From Bench to Bedside: Transforming Cancer Therapy with Protease Inhibitors

Abstract

Proteases play a pivotal role in cancer progression, facilitating processes such as extracellular matrix degradation, angiogenesis, and metastasis. Consequently, protease inhibitors have emerged as promising therapeutic agents in oncology. This review provides a comprehensive overview of the mechanisms by which protease inhibitors modulate cancer biology, categorizing inhibitors by their target protease classes, including matrix metalloproteinases, cysteine proteases, and serine proteases. We discuss the therapeutic potential of both synthetic and natural protease inhibitors, highlighting their applications in preclinical and clinical settings. Furthermore, challenges such as specificity, toxicity, and resistance mechanisms are addressed, alongside strategies to overcome these limitations through innovative drug designs and combination therapies. The future of protease inhibitors in cancer treatment lies in precision medicine, leveraging proteomic profiling to tailor therapies to individual tumors. This review underscores the importance of ongoing research and the development of novel approaches to harness protease inhibitors effectively for cancer management.

1. Introduction

Cancer is a multifaceted disease characterized by uncontrolled cell proliferation, the evasion of apoptosis, sustained angiogenesis, and the ability to invade surrounding tissues and metastasize to distant organs [1]. These hallmarks of cancer are supported by intricate biochemical and molecular pathways, many of which involve proteases—enzymes that catalyze the breakdown of proteins by hydrolyzing peptide bonds [2]. Initially recognized for their role in protein turnover and extracellular matrix remodeling, proteases are now understood to be key regulators of several processes integral to cancer progression, including tumor invasion, angiogenesis, immune modulation, and the activation of pro-tumorigenic signaling pathways [3].

Proteases are classified into distinct families based on their catalytic mechanisms (Figure 1), including serine proteases, cysteine proteases, aspartic proteases, and metalloproteases [2,4]. These enzymes are often dysregulated in cancer, with their overexpression or aberrant activity contributing to tumor growth and metastasis [5]. For instance, matrix metalloproteinases (MMPs) degrade components of the extracellular matrix, creating a permissive environment for cancer cells to invade and metastasize [6]. Similarly, cysteine proteases such as cathepsins facilitate tumor invasion and modulate immune responses. These observations underscore the pivotal role of proteases in the tumor microenvironment and their potential as therapeutic targets.

Protease inhibitors, a diverse group of molecules that block protease activity, have garnered significant interest as anticancer agents; they can be classified based on their structure, origin, or mode of action and include small-molecule inhibitors, monoclonal antibodies, and endogenous protease inhibitors [7]. The therapeutic promise of protease inhibitors lies in their ability to selectively target pathological protease activity, thereby impeding processes such as tumor invasion, angiogenesis, and metastasis [8]. However, the clinical development of protease inhibitors has faced significant challenges, including off-target effects, toxicity, and the development of resistance mechanisms by cancer cells [9].

Protease inhibitors, once heralded as transformative cancer therapies, have faced considerable setbacks in clinical development due to challenges in balancing efficacy and safety [10]. MMP inhibitors exemplify these challenges. Designed to target MMPs critical for tumor invasion and metastasis, these inhibitors showed promise in preclinical studies; however, clinical trials revealed limited therapeutic efficacy in many cancers, as tumors adapted by activating compensatory pathways or utilizing non-MMP proteases for progression [11]. Furthermore, the broad-spectrum nature of many early MMP inhibitors led to significant off-target effects, including musculoskeletal toxicity, which manifested as joint stiffness and pain in patients, and this toxicity often required dose reductions, compromising the inhibitors’ therapeutic potential and ultimately resulting in the discontinuation of several candidates [12].

Another factor contributing to the failure of protease inhibitors in cancer therapy is the complexity of protease function in tumor biology. Proteases such as MMPs, urokinase plasminogen activators (uPAs), and cathepsins have dual roles in promoting and suppressing tumors, depending on the tumor microenvironment, and this functional duality made it challenging to predict patient responses and stratify those who might benefit from protease-targeted treatments [13]. In addition, early protease inhibitors were developed without a precise understanding of the protease profiles unique to different cancers, leading to suboptimal outcomes [14]. Advances in precision medicine and biomarker-driven approaches are now guiding the development of next-generation protease inhibitors, aiming to overcome the limitations that plagued earlier efforts [15]. Nonetheless, the history of protease inhibitors underscores the importance of specificity, biomarker identification, and a deeper understanding of protease networks in successful cancer therapy development.

This review provides a detailed examination of the role of proteases in cancer biology and the therapeutic potential of protease inhibitors. It explores the molecular mechanisms underlying protease activity in cancer, the design and classification of protease inhibitors, and their current status in clinical and preclinical research. Furthermore, this review addresses the limitations associated with protease inhibitors, including issues of selectivity and toxicity, and highlights emerging strategies to overcome these obstacles, such as the use of advanced drug delivery systems and combination therapies. Finally, the potential for precision medicine to tailor protease inhibitor therapies to individual tumor profiles is discussed, offering a roadmap for future advancements in this field.

