Studies have revealed that inhibiting NEP activity enhances insulin secretion stimulated by glucose in islet tissues extracted from C57BL/6 mice (Esser et al., 2018). Furthermore, in vitro experiments showed that NEP-deficient mouse islet tissues are protected from insulin secretion dysfunction (Zraika et al., 2013). In vivo studies have shown that NEP-deficient mice fed a high-fat diet demonstrate enhanced insulin sensitivity, improved β-cell function, better glucose tolerance, and increased β-cell proliferation (Willard et al., 2017; Parilla et al., 2018). Study reveals that under acute physiological conditions, selectively inhibiting intestinal neprilysin boosts insulin secretion in response to oral glucose, and this impact is mediated through the GLP-1 receptor, thereby establishing a novel role for intestinal neprilysin in the regulation of beta-cell function (Esser et al., 2023). NEPIs are also effective in the management of type 2 diabetes mellitus (T2DM) by increasing the circulating level of GLP-1, which is degraded by NEP (Esser and Zraika, 2019). However, the influence of other enzymes, such as DPP-4 inhibitors, on NEP substrates can diminish the efficacy of NEPI or adversely affect insulin sensitivity and β-cell function (Esser et al., 2022). As a result, the levels of NEP substrates may rise, suggesting that a combined NEPI treatment approach for type 2 diabetes could be more efficacious (AlAnazi et al., 2023).

Diabetic nephropathy (DN) is recognized as a prevalent cause of end-stage renal disease, driven significantly by chronic hyperglycemia activating the renin-angiotensin system (RAS). Therefore, blocking RAS can help delay the progression of DN (Gallagher and Suckling, 2016). The persistent hyperactivation of the RAS is implicated in the progression of diabetic nephropathy. Concurrently, the natriuretic peptide system (NPS) is a counterbalancing negative feedback mechanism (Goru et al., 2017; Malek and Gaikwad, 2017). NEPI can enhance the bioavailability of natriuretic peptides, which may contribute to mitigating chronic kidney disease (CKD) progression (Lai et al., 2023). Clinically, urinary NEP levels are increased in diabetic patients, and NEPI can delay DN progression. Furthermore, research indicates that treatment with angiotensin receptor-neprilysin inhibitors (ARNIs) can lead to improvements in renal function, reduce glomerular and tubulointerstitial fibrosis, and weaken inflammation, pro-fibrotic and apoptotic signaling, ultimately delaying DN onset (Davis et al., 2022).

Diabetic retinopathy (DR) stands as the leading cause of vision loss among individuals with diabetes. DR is recognized as a progressive neurovascular condition affecting the retina, with its severity and progression closely linked to the duration of diabetes. Medications such as angiotensin receptor blockers (ARBs) and ARNIs have demonstrated the ability to significantly attenuate NEP activity (Dahrouj et al., 2013). Inhibiting retinal NEP activity and increasing retinal natriuretic peptide levels can improve DR treatment outcomes, and ARNIs can improve neurovascular lesions in the retinas of diabetic rats (Prasad et al., 2016).

Recent advancements in the field of diabetic complications have shed light on the potential therapeutic roles of neprilysin inhibitors, both as monotherapy and in combination with other agents, in the management of diabetic nephropathy (DKD) and diabetic cardiomyopathy (DCM). In a study, the combination of SGLT2 inhibitors with neprilysin inhibitors demonstrated promising results in improving glycemic control and reducing inflammation and oxidative stress, which are key factors in the progression of DKD (Tarun et al., 2024). Furthermore, the synergistic effects of these drugs on lowering glomerular pressure and proteinuria offer a novel approach to DKD management.

Residing on the cell surface of the corneal epithelium, where it remains undisturbed, NEP plays a pivotal role in controlling the chromatic interactions between corneal nerve fibers and the epithelial cells themselves through the modulation of peptides involved in these communications. Studies have shown that NEPI combined with topical ascorbic acid, citric acid, antibiotics, or steroids helps improve epithelial replanting on the corneal surface (Genova et al., 2018). Although a previous study has documented the presence of NEP in the human corneal epithelium, the exact physiological functions of this enzyme in this context have yet to be fully elucidated (Hasby and Saad, 2013).

