phenyl group inserted into the S1‘ pocket, whereas the terminal isopropyl (compound 80, Supplementary Figure S20) and benzyl (compound 81, Supplementary Figure S20) groups were located closer toward the S2 pocket. However, it is important to note that though these compounds contain ester and hydroxamate functions, these are not involved in zinc binding. Compound 81 fitted well at the MMP-2 active site (PDB ID: 8H78) to form a chelation with the catalytic Zn2+ ion. Both these compounds formed hydrogen bonding interactions with Ala84 and π-π stacking interactions with His121 and the phenyl ring associated with the quinoxaline ring. The most effective compound 81 (HCT-116 IC50 = 65.48 µM) was further subjected to an apoptotic assay, showing reduction of cell number and size with shrinkage, fragmentation, and a decrease in overall cell population, suggesting good apoptotic character of compound 81. Yang and coworkers [182] identified some possible MMP-2 inhibitors from a set of 4225 natural products of the ChEMBL database through advanced machine learning-based chemoinformatic approaches. The machine learning based methods (random Forest, support vector machine, artificial neural network, and stacking) produced statistically significant outcomes, and model scores greater than 0.7 resulted in 1058 screened hits from the zinc database. After ADMET screening, 17 molecules were found to possess good pharmacokinetic properties and were subjected to molecular docking and MD simulation analysis. Among these molecules, three compounds were finally selected as possible hit candidates against MMP-2. The final hit compounds, i.e., ZINC15858504, ZINC712249, and ZINC4270723, exhibited good binding affinity of −8.9 kcal/mol, −8.6 kcal/mol, and −8.4 kcal/mol, respectively, while binding to the MMP-2 active site. ZINC15858504 and ZINC712249 formed hydrogen bonding interactions with Leu83 and Ala84. However, ZINC712249 formed additional hydrogen bonding with Tyr142 as well as hydrophobic interactions with Leu82, Leu83, His120, Tyr142, and Ala84, along with π-staking with His120. Apart from that, for the last several years, a variety of ligand-based and structure-based molecular modeling studies on several MMP-2 inhibitors were conducted by several groups of researchers that may be effective in the design and discovery of novel small-molecule MMP-2 inhibitors. The results of those ligand-based and structure-based molecular modeling studies are listed in Table 2. Table 2. | Sl. No. | Scaffold/Compound | QSAR Techniques | Observation and Finding | Ref. | |---|---|---|---|---| | 1 | Diverse molecules | Bayesian classification, Recursive partitioning, and SARpy | Hydroxamate, carboxylic acid, biphenyl/phenoxy phenyl sulfonamide, and larger hydrophobic aryl groups were found to be very crucial for effective MMP-2 inhibition | [183] | | 2 | Pyrrolidine derivatives | CoMFA, CoMSIA, and, HQSAR | Steric and electrostatic groups were very important for MMP-2 inhibition; the methoxy group decreased the activity. | [184] | | 3 | Sulfonamide hydroxamate derivatives | Topomer CoMFA | The bulkier group with a positive charge at the 4th position of the pyrrole ring and a negatively charged substitution at the 2nd position of the pyrrole ring increased the MMP-2 inhibitory activity. | [185] | | 4 | Arylsulfonyl L-(+) glutamine derivatives | CoMFA and CoMSIA | Steric, electrostatic, and hydrophobic groups were found to be better for MMP-2 inhibition | [186] | | 5 | Alkyl/arylsulfonyl pyrrolidine derivatives | HQSAR, Bayesian classification, and CoMSIA | The hydroxamate group, p-Tosyl group, and sulfonyl pyrrolidine groups were important for the MMP-2 inhibition. | [187] | | 6 | Diverse molecules | Bayesian classification, SVM, and ANN | The arylsulfonamido hydrophobic function was one of the important structures favorable for MMP-2 inhibitory activity. | [188] | | 7 | Arylsulphonamide-based derivatives | S-MLR, SVM, ANN, Topomer CoMFA, and Bayesian classification | The ortho-carboxylate and the para-substitution by acetamido, amino, methoxy, and nitro substitutions at the phenyl ring are favorable for MMP-2 inhibition. | [189] | 6. Future perspectives MMPs are a superfamily of Zn2+-dependent metalloenzymes contributing to diverse pathophysiological and disease conditions. Their proteolytic activity on ECM substrates is also pivotal for numerous physiological conditions, including tissue remodeling and repair, homeostasis, and