2. Proteases in Cancer Biology

Proteases, also known as proteinases or peptidases, are enzymes responsible for the hydrolysis of peptide bonds in proteins, playing essential roles in a variety of physiological and pathological processes [16]. In cancer, these enzymes are key regulators of tumor development and progression, contributing to processes such as extracellular matrix (ECM) remodeling, angiogenesis, immune modulation, apoptosis evasion, and metastasis [5]. Dysregulated protease activity is a hallmark of many cancers, with elevated protease expression or activity often correlating with aggressive tumor behavior and poor prognosis [17].

Proteases are classified into major families based on their catalytic mechanisms and active site residues. Serine proteases, which utilize a serine residue for catalysis, are involved in tissue remodeling, the activation of growth factors, and ECM degradation [18]. Examples include urokinase plasminogen activator (uPA) [19] and kallikreins [20], both of which have well-established roles in cancer progression. Cysteine proteases, characterized by a cysteine residue at their active site, include enzymes such as cathepsins which are implicated in tumor invasion, angiogenesis, and immune modulation, with cathepsins B and L frequently overexpressed in various malignancies [21]. Metalloproteases, which depend on a metal ion such as zinc for activity, represent another key protease family in cancer which includes matrix metalloproteinases (MMPs), the most studied metalloproteases, and are central to ECM degradation and metastatic dissemination [22]. Finally, aspartic proteases, which use aspartic acid residues in their catalytic site, include cathepsin D, an enzyme involved in tumor invasion and metastasis [23].

The functional roles of proteases in cancer biology are diverse and integral to tumor progression. Proteases such as MMPs are critical for ECM degradation, allowing cancer cells to invade surrounding tissues and migrate to distant sites [24]. In addition to facilitating invasion, proteases promote angiogenesis by releasing and activating pro-angiogenic factors, including vascular endothelial growth factor (VEGF) [25]. Furthermore, proteases contribute to apoptosis evasion by degrading apoptotic mediators or activating survival pathways, enabling cancer cells to resist programmed cell death [26]. Proteases also play a pivotal role in metastasis, aiding in the detachment of tumor cells from the primary tumor, intravasation into blood vessels, and colonization of distant organs [27]. Additionally, proteases modulate the immune response, often suppressing immune surveillance by cleaving immune regulatory molecules, thus enhancing tumor immune evasion [28].

Proteases play a critical role in modulating apoptotic pathways by interacting with key regulators such as Bcl-2, p53, and caspases [26]. For instance, MMP-7 has been implicated in the cleavage of Fas ligands, thereby inhibiting Fas-mediated apoptosis in cancer cells [29]. Similarly, cathepsin B can degrade pro-apoptotic proteins, enhancing tumor cell survival under stress conditions [30]. The intricate relationship between proteases and apoptotic regulators suggests that combining protease inhibitors with apoptosis-inducing agents, such as Bcl-2 antagonists, may enhance therapeutic efficacy in resistant cancers [31].

Several proteases are particularly noteworthy for their roles in cancer. MMPs, such as MMP-2 and MMP-9, degrade type IV collagen in the basement membrane, a critical step in tumor invasion and metastasis, and the overexpression of these proteases is associated with advanced stages of cancer and poor clinical outcomes [32]. Cathepsins, another prominent group, are involved in ECM remodeling and the activation of pro-angiogenic factors and cathepsin B and cathepsin L are frequently upregulated in aggressive cancers [33,34], while cathepsin D, an aspartic protease, is implicated in enhanced metastatic potential, particularly in breast cancer [35]. The uPA, a serine protease, converts plasminogen to plasmin, a protease that degrades fibrin and ECM components, and is closely linked to cancer cell invasion and metastasis [36]. The overactivation of these and other proteases underscores their critical role in cancer biology and highlights their potential as therapeutic targets.

The tumor microenvironment (TME) plays a critical role in determining whether proteases act as tumor-promoting or tumor-suppressing factors [37]. In hypoxic conditions, certain proteases such as MMPs are upregulated to facilitate angiogenesis and invasion [38]. Conversely, in a more immune-infiltrated microenvironment, proteases like MMP-12 may enhance anti-tumor immunity by promoting macrophage-mediated responses [39]. Additionally, cancer-associated fibroblasts can alter extracellular pH, influencing protease activity and shifting their role toward either matrix degradation or immune suppression [40]. Understanding these context-dependent functions is crucial for optimizing the therapeutic application of protease inhibitors

In summary, proteases are central players in the molecular and cellular mechanisms that drive cancer progression. Their diverse functional roles and frequent dysregulation in tumors make them attractive targets for therapeutic intervention. Understanding the complex biology of proteases provides the foundation for developing inhibitors that can mitigate their pro-tumorigenic effects and improve clinical outcomes for cancer patients.