In hypertensive animal models, long-term administration of omapatrilat has been observed to effectively halt the advancement of renal complications, including glomerulosclerosis, tubulointerstitial fibrosis, and overall kidney injury (Cao et al., 2001). In a 5/6 nephrectomy model, treatment with AVE 7688 has demonstrated significant efficacy in Mitigating proteinuria and ameliorating the severity of glomerulosclerosis and tubulointerstitial fibrosis (Benigni et al., 2004). AVE 7688 increased renal nitric oxide compounds, reduced endothelin-1 synthesis, decreased renal vascular contraction, and raised tubular ANP release (Benigni et al., 2004). In another 5/6 nephrectomy model, omapatrilat lowered systolic blood pressure and glomerular capillary pressure, reduced proteinuria, and mitigated glomerulosclerosis (Taal et al., 2001). Therefore, combined NEPI can improve CKD treatment outcomes.

Ulcerative Colitis is an idiopathic chronic inflammatory condition that targets the colon’s mucosal lining, leading to relapsing and remitting symptoms. The absence or inhibition of NEP can exacerbate Clostridium difficile toxin A-induced intestinal inflammation (Sargın et al., 2017). Additionally, recombinant NEP can prevent inflammation in NEP knockout mice. A significant deficiency in NEP activity could contribute to a pro-inflammatory environment within the colonic mucosa (Ter Beek et al., 2007). The inflammatory state is linked to NEP and substance P (SP) loss. The pro-inflammatory action of low SP levels may be related to bioactive fragments. In UC, the metabolism of peptides that act as pathogenic agents is likely impaired due to a substantial decrease in the activity of the principal enzyme that catalyzes their hydrolysis.

Additional investigative efforts are warranted to elucidate the underlying reasons for the observed decline in peptide levels. Vu et al.'s research confirms Vasoactive Intestinal Peptide (VIP) as an anti-inflammatory agent, showing reduced DSS-induced Colitis in both VIP knockout mice and wild-type mice treated with VIP antagonists (Vu et al., 2014), NEP Deficiency might dampen VIP anti-inflammatory-action-by-affecting production of its active VPAC1-binding fragments (Pavlovic et al., 2011). In the colonic tissues of UC patients, the expression of SP and VIP significantly decreases, and NEP expression is markedly reduced.

Alzheimer’s disease manifests as a neurodegenerative condition with hallmark pathologies: the intracellular presence of phosphorylated tau and the extracellular presence of β-amyloid plaques (Nalivaeva et al., 2020; Yiannopoulou and Papageorgiou, 2020). NEP is associated with the degradation and clearance of Aβ. NEP, a crucial metalloprotease, plays a significant role in the degradation and clearance of Aβ peptides (Grimm et al., 2014), and its presynaptic neuronal localization aids in Aβ removal (Iwata et al., 2004). In late-onset AD patients, there is a selective reduction in NEP mRNA expression and protein levels in brain areas. The decline in NEP levels and activity with age may contribute to late-onset Alzheimer’s disease in both humans and rodents (Iijima-Ando et al., 2008). Preclinical research suggests that the administration of NEPI may trigger Alzheimer ‘s-like pathologies in animal models (Liu et al., 2010). In mice (Poirier et al., 2006), NEPI exacerbates AD progression. A deficiency in NEP leads to impaired degradation of exogenous Aβ and disruptions in the metabolic regulation of endogenous Aβ levels. In the brains of NEP-deficient mice, Aβ levels are highest in the hippocampus, followed by the cortex. The thalamus/striatum, and lowest in the cerebellum, mirroring the pattern of vulnerability to Aβ deposition observed in the brains of AD patients (Liu et al., 2009; Liu et al., 2010; Pennant et al., 2014). Observations indicate (Marr and Spencer, 2010) that even a slight reduction in NEP activity can lead to AD development by facilitating the buildup of Aβ peptides. Studies (Ali et al., 2024) have suggested that NEPI might offer protective effects against the development of AD by increasing the levels of GLP-1, neuropeptide Y (NPY), and substance P. However, NEPI might contribute to AD development by increasing levels of BK and NPs (Bavishi et al., 2015). The contradictory findings on NEPIs' impact on AD progression are yet to be conclusively established in clinical settings.

