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. 2025 Jul 4;10(27):28643–28669. doi: 10.1021/acsomega.5c02725

Targeting Urease: A Promising Adjuvant Strategy for Effective Helicobacter pylori Eradication

Shivani Kunkalienkar †, Neha S Gandhi ‡, Ashutosh Gupta †, Moumita Saha †, Aravinda Pai §, Shiran Shetty ∥, Abhishek Gupta ⊥, Namdev Dhas #, Raghu Chandrashekar Hariharapura , K Nandakumar ∇, Nagalakshmi Narasimhaswamy ○, Sudheer Moorkoth †,*
PMCID: PMC12268749  PMID: 40687008

Abstract

Helicobacter pylori, a Gram-negative bacterium, exhibits unique adaptations to thrive in an acidic gastric environment. Urease enzyme present in the bacteria converts urea to ammonia and carbon dioxide, making the surrounding acidic environment of the bacteria neutral. This adaptation helps the bacteria to survive and travel further to gastric epithelial cells, where it attaches to mucin and damages the tissues, leading to gastritis, peptic ulcer, and ultimately, cancer. Physicians typically prescribe first-, second-, and third-line antibiotic therapies to eliminate the bacterium, but these treatments frequently fail to achieve complete eradication. This failure, driven by factors such as the coccoid form, high bacterial load, and biofilm formation, contributes to the growing problem of antibiotic resistance. Targeting urease activity presents a promising strategy to reduce the H. pylori pathogenicity and enhance its susceptibility to antibiotics. Inhibiting urease enzyme activity would be an option to make the bacteria less pathogenic and more prone to antibiotic treatment. Including the urease inhibitors as an adjuvant with the current antibiotic treatment regimen would effectively eradicate the bacteria. This comprehensive review discusses the structural characteristics of the urease enzyme and its role in pathogenesis and the available urease inhibitors along with their pharmacophoric features. An elaborative pharmacophore-based screening and docking study on scaffolds such as chlorogenic acid, catechol, and hydroxamic acid to discover a potent urease inhibitor is a future scope identified in this review.

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1. Introduction

Helicobacter pylori (H. pylori) is a rod-shaped, multiflagellate, microaerophilic, and Gram-negative bacteria infecting greater than half of the world’s population. , The bacterium was first recognized as a significant cause of gastric ulcers in 1982 by two Australian physicians, Barry Marshall and Robin Warren. Their groundbreaking discovery led to their recognition with the Noble Prize in Physiology or Medicine in 2005, highlighting the association of H. pylori in gastrointestinal health. The bacterium causes diseases like chronic gastritis, peptic ulcer disease, MALT lymphoma, nonulcer dyspepsia, inflammatory bowel disease, gastroesophageal reflux disease, diffuse duodenal nodular lymphoid hyperplasia, antral polyp, and gastric cancer. − H. pylori can be transmitted through oral–oral, fecal–oral, or sexual route. Several risk factors are associated with infection by H. pylori, including, but not limited to, lower socio-economic status (SES), overcrowded living conditions, inadequate sanitation and hygiene, close contact with infected individuals, consumption of contaminated food or water, smoking, alcohol use, dental conditions such as dental caries, and certain dietary habits like consumption of undercooked meat and spicy foods. −

In addition to SES, the prevalence of H. pylori varies with gender, age, and geographic location. The infection is most widespread in Africa, where prevalence rates often reach or exceed 70%. In West Africa, Nigeria has reported a wide range of prevalence ranging from 38% to as high as 92%, with children being the most affected group. Within Nigeria, the northern region reported a prevalence of 87.8%, while the southern region showed rates ranging from 28% to 51.4%. Other West African countries, such as Togo (93.1%), Cameroon (73.2%), the Republic of Benin (71.5%), and Congo-Brazzaville (70.41%) also reported high prevalence rates. In countries like Côte d’Ivoire, Ghana, and Senegal, the H. pylori infection rate was approximately 50%. In East Africa, Burundi and Rwanda had a prevalence of 70.8% and 75% respectively. Outside Africa, high seroprevalence rates were observed in Bangladesh (92%), India (79%), and Vietnam (74.6%). Overall, developing countries have shown a higher prevalence of H. pylori infection (50.8%) compared to developed countries (34.7%).

Age-related differences were also noted, with higher infection rates found in young adults (48.6%) compared to children (32.6%). India has a large population, with more than 70% of its people under the age of 35 years. As a result, the country is particularly vulnerable to the burden of H. pylori infection. This public health challenge could have a substantial impact on national development and progress.

Treating H. pylori is very crucial for preventing conditions like peptic ulcer and gastric cancer. Most clinical guidelines recommend triple therapy as the first-line treatment for management of H. pylori. ,− This typically includes PPI and two