3. Protease Inhibitors Classification and Mechanisms

Protease inhibitors represent a diverse group of molecules designed to modulate the activity of proteases, which are often dysregulated in cancer [41]. These inhibitors are categorized based on their origin, molecular structure, and mode of action. Synthetic inhibitors are chemically designed molecules that are highly specific to their target proteases; for instance, batimastat and marimastat are synthetic inhibitors of MMPs, developed to block the degradation of the extracellular matrix and prevent tumor invasion and metastasis [42]. Natural inhibitors, on the other hand, are derived from biological sources such as endogenous proteins or microbial products and include engineered tissue inhibitors of metalloproteinases (TIMPs) [43], which regulate MMP activity under normal physiological conditions, and compounds such as epoxomicin, a natural cysteine protease inhibitor [44]. Monoclonal antibodies have also emerged as a powerful class of inhibitors, providing specificity and versatility in targeting proteases, where they can bind to proteases with high affinity, and neutralizing their activity; for example, monoclonal antibodies targeting uPA [45] or cathepsin proteins [46] have shown promise in preclinical studies. Small-molecule inhibitors, which are low-molecular-weight compounds, are particularly attractive due to their ability to penetrate tissues and cells effectively. Examples include inhibitors of cysteine proteases such as cathepsins [47] and compounds targeting serine proteases like uPA [48].

Protease inhibitors function through several distinct mechanisms, depending on how they interact with the enzyme’s active or regulatory sites. The three primary reversible inhibition mechanisms include competitive, non-competitive, and uncompetitive inhibition, while some inhibitors act irreversibly by covalently modifying the enzyme. The mechanisms of action of protease inhibitors depend on their interaction with the enzyme’s active or regulatory sites. Competitive inhibition, one of the most common mechanisms, involves the inhibitor binding to the protease’s active site, thereby preventing substrate access and enzymatic activity [49]. This mechanism is exemplified by batimastat and marimastat, which mimic the substrate structure to occupy the active site of MMPs [42]. Non-competitive inhibitors bind to an allosteric site (a region distinct from the active site), inducing conformational changes in the enzyme that reduce its catalytic efficiency. Unlike competitive inhibitors, non-competitive inhibitors do not compete with the substrate, meaning they can inhibit enzyme activity regardless of substrate concentration. This mechanism provides an advantage in maintaining inhibition even when substrate levels fluctuate. An example is the inhibition of certain serine proteases through allosteric modulation, reducing their proteolytic activity [50]. Additionally, irreversible inhibitors form covalent bonds with the enzyme, permanently modifying its active site or regulatory regions. These inhibitors, such as CA-074 as an irreversible inhibitor of cathepsin B, lead to sustained enzyme inactivation, requiring new enzyme synthesis to restore function. This mechanism is advantageous in long-term therapeutic applications, such as cancer treatment. These inhibition strategies highlight the diverse mechanisms through which protease inhibitors regulate enzymatic activity, and a detailed understanding of these mechanisms is essential for designing selective and potent inhibitors for therapeutic applications [51].

Several protease inhibitors have been explored in research and clinical trials for cancer therapy. Batimastat and marimastat, as early MMP inhibitors, demonstrated efficacy in reducing tumor invasion and metastasis in preclinical models but faced limitations in clinical applications due to their toxicity and lack of specificity [52]. Efforts to overcome these challenges have led to the development of more selective inhibitors, such as those targeting specific cysteine proteases like cathepsin B and L, which are critical in tumor invasion and metastasis [8]. Endogenous inhibitors such as TIMPs have also been investigated for therapeutic use, with strategies focusing on enhancing their selectivity and delivery [43]. Small-molecule inhibitors targeting serine proteases, including uPA inhibitors, have shown potential in modulating plasminogen activation pathways critical for cancer cell invasion [53]. Overall, protease inhibitors, with their diverse structures and mechanisms, remain a promising class of therapeutic agents in cancer research, although overcoming the challenges of specificity, toxicity, and resistance remains a key focus for their successful clinical translation.

4. Clinical Applications and Therapeutic Potential

Protease inhibitors have demonstrated significant potential in cancer therapy, with preclinical and clinical studies highlighting their effectiveness in various cancer types [54] (Table 1). Their role in mitigating tumor progression, invasion, and metastasis underscores their therapeutic importance. However, the translation of these findings into clinical practice has been met with both successes and challenges, necessitating further refinement and innovation [10]. Protease inhibitors have been evaluated across multiple cancer types, with varying degrees of success. In breast cancer, inhibitors of MMPs such as marimastat have shown promise in reducing metastatic spread by limiting ECM degradation [55]. Similarly, the uPA system, frequently dysregulated in multiple kinds of cancer, has been targeted with serine protease inhibitors to suppress invasion and migration [19]. In prostate cancer, protease inhibitors targeting cathepsins and MMPs have demonstrated the ability to reduce tumor growth and metastasis in preclinical models [56]. Cathepsin B and L inhibitors have been explored for their role in mitigating invasive potential in aggressive forms of prostate cancer and melanoma [57,58]. In colorectal cancer, MMP inhibitors have been tested for their ability to prevent angiogenesis and invasion [59]. Although the results have been promising in animal models, challenges with toxicity and efficacy have limited their broader application in human trials. Despite the challenges faced by broad-spectrum MMP inhibitors, certain protease inhibitors have successfully reached clinical use. Notably, proteasome inhibitors such as bortezomib (Velcade) and carfilzomib (Kyprolis) have been approved for the treatment of multiple myeloma, demonstrating significant survival benefits [60,61]. These inhibitors selectively target the 26S proteasome, leading to the accumulation of misfolded proteins and induction of apoptosis in rapidly dividing cancer cells [62]. Additionally, nelfinavir, originally an HIV protease inhibitor, has shown promise in combination therapies for pancreatic and cervical cancers by inhibiting stress response pathways [63]. The success of these inhibitors underscores the importance of target specificity and mechanism-based design in overcoming toxicity and resistance challenges in clinical settings.