HF represents the end stage of various cardiac diseases. Inhibiting the excessive activation of the neuroendocrine system is a crucial measure in the treatment of HF. However, traditional drug treatments based on angiotensin-converting enzyme inhibitors (ACEI) and angiotensin II receptor blockers (ARB) have been found to exhibit an “upper limit effect,” where the effectiveness of the drugs diminishes or is even lacking in some long-term users, leading to reduced clinical outcomes (Arundel et al., 2020). NEP plays a significant role in the pathogenesis and development of HF by affecting the natriuretic peptide system, the RAAS, and the kallikrein-kinin system (Domenig et al., 2016; Ponikowski et al., 2016). In 2006, the first ARNI—sacubitril/valsartan was introduced. Extensive clinical trials have confirmed that ARNI offers significant benefits in the treatment of heart failure with reduced ejection fraction (HFrEF), new-onset HF, and acute decompensated HF, as well as in improving prognosis (Seferovic et al., 2019). Furthermore, the apelin/label system plays a crucial role in the cardiovascular system including the regulation of vascular tension, blood pressure, and fluid homeostasis (Kuba et al., 2019). Recent studies have indicated that neprilysin can cleave and inactivate apelin peptides, suggesting that neprilysin may indirectly modulate the function of the apelin/label system by affecting the stability and activity of apelin peptides (Chapman et al., 2021).

Fasidotril (Compound 12, Figure 4A) is a diester prodrug developed by Bioproject. Its active metabolite, Fasidotrilat (Compound 13, Figure 4A), effectively inhibits angiotensin I-converting enzyme (ACE IC₅₀ = 9.8 nM) and neutral endopeptidase (NEP IC₅₀ = 5.1 nM) (C Marie et al., 1995). In heart failure models, long-term treatment with fasidotril improves myocardial hypertrophy and increases survival rates without lowering blood pressure (C Marie et al., 1995). Studies have shown that in clinical studies, fasidotril strongly affects renin-dependent and volume-dependent hypertension models and can sustainably reduce blood pressure (C Marie et al., 1995). Fasidotril inhibits NEP, which reduces the degradation of ANP and BNP, enhancing their natriuretic and vasodilatory actions, thereby helping to alleviate the symptoms of heart failure (Pathadka et al., 2021). Additionally, by blocking ACE, fasidotril decreases the production of Ang II, mitigating its vasoconstrictive and pro-inflammatory actions, and offering therapeutic benefits for hypertension and myocardial infarction (Tsutsui et al., 2021). This makes fasidotril a promising candidate for treating myocardial infarction and congestive heart failure.

Sampatrilat (Compound 14, UK 81252, Figure 4B) functions as a dual-action inhibitor targeting both ACE and NEP and it holds promise for therapeutic use in managing conditions such as hypertension and congestive heart failure (Wallis et al., 1998; Venn et al., 1998b; Venn et al., 1998a). Given its dual action, sampatrilat may offer more excellent benefits in treating chronic heart failure than traditional ACE inhibitors (Norton et al., 1999).

SamASP (Compound 15, Figure 4B) interacts with NEP akin to sampatrilat, securing its position in the S2' and S1' subsites, the region surrounding the zinc ion, and extending its reach into the Non-prime subsites (Sharma et al., 2020). In the non-prime region, the secondary amide and aspartic acid-like side chains on the C5 position of SamASP offer a fitting interaction that aligns well with the enzyme’s binding requirements, with the carboxyl group of the tartaric ester located in the densest electron density regions. The secondary amide’s strategic placement enables its C1 and C2 atoms to engage in hydrophobic interactions with the Val-710 residue (Sharma et al., 2020).