modulation of innate immunity. Though several MMPIs with extreme efficacy entered numerous phases of clinical trials, these were terminated or withdrawn due to various adverse toxicities, and none of them have been on the market. Still, these MMPs are promising druggable targets, and there is an unmet requirement for developing target-specific and selective MMPIs. However, homological similarities among various MMP structures make the development approaches extremely challenging. Among all MMP subtypes, MMP-2 (gelatinase A) is taken into consideration as a key biomolecular target due to its strong association with cancer progression, metastasis, and angiogenic events. Apart from that, MMP-2 modulates several major diseases and thus, it is an extremely crucial target for drug development against these disease conditions. However, there is still a large gap in identifying selective MMP-2 inhibitors to date as promising drug candidates in the market despite a long journey of the last five decades [6]. Over the past few decades, numerous efforts have been taken into consideration to develop potential MMP-2 inhibitors as anticancer agents, and numerous molecules (such as batimastat, marimastat, prinomastat, rabimastat, doxycycline, minocycline, etc.) have undergone various phases of clinical trials but failed to come into the market either due to poor bioavailability, dose-related toxicities, poor pharmacokinetic profiles or broad-spectrum nonselective type of inhibition with other MMP isoforms as well as variable outcomes among human and animal models along with failure to treat advanced stages of cancers. Still, MMP-2 remains a crucial target for the development of novel drug candidates, and thus, developing specific MMP-2 inhibitors is an urgent requirement, though it is really challenging and time-consuming [6]. Recently, some studies disclosed that MMP-2 can be a target for developing antileukemic drug candidates [6,19,155–157,159] which could be a breakthrough in this field of antileukemic therapy since it is noteworthy that most MMPIs were evaluated against solid tumors and none of them were tried against hematological cancers in clinical studies [6,11,19]. Apart from that, there is still a huge opportunity to design and develop specific MMP-2 inhibitors not only against cancers but also in other diseases, since most of these MMP-2 inhibitors were evaluated extensively for cancer treatment. This approach may aid a new avenue of novel, specific MMP-2 inhibitors in versatile disease conditions clinically, apart from cancer, in modern therapeutics. In this context, it may be well-apprehended that novel small-molecule MMP-2 inhibitors may be a valuable tool for achieving newer therapeutics not only in cancers but also in other life-threatening diseases after thorough clinical investigations. Therefore, considering the structural homology among various MMPs, some key aspects should be taken into consideration for developing novel and specific MMP-2 inhibitors. The P1‘ substituents should be selected in such a way that they could be specific for fitting into the S1’ pocket of MMP-2 without any interactions with other medium-sized or large-sized S1’ pocket containing MMPs. Again, ZBG groups may play a pivotal role in achieving selectivity. Apart from strong ZBGs (such as hydroxamates), comparatively moderate or weak ZBGs (namely carboxylic acid, phosphonates, etc.) may also be beneficial for binding differently to the catalytic Zn2+ ion that may be effective for achieving selectivity toward specific MMPs [6,19]. From this current study, the comparative structural analysis of MMP-2 catalytic site with the same of the other MMP isoforms have shown that there are a few key factors such as the shape and volume of the hydrophobic S1‘ pocket, presence of variable amino acids with different side chain residues at the active site, S1‘ pocket, and the Ω-loop, their proximity, intra-amino acid interactions, and interactions with the functional groups of the ligands can hugely affect the conformation of the active site (specifically the S1 and the Ω-loop) and therefore affecting/altering the isoform selectivity. Thus, during the design of potent and selective MMP-2 inhibitors (both zinc-binding and non-zinc binding molecules), the conformation, volume of the P1‘ substituents for the S1‘ pocket, the presence of an effective functional group for better interaction with active site