Despite the mixed clinical success of protease inhibitors, several cancer types and molecular subtypes have shown promising responses to these agents. Multiple myeloma and hematologic malignancies have benefited from proteasome inhibitors such as bortezomib and carfilzomib, which selectively block the degradation of pro-apoptotic proteins, leading to tumor cell death [64]. In triple-negative breast cancer (TNBC), protease inhibitors targeting cathepsin B and uPA have demonstrated preclinical efficacy in reducing metastasis [65,66]. Pancreatic cancer has been another area of active research, with uPA inhibitors in combination with chemotherapy showing potential in reducing tumor invasion [67]. These findings suggest that protease inhibitors may be most effective in cancers where proteolytic remodeling is a dominant driver of disease progression.

Table 1. Protease inhibitors clinical status in cancer research.

Name	Cancer Type	Clinical Trial Status (ClinicalTrials.gov ID)	Type of Protease Inhibited	Ref.
Bortezomib (Velcade)	Multiple Myeloma	FDA approved (NCT00006362)	Proteasome	[60,68]
Carfilzomib (Kyprolis)	Multiple Myeloma	FDA approved (NCT00461045)	Proteasome	[69]
Ixazomib (Ninlaro)	Multiple Myeloma	FDA approved (NCT00963820)	Proteasome	[70]
Andecaliximab (GS-5745)	Colorectal Carcinoma	Not FDA-Approved. (NCT01803282)	MMP-9	[71]
CA-074	Breast Cancer	Preclinical	Cathepsin B	[72]
CA-030	Breast Cancer	Preclinical	Cathepsin B	[73]
CLIK-148	Breast Cancer	Preclinical	Cathepsin L	[74,75]
CLIK-195	Breast Cancer	Preclinical	Cathepsin L	[76]
L-235	Breast Cancer Bone Metastasis	Preclinical	Cathepsin K	[77]
Fsn0503 (anti-CtsS antibody)	Colorectal Carcinoma	Preclinical	Cathepsin S	[78]
Marimastat	Various Cancers	Not FDA-Approved. (NCT00002911)	MMPs	[79]
Prinomastat	Various Cancers	Not FDA-Approved. (NCT00004199)	MMPs	[80]
Neovastat	Various Cancers	Not FDA-Approved. (NCT00026117)	MMPs	[81]
Rebimastat	Various Cancers	Not FDA-Approved. (NCT00040755)	MMPs	[82]
COL-3	Sarcomas	Not FDA-Approved. (NCT00020683)	MMP-2 and MMP-9	[83]
WX-671	Pancreatic cancer	Phase II (NCT00499265)	uPA	[84]
AE-941	NSCLC	Not FDA-Approved. (NCT00005838)	MMP-2 and MMP-9	[85]
Bowman–Birk inhibitor	Various Cancers	Phase II (NCT00330382)	Serine protease	[86]
AG3340	NSCLC	Not FDA-Approved. (NCT00004199)	MMPs	[87]
BMS-275291	NSCLC	Not FDA-Approved. (NCT00006229)	MMPs	[88]
Nelfinavir	Pancreatic cancer	Phase II (NCT02024009)	HIV protease	[89]

Table 1. Protease inhibitors clinical status in cancer research.

Name	Cancer Type	Clinical Trial Status (ClinicalTrials.gov ID)	Type of Protease Inhibited	Ref.
Bortezomib (Velcade)	Multiple Myeloma	FDA approved (NCT00006362)	Proteasome	[60,68]
Carfilzomib (Kyprolis)	Multiple Myeloma	FDA approved (NCT00461045)	Proteasome	[69]
Ixazomib (Ninlaro)	Multiple Myeloma	FDA approved (NCT00963820)	Proteasome	[70]
Andecaliximab (GS-5745)	Colorectal Carcinoma	Not FDA-Approved. (NCT01803282)	MMP-9	[71]
CA-074	Breast Cancer	Preclinical	Cathepsin B	[72]
CA-030	Breast Cancer	Preclinical	Cathepsin B	[73]
CLIK-148	Breast Cancer	Preclinical	Cathepsin L	[74,75]
CLIK-195	Breast Cancer	Preclinical	Cathepsin L	[76]
L-235	Breast Cancer Bone Metastasis	Preclinical	Cathepsin K	[77]
Fsn0503 (anti-CtsS antibody)	Colorectal Carcinoma	Preclinical	Cathepsin S	[78]
Marimastat	Various Cancers	Not FDA-Approved. (NCT00002911)	MMPs	[79]
Prinomastat	Various Cancers	Not FDA-Approved. (NCT00004199)	MMPs	[80]
Neovastat	Various Cancers	Not FDA-Approved. (NCT00026117)	MMPs	[81]
Rebimastat	Various Cancers	Not FDA-Approved. (NCT00040755)	MMPs	[82]
COL-3	Sarcomas	Not FDA-Approved. (NCT00020683)	MMP-2 and MMP-9	[83]
WX-671	Pancreatic cancer	Phase II (NCT00499265)	uPA	[84]
AE-941	NSCLC	Not FDA-Approved. (NCT00005838)	MMP-2 and MMP-9	[85]
Bowman–Birk inhibitor	Various Cancers	Phase II (NCT00330382)	Serine protease	[86]
AG3340	NSCLC	Not FDA-Approved. (NCT00004199)	MMPs	[87]
BMS-275291	NSCLC	Not FDA-Approved. (NCT00006229)	MMPs	[88]
Nelfinavir	Pancreatic cancer	Phase II (NCT02024009)	HIV protease	[89]