Omapatrilat (Compound 16, Vanlev, NEP IC₅₀ = 8.0 nM, PDB ID: 6SUK, Figure 4C) is an investigative pharmaceutical that functions as an inhibitor for both NEP and ACE (Sharma et al., 2020). In clinical studies, omapatrilat has been extensively investigated as a dual ACEI/NEPI (Cozier et al., 2018; Robl et al., 1997), and has proven significant efficacy in lowering blood pressure among individuals with hypertension (Azizi et al., 2000; Kostis et al., 2004). However, compared to placebo, omapatrilat is associated with a notably higher occurrence of side effects such as coughing, skin flushing, short-term facial redness, gastrointestinal disturbances, and potentially life-threatening adverse reactions such as angioedema (Arendse et al., 2019). These adverse safety profiles have hindered the advancement of this promising vasopeptidase inhibitor.

Omapatrilat was crafted to mimic a tripeptide, allowing it to engage with the S1, S1', and S2' subsites of the target metalloproteases, a design intended to enhance its binding specificity and efficacy (Sharma et al., 2020). The primary binding occurs in the NEP complex structure at the S1' and S2' subsites. At the same time, a segment of the bicyclic group protrudes into the S3' region, facilitating a more extensive interaction with the enzyme’s binding pockets (Sharma et al., 2020). The thiol group of omapatrilat forms a coordination complex with the zinc ion and two water molecules, facilitating interactions with His711 and the backbone of Ala543 (Sharma et al., 2020). The phenyl moiety of omapatrilat delves into the S1' pocket, creating many hydrophobic contacts with Phe106, Phe563, Val580, and Trp693. Moreover, its Cα-analogous atom also participates in hydrophobic interactions with His583, thereby enhancing the compound’s binding to the NEP enzyme (Sharma et al., 2020). The P1′ carbonyl of omapatrilat has hydrophobic interactions with His-711 at C11 and bidentate interactions with Arg-717 at O4 (Sharma et al., 2020).

M100240 (Compound 17, Figure 4C), a sulfur ester of MDL 100173, is a dual inhibitor targeting both angiotensin I-converting enzyme (ACE IC₅₀ = 0.08 nmol/L) and neutral endopeptidase (NEP IC₅₀ = 0.11 nmol/L) (Rousso et al., 1999). M100240 has been demonstrated in clinical research to decrease ACE activity and the concentration of angiotensin II. Simultaneously, it raises plasma renin levels and potentiates the beneficial actions of atrial natriuretic peptides (Rousso et al., 1999). This could offer distinctive therapeutic advantages in conditions marked by heightened vascular volume or sodium surplus and increased venous pressure (Schiering et al., 2016). Studies further suggest that M100240 exhibits safety and good tolerability in individuals, whether in a fed or fasting state (Schiering et al., 2016).

Lauren B. and the research team created three dipeptides with a carboxyl-3-phenyl propyl group at the terminal end and N-terminal nitrogen inspired by the structure of LisW—an ACE inhibitor. This synthesis was conducted to investigate the structural prerequisites for achieving dual inhibition of both ACE and NEP (Arendse et al., 2022). Removal of the P1′ amine from LisW to create AD 011 (Compound 18, Figure 4D) improved NEPI potency but showed poorer NEP affinity compared to ACE affinity (Oefner et al., 2000b; Arendse et al., 2022). Substituting the P2′ tryptophan residue in AD 011 with tyrosine to produce AD 012 (Compound 19, Figure 4D) further enhanced binding to NEP (Oefner et al., 2004; Arendse et al., 2022). In contrast to the earlier reported 2-mercapto-3-phenylpropionyl derivatives that possessed identical P1′ and P2′ groups, compound AD 013 (Compound 20, Figure 4D) exhibited a diminished binding affinity for NEP and the catalytic domain of cACE (Sharma et al., 2020). For inhibitors lacking suitable P1′ side chains for NEP S1′ subsite binding, those with a 2-mercapto-3-phenylpropionyl N-terminal may adopt a more selective binding orientation in NEP (Arendse et al., 2022).