residues of MMP-2 and other MMP isoforms should be considered apart from the Zn2+ chelation. Nevertheless, to avoid off-target side effects, non-zinc chelating, pocket-specific, and mechanism-based inhibitors can be an effective approach. Thus, MMP-2 inhibitors can be designed in such a way that they specifically interact with the conserved or variable Ω-loop and S1‘ pocket amino acids [19]. Nowadays, to reduce the time for drug designing, several in silico approaches (namely ligand-dependent and structure-dependent drug designing strategies) through various computational tools have been successfully utilized. Already, a number of such strategies have been carried out earlier. Further extensive investigations and careful observations of the binding mode of interactions between enzyme-ligand complexes might help in the design strategy of novel MMP-2-specific inhibitors. In this context, crystallographic data and NMR solution structures may also play pivotal roles in designing novel MMP-2 inhibitors [11,19]. Here, in this article, the development of recent small-molecule MMP-2 inhibitors comprising various ZBGs and diverse scaffolds, as well as their structural information along with their in-depth biological implications in cancers and other diseases, has been discussed in detail. These observations reinforce the importance of selective MMP-2 inhibition as a therapeutic approach, paving the way for future research into optimizing small-molecule MMP-2 inhibitors for clinical applications. Again, looking at the progress of MMP-2 inhibitors in recent years, several newer approaches like successful addition of drugs that inhibit MMP-2 and opting for nanoformulation (as discussed before), which might help to reduce toxicity and enhance the selectivity and bioavailability [176]. Furthermore, apart from the synthetic and peptidomimetic compound, the compounds from the natural sources like theaflavins, rosmarinic acid, *-octadecatrienoic acid, lithospermic acid, as well as curcumin showed potential MMP-2 inhibition. Therefore, application of drug repurposing from natural sources while applying in silico techniques, high throughput screening, and semi-synthetic approaches for selective MMP-2 inhibitor development can not only improve the potency but also reduce the MSS toxicity and other adverse effects. Besides, adaptation of newer approaches including nanocarrier-based targeted systems, Proteolysis Activating Chimera (PROTAC)-based targeting and ubiquitination of MMP-2 by utilizing known selective MMP-2 inhibitors can also surpass the toxicity, selectivity, and pharmacokinetic barriers associated with selective MMP-2 inhibitor development. In a nutshell, as the development of these MMP-2 inhibitors advances, further in vivo studies and structure-activity relationship optimizations will be essential to translate these promising results into viable therapeutic options for several cancers and other life-threatening diseases. Supplementary Material Acknowledgments SB is thankful to the Department of Medicinal Chemistry, School of Pharmacy, The Neotia University, for providing the necessary research facilities. NA is thankful to the authority of Jadavpur University for providing research facilities to conduct the research work. The authors are sincerely thankful to the Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India, for providing the necessary research facilities. The authors declare that they do not have any conflict of interest. No writing assistance was utilized in the production of this manuscript. Funding Statement This paper was not funded. Article highlights MMP-2 is a key biomolecular target associated with cancer progression, metastasis, and angiogenesis. Recent small-molecule MMP-2 inhibitors comprising various ZBGs and diverse scaffolds have been discussed. Recent molecular modeling approaches on MMP-2 inhibitors have been discussed. Novel small-molecule selective MMP-2 inhibitors can provide effective clinical outcomes for various life-threatening diseases including several cancers. Disclosure statement Peer reviewers on this manuscript have no relevant financial or other relationships to disclose. Writing assistance No writing assistance was utilized in the production of this manuscript Supplementary material Supplemental data for this article can be accessed online at https://doi.org/10.1080/17568919.2025.2570970

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