4.1. Clinical Trials

Protease inhibitors have shown promise in oncology by targeting key enzymes involved in tumor growth, invasion, and survival. In oncology, WX-UK1, a uPA inhibitor, is being developed for its role in reducing tumor invasion and metastasis. Its oral prodrug, upamostat (WX-671), is under clinical evaluation for cancers such as pancreatic cancer [84,90]. This serine protease inhibitor disrupts tumor metastasis by inhibiting the uPA system, crucial for extracellular matrix degradation. Despite preclinical efficacy and early clinical evaluations, no data on regulatory approval or large-scale success are currently available.

Proteasome inhibitors are well-established in oncology, and bortezomib, approved for multiple myeloma and mantle cell lymphoma, targets the 26S proteasome, inducing apoptosis in rapidly dividing cancer cells [91]. Clinical trials demonstrated improved progression-free survival, but common side effects include peripheral neuropathy, fatigue, and hematological toxicities [92,93]. Other threonine protease inhibitors, such as ER-807446 [94] and MLN-519 [95], showed preclinical promise in reducing tumor growth, but further development has been limited due to toxicity or lack of efficacy in advanced trials. TMC-95A, a natural proteasome inhibitor with a unique non-covalent binding mechanism, remains a research tool, without clinical translation [96,97].

MMP inhibitors specifically targeting cancer have had mixed outcomes. Marimastat, a broad-spectrum MMP inhibitor, reached Phase III for various cancers but was discontinued due to dose-limiting musculoskeletal toxicity [98]. Similarly, Rebimastat failed to secure approval after Phase III trials. These failures highlight the challenge of balancing therapeutic efficacy with minimizing side effects such as joint pain and systemic toxicity which arise due to the broad physiological roles of MMPs [99]. Prinomastat is a synthetic, hydroxamic acid-based MMP inhibitor designed to target MMPs like MMP-2, MMP-9, and MMP-14, which are involved in tumor invasion and metastasis. It was evaluated in Phase III clinical trials for non-small cell lung cancer (NSCLC) and despite promising preclinical data, the trials demonstrated limited efficacy in improving survival or disease progression and revealed significant side effects, including musculoskeletal pain, due to the off-target inhibition of MMPs involved in normal tissue remodeling [100]. S-3304 is an orally administered MMP inhibitor, developed by Shionogi & Co., Ltd., (Osaka, Japan), primarily targeting MMP-2 and MMP-9, and as of 2024, S-3304 has not received regulatory approval for cancer treatment. The lack of substantial efficacy in clinical trials, despite a favorable safety profile, has resulted in the discontinuation of its development for oncological indications. No ongoing clinical trials or further investigations involving S-3304 are currently registered [101,102]. SC-77964 is a small-molecule MMP inhibitor developed by Pfizer Inc. designed to target specific MMPs involved in cancer progression. Regardless of encouraging preclinical results, SC-77964 has not advanced beyond the preclinical stage. There are no registered clinical trials involving SC-77964 and it has not received regulatory approval for any indication [103]. Ro 28-2653 is a synthetic MMP inhibitor developed by Roche, exhibiting high selectivity for MMP-2, MMP-9, and MT1-MMP. Ro 28-2653 has not advanced to clinical trials and remains in the preclinical stage of development. The lack of clinical progression may be attributed to various factors, including challenges in demonstrating sufficient efficacy, potential safety concerns, or strategic decisions by the developing company [104,105].

One of the key reasons for the failure of MMP inhibitors in clinical trials is the ability of tumors to compensate for MMP blockade by activating alternative proteolytic pathways. Studies have shown that in response to MMP inhibition, tumors can upregulate ADAM proteases, which facilitate invasion through alternative extracellular matrix degradation mechanisms [2]. Additionally, certain tumors rely on plasminogen activator (uPA) systems or serine proteases to maintain metastatic potential [36]. Recent preclinical investigations have explored dual-targeting strategies where MMP inhibitors are co-administered with urokinase inhibitors or cathepsin inhibitors to prevent tumor escape mechanisms. For example, a combination of batimastat (MMP inhibitor) with serine protease inhibitor (aprotinin) has effectively inhibited the degradation of collagen IV and casein by tumor cells [106].

Among these, bortezomib, as a proteasome inhibitor, remains the only cancer protease inhibitor fully approved for clinical use. It has transformed the treatment landscape for multiple myeloma and mantle cell lymphoma. Other inhibitors, including WX-UK1 and upamostat, are still in various stages of clinical development, while compounds like marimastat and rebimastat have been discontinued due to adverse effects. The field continues to evolve, with ongoing research focused on improving specificity, minimizing side effects, and expanding therapeutic applications.

4.2. Combination Therapies

To address the limitations of protease inhibitors as monotherapies, combination strategies have emerged as a promising approach [107]. Combining protease inhibitors with chemotherapy has shown synergistic effects, with inhibitors reducing ECM remodeling and enhancing drug penetration into tumors [8]. For example, MMP inhibitors have been paired with cytotoxic agents to target both the tumor microenvironment and cancer cells directly [108]. Similarly, the combination of protease inhibitors with immunotherapy has gained traction, particularly in enhancing the efficacy of immune checkpoint inhibitors [109]. While still in early stages, combining protease inhibitors with immunotherapy agents is an area of active research. Studies are exploring the potential of combining proteasome inhibitors with immune checkpoint inhibitors to enhance anti-tumor immune responses [110]. The synergistic effects observed in these combination therapies can be attributed to several mechanisms. Protease inhibitors can sensitize cancer cells to apoptosis induced by chemotherapy agents [111]. Some protease inhibitors, like nelfinavir, can inhibit multidrug efflux pumps, potentially overcoming drug resistance [112]. Protease inhibitors can induce endoplasmic reticulum (ER) stress, which may sensitize cancer cells to other therapies [113]. Combining different inhibitors can target multiple oncogenic pathways simultaneously, leading to more robust anti-tumor effects [114]. Several clinical trials have explored the potential of protease inhibitor combinations in cancer therapy. A phase II trial demonstrated that indinavir combined with chemotherapy improved outcomes in elderly patients with Kaposi sarcoma, without additional toxicity [115]. Ongoing trials are investigating the combination of nelfinavir with various chemotherapy regimens in different cancer types [116].

Several studies have demonstrated the potential of combining protease inhibitors with chemotherapy or immunotherapy to improve therapeutic efficacy and overcome resistance. For example, the uPA inhibitor WX-UK1 has shown promise in combination with gemcitabine for pancreatic cancer, with early clinical trials suggesting a reduction in tumor metastasis [84]. Similarly, proteasome inhibitors like bortezomib have been successfully paired with immune checkpoint inhibitors in multiple myeloma, enhancing T-cell responses and improving survival rates [117]. The combination of MMP inhibitors such as marimastat with cytotoxic agents has also been explored, with mixed results due to toxicity challenges but promising synergistic effects in specific cancer types [118,119]. These studies highlight the importance of combinatorial approaches in enhancing the effectiveness of protease inhibitors. Protease inhibitors have been increasingly recognized for their ability to enhance immune checkpoint therapy by modulating the tumor immune microenvironment [120,121]. MMP inhibitors can improve T-cell infiltration by reducing the physical barriers imposed by excessive extracellular matrix deposition, making tumors more susceptible to anti-PD-1 and anti-PD-L1 therapies [122]. Additionally, cathepsin S inhibition has been shown to enhance antigen presentation by dendritic cells, thereby improving immune surveillance [123]. These mechanisms highlight the potential for protease inhibitors to be used in combination with immune checkpoint blockade to enhance anti-tumor immunity.

HIV protease inhibitors have demonstrated the ability to enhance the effects of traditional chemotherapy agents. For example, ritonavir has been shown to potentiate the antiproliferative and proapoptotic effects of docetaxel in hormone-independent prostate cancer cells [124]. Indinavir combined with chemotherapy has shown promise in elderly patients with advanced progressive classic Kaposi sarcoma, potentially boosting and extending the duration of chemotherapy effects [115]. Proteasome inhibitors, particularly when combined with other targeted therapies, have shown significant potential. Carfilzomib combined with nelfinavir or lopinavir has demonstrated increased efficacy in triple-negative breast cancer models, potentially overcoming resistance mechanisms [112]. Bortezomib, when used in combination with DNA-damaging drugs like doxorubicin and melphalan, has shown promise in the treatment of multiple myeloma [125].

By modulating the tumor microenvironment, protease inhibitors can improve immune cell infiltration and activity. Furthermore, the integration of protease inhibitors with radiotherapy has been explored to enhance the effects of radiation by disrupting tumor stroma and improving oxygenation, thereby increasing tumor radiosensitivity [126]. Protease inhibitors continue to hold significant potential in cancer therapy, with advancements in understanding their mechanisms and refining their applications leading to promising results. The focus on combination therapies and innovative delivery systems offers hope for overcoming current limitations and realizing their full therapeutic potential.

5. Challenges and Limitations

While protease inhibitors hold great promise as therapeutic agents in cancer treatment, their clinical development and application have been met with several significant challenges. These limitations include issues related to target specificity, toxicity, and the development of resistance mechanisms, which have collectively hindered their broader adoption and effectiveness in oncology [127].

One of the primary challenges in the development of protease inhibitors is achieving target selectivity. Proteases are ubiquitous enzymes with diverse physiological functions, and many shares structural similarities within their catalytic domains. This overlap often results in inhibitors affecting multiple proteases, leading to unintended off-target effects [128]. For example, MMP inhibitors such as batimastat and marimastat were found to lack sufficient specificity, inadvertently inhibiting multiple MMPs and disrupting normal physiological processes. This lack of selectivity not only reduces therapeutic efficacy but also increases the risk of adverse effects, necessitating the development of inhibitors with enhanced specificity and refined targeting mechanisms [42].

Toxicity represents another significant hurdle in the clinical use of protease inhibitors. Off-target effects arising from poor selectivity can lead to adverse reactions, as seen in early clinical trials of MMP inhibitors. Musculoskeletal toxicity, characterized by joint pain and stiffness, was a prominent side effect that limited the tolerability and use of these agents in patients [129]. Additionally, the systemic inhibition of proteases that play critical roles in normal tissue homeostasis can result in unintended consequences, further complicating their clinical application. Efforts to mitigate toxicity have included optimizing dosing regimens, exploring localized delivery methods, and designing next-generation inhibitors with improved pharmacological profiles [130].

Tumor resistance to protease inhibitors poses another major challenge. Tumors are highly adaptive and capable of developing mechanisms to circumvent the effects of targeted therapies [131]. In the case of protease inhibitors, cancer cells may upregulate alternative proteases or signaling pathways to maintain tumor progression despite the inhibition of a specific target [132]. For instance, inhibiting one MMP may lead to compensatory activity by other MMPs or protease families, rendering the treatment less effective over time. Furthermore, changes in the tumor microenvironment, such as altered ECM composition or the increased expression of protease activators, can contribute to resistance. Addressing these resistance mechanisms requires a combination of approaches, including multi-target therapies, combination treatments with other agents, and strategies to anticipate and counteract tumor adaptation.

The dual role of proteases in both promoting and suppressing tumor progression presents a significant challenge in the development of protease inhibitors. While many proteases facilitate tumor invasion and metastasis, others contribute to immune surveillance and tumor suppression in specific contexts. For instance, MMP-8 has been shown to exert protective effects against cancer progression by modulating the extracellular matrix [133]. Consequently, recent efforts have shifted toward biomarker-driven approaches to selectively inhibit pathological protease activity while preserving physiological functions [134]. Advances in single-cell transcriptomics and proteomic profiling allow for the stratification of patients based on tumor-specific protease expression, thereby reducing unintended effects and improving therapeutic outcomes [134].

To overcome the selectivity challenges that contributed to the failure of early MMP inhibitors, recent advancements in computational drug design, proteomics, and structural biology have enabled the development of more precise inhibitors [135,136,137]. Structure-based drug design (SBDD) has been employed to identify inhibitors that selectively target disease-associated MMPs while sparing physiologically important ones [138]. Additionally, peptidomimetic inhibitors, which mimic natural protease substrates but resist degradation, have shown increased specificity [7]. Furthermore, advances in activity-based profiling using proteomics have facilitated the identification of cancer-specific protease signatures, guiding patient stratification for targeted therapy.

The challenges of selectivity, toxicity, and resistance underscore the complexity of translating protease inhibitors into effective cancer therapies. Continued research and innovation are essential to address these limitations and unlock the full therapeutic potential of protease inhibitors in oncology. The future of protease inhibitors in cancer therapy lies in addressing the challenges of specificity, toxicity, and resistance while leveraging innovative research and emerging technologies. Advancements in drug delivery, the identification of novel protease targets, and the integration of personalized medicine approaches are pivotal for optimizing the clinical potential of protease inhibitors.

6. Research Innovations

Despite these challenges, advances in drug design and delivery offer hope for overcoming these limitations. The development of highly selective small-molecule inhibitors, monoclonal antibodies, and peptide-based inhibitors has improved the precision of targeting specific proteases [139]. Novel drug delivery systems, such as nanoparticles and antibody–drug conjugates, have enabled the localized and controlled release of inhibitors, reducing systemic toxicity [140]. Additionally, combining protease inhibitors with other therapeutic modalities, such as chemotherapy, immunotherapy, or radiotherapy, has shown promise in enhancing efficacy and preventing resistance [141,142,143]. Nanoparticle-based delivery systems, for instance, allow for the encapsulation of protease inhibitors, enabling targeted delivery to tumor sites while sparing healthy tissues. These systems can be engineered to release their payload in response to specific stimuli within the tumor microenvironment, such as pH changes or enzymatic activity, further improving selectivity [144,145]. Bortezomib and carfilzomib (proteasome inhibitors) have been loaded into PEGylated lipid micelles, liposomes, gold nanoparticles, and mesoporous silica nanoparticles. These formulations demonstrated superior therapeutic effects, including higher efficacy, increased circulation time, and decreased systemic toxicity compared to free drug formulations in breast cancer, non-small cell lung cancer, and colon carcinoma models [146,147]. Doxorubicin and epoxomicin (a proteasome inhibitor) have been co-encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles. This formulation showed enhanced anticancer efficiency, reduced drug resistance, and decreased toxicity to normal cells compared to free drugs [148].

Additionally, antibody–drug conjugates (ADCs) offer a promising approach by linking protease inhibitors to monoclonal antibodies that specifically bind to tumor-associated antigens, achieving both precision and efficacy [149]. ADCs offer several potential benefits for cancer therapy, including improved selectivity, enhanced efficacy, and reduced systemic toxicity [150]. By targeting tumor-associated antigens, ADCs can deliver cytotoxic agents specifically to cancer cells while sparing healthy tissues. The combination of antibody specificity and potent payloads allows for the delivery of highly cytotoxic agents that would be too toxic for systemic administration, potentially improving the therapeutic index [151].The concept of using protease inhibitors as ADC payloads aligns with the general principles of ADC design, including tumor microenvironment targeting, payload activation, and versatility. When developing protease inhibitor ADCs, several factors need to be considered, such as target selection, linker design, payload potency, and optimization, to achieve the desired balance of efficacy and safety [152].

Emerging technologies like CRISPR-Cas9 genome editing also hold promise for targeting proteases in cancer. By selectively knocking out or modulating the expression of genes encoding dysregulated proteases, CRISPR-based approaches can provide highly specific and durable therapeutic effects. These strategies may complement traditional protease inhibitors, particularly in addressing resistance mechanisms driven by compensatory upregulation of alternative proteases [153]. Despite its promise, the clinical application of CRISPR-Cas9 in cancer therapy faces several challenges which must be addressed before clinical implementation. One major concern is the off-target effects of CRISPR-Cas9, where unintended genetic modifications can lead to unpredictable consequences, raising safety and regulatory issues [154]. Additionally, the potential immunogenicity of Cas9 proteins poses a challenge for in vivo applications, necessitating further research into reducing immune responses against these components. Another significant challenge is the regulatory and ethical barriers associated with personalized medicine, as patient-specific protease inhibitor therapies require extensive validation, making clinical translation costly and time-consuming. Addressing these challenges will be essential for advancing the integration of these cutting-edge technologies into clinical practice [155].

The ongoing identification of novel proteases implicated in cancer progression opens new avenues for therapeutic intervention. While traditional efforts have focused on well-studied proteases such as MMPs, cathepsins, and uPA, recent research has uncovered additional protease families with significant roles in tumor biology. For example, the ADAM (a disintegrin and metalloprotease) family has been implicated in cell signaling and adhesion processes critical for tumor growth and metastasis [156]. Similarly, proteases involved in the unfolded protein response (UPR), such as those in the calpain family, are emerging as potential targets due to their role in regulating cancer cell survival under stress conditions [157]. Targeting these novel proteases may provide opportunities to disrupt cancer-specific pathways while avoiding the toxicity associated with inhibiting ubiquitously expressed proteases.

The integration of protease inhibitors into personalized medicine paradigms has the potential to transform cancer treatment. By leveraging proteomic profiling technologies, it is now possible to identify the specific protease expression patterns within individual tumors and this information can guide the selection of the most appropriate protease inhibitor for each patient, optimizing therapeutic outcomes and minimizing side effects [158]. For example, tumors with high expression of MMP-9 may benefit from targeted MMP inhibitors, while those with elevated cathepsin activity could respond better to cysteine protease inhibitors. Personalized approaches may also involve combining protease inhibitors with other targeted therapies based on the molecular characteristics of the tumor microenvironment.

The implementation of precision medicine in protease inhibitor development hinges on the identification of predictive biomarkers that determine patient responses. Technique such as mass spectrometry-based proteomics has been employed to analyze protease expression in tumor tissues [159]. For example, high MMP-9 expression in colorectal cancer has been correlated with responsiveness to MMP-targeted therapies [160], whereas elevated cathepsin B levels have been linked to aggressive breast cancer subtypes [161]. Furthermore, liquid biopsy-based protease activity assays are emerging as a non-invasive approach to monitor tumor responses to therapy in real time [162]. These advancements are paving the way for more stratified clinical trials that match protease inhibitors to patients most likely to benefit from them.

Additionally, advances in bioinformatics and artificial intelligence (AI) are facilitating the development of predictive models for treatment response. These tools can integrate data from genomic, transcriptomic, and proteomic analyses to predict which patients are most likely to benefit from specific protease inhibitors, enabling a more tailored and effective approach to cancer therapy [163,164,165].

The continuous exploration of protease inhibitors’ mechanisms and therapeutic applications remains crucial for advancing cancer treatment. By integrating novel drug delivery systems, personalized medicine approaches, and AI-driven predictive models, future therapies may achieve enhanced specificity and minimized toxicity. Ongoing clinical trials and combination strategies will play a key role in determining the success of protease inhibitors in oncology. Further interdisciplinary research is essential to fully realize their therapeutic potential.

In conclusion, the future of protease inhibitors in oncology is poised for transformation through innovative research and emerging technologies. Advanced drug delivery systems, novel protease targets, and personalized medicine approaches offer the potential to overcome current limitations and unlock new therapeutic possibilities. As these strategies continue to evolve, they hold the promise of improving the specificity, efficacy, and safety of protease inhibitors, ultimately enhancing their impact on cancer treatment. Protease inhibitors remain a promising yet challenging frontier in cancer treatment. Continued research and collaboration will be essential to unlock their potential and improve outcomes for cancer patients worldwide.