Daglutril (Compound 21, SLV-306, Figure 5A) represents an innovative dual-action inhibitor targeting both NEP and ECE (Seed et al., 2012). The fundamental principle of this drug is to inhibit both neprilysin and endothelin-converting enzymes (Seed et al., 2012). The dual-action inhibitor’s benefit is bypassing the compensatory actions that occur with single enzyme inhibition, potentially offering better efficacy by blocking both enzymes simultaneously (Ferro et al., 1998). Following oral intake, Daglutril is metabolized into its active form (Dickstein et al., 2004), which then inhibits the systemic transformation of Big-ET1 and boosts circulating levels of ANP (Seed et al., 2012). In a compact, randomized, crossover, double-masked, placebo-controlled study, Daglutril was found to lower blood pressure in individuals with type 2 diabetic nephropathy throughout the 8-day treatment period, yet it did not demonstrate a reduction in albuminuria (Parvanova et al., 2013).

Dipeptidyl peptidase IV (DPP-IV) is a versatile type II transmembrane serine protease that functions as a glycoprotein. It comprises 766 amino acids: 6 amino acids are located within the cytoplasm, 22 amino acids comprise the portion that spans the plasma membrane, and the remaining 738 amino acids constitute the extracellular domain (Misumi et al., 1992). Oefner et al. proposed that DPP-IV inhibitors could be connected to NEPIs through their terminal unreacted substituents, residues, or C-terminal portions (Oefner et al., 2004). Following this design strategy, they synthesized NEP/DPP-IV dual inhibitors MCB 3937 (Compound 22, DPP-IV IC₅₀ = 0.3 μM, NEP IC₅₀ = 0.45 μM, PDB ID: 2QPJ, Figure 5B) and MCB 4241 (Compound 23, DPP-IV IC₅₀ = 0.2 μM, NEP IC₅₀ = 0.6 μM, Figure 5B) (Oefner et al., 2004). The benzyl group of the Inhibitors engages with the S1′ subsite of the enzyme, enhancing the binding affinity for NEP. This interaction is mainly facilitated by the aromatic or bulky hydrophobic residues present in the subsite (Oefner et al., 2004). The concept of dual NEP/DPP-IV inhibition can, in theory, be extended to a range of established enzyme inhibitor classes to potentially enhance their inhibitory efficacy.

ARNIs are a novel class of cardiovascular drugs that have emerged as a significant advancement in the treatment of heart failure and hypertension. ARNIs exert their effects by inhibiting the angiotensin receptor and neprilysin, a neutral endopeptidase that degrades various vasoactive peptides, including natriuretic peptides (Yamamoto and Rakugi, 2021).

The first-in-class ARNI, sacubitril/valsartan, has demonstrated superior cardioprotective effects compared to traditional renin-angiotensin system inhibitors (RAS-Is) in large clinical trials such as the PARADIGM-HF trial (Tsukamoto et al., 2023). This drug combines the neprilysin inhibition of sacubitril with the angiotensin receptor blockade of valsartan, enhancing the levels of beneficial peptides like natriuretic peptides while blocking the effects of angiotensin II (Kario and Williams, 2022).

ARNIs are effective in reducing cardiovascular mortality and heart failure hospitalizations by 20% in ambulatory patients with heart failure and reduced ejection fraction, as reported in the PARADIGM-HF trial (Mann et al., 2022). They have also been suggested to provide renoprotective effects beyond those of RAS-Is in patients with heart failure (Tsukamoto et al., 2023).

Furthermore, ARNIs have been studied for their efficacy in blood pressure management. Accumulating evidence suggests that sacubitril/valsartan is superior to conventional RAS Blockers in lowering blood pressure in patients with hypertension (Bozkurt et al., 2023). This class of drugs is effective in improving renal outcomes and has a lower risk of renal dysfunction and a higher estimated glomerular filtration rate (eGFR) compared to RAS-Is, without an increased risk of hyperkalemia (Chopra et al., 2023).

Phosphoramidon (Compound 24, Figure 5C) is a microbial metabolite and a specific metalloprotease thermolysin inhibitor. Phosphoramidon inhibits neprilysin (NEP, IC₅₀ = 0.034 μM, PDB ID: 1DMT), as well as an endothelin-converting enzyme (ECE, IC₅₀ = 3.5 μM) and angiotensin-converting enzyme (ACE, IC₅₀ = 78 μM) (Oefner et al., 2000b). The rhamnose group at the P1 position of the inhibitor is mainly in contact with the solvent, allowing the majority of the phosphoramidon surface to engage with the enzyme, thereby providing a secondary stabilization effect on the S1 subsite (Gaucher et al., 1999; Ksander et al., 1997b). The NEP’s1 subsite creates a substantial hydrophobic pocket that selectively binds with aromatic or bulky hydrophobic moieties (Tiraboschi et al., 1999). The S2′ subsite, notably spacious, reaches into the solvent environment through the junctional helix A3, approaching the side chains of residues R102, D107, and R110. This observation verifies that the S2′ subsite of NEP exhibits lower specificity and can accommodate larger side chains. These polar residues are not involved in the binding process with the inhibitor (Lambert et al., 1993).

As a NEPI, Racecadotril can rapidly metabolize into the effective Thiorphan (compound 25, IC₅₀ = 1.8 nM, PDB ID = 5V48, Patent: US20110319656, Figure 5C) in the body (Roques et al., 1980; Lambert et al., 1993; Labiuk et al., 2019; Schwartz, 2000; Eberlin et al., 2012). In contrast, its inhibitory effects on ACE and Endothelin-Converting Enzyme 1 (ECE1) are significantly weaker, with Ki values exceeding 0.1 and 10 μM, respectively. Thiorphan exerts its inhibitory effect on NEP by binding to the zinc ion at the enzyme’s active site via its thiol group, a critical interaction for its activity (Labiuk et al., 2019). This coordination interferes with the catalytic mechanism of NEP, thereby inhibiting its peptidase activity (Labiuk et al., 2019). Additionally, the phenyl ring structure of Thiorphan penetrates the central cavity of NEP and interacts hydrophobically with surrounding residues such as Phe107, Ile559, Phe564, Val581, and Trp694. These hydrophobic interactions are essential for Thiorphan’s stable binding in the NEP active site, contributing to its inhibitory potency (Labiuk et al., 2019). These features make Thiorphan a promising candidate drug, warranting further research and development to explore its potential in treating related diseases.

The three-dimensional model of thermolysin (TLN) has been widely used for identifying the active sites of NEP (Tiraboschi et al., 1999) and for inhibitor design (Ksander et al., 1997a). Previous research has identified R-Mercaptoacyl dipeptides such as compound Ala-Pro (Coric et al., 1996) and compound Gly-(5-Ph)Pro (Fournie-Zaluski et al., 1996), which bind to NEP through thiol-induced Zn2+ ion coordination and interactions with the enzyme’s1′ and S2′ subsites (Gaucher et al., 1999; Holmes and Matthews, 1981). Based on this discovery, they further validated the binding activity of three R-Mercaptoacyl dipeptides to NEP: [(2S)-2-sulfanyl-3-phenylpropanoyl]Gly-(5-Ph)Pro26 (TLN IC₅₀ = 1200 ± 200 nM; NEP IC₅₀ = 1.6 ± 0.3 nM; ACE IC₅₀ = 0.55 ± 0.05 nM, Figure 5D), [(2S)-2-sulfanyl-3-phenylpropanoyl]Phe-Tyr27 (TLN IC₅₀ = 42 ± 1 nM; NEP IC₅₀ = 2.9 ± 0.6 nM; ACE IC₅₀ = 5.6 ± 0.6 nM, Figure 5D), and [(2S, R)-2-sulfanylheptanoyl]-Phe-Ala28 (TLN IC₅₀ = 48 ± 5 nM, PDB ID: 1QF1; NEP IC₅₀ = 2.9 ± 0.5 nM, Figure 5D) (Gaucher et al., 1999). Compounds 27 and 28 exhibit higher affinity than compound 26. Comparing the inhibitory potency of these compounds and their various analogs against TLN, NEP, and ACE provides insights that confirm their binding modes with the latter two enzymes (Gaucher et al., 1999). A novel binding mode for thermolysin recognition, distinguished by the bidentate coordination of the zinc atom through thiol and oxygen groups, has been suggested. This concept opens up new avenues for designing inhibitors modeled after this interaction (Gaucher et al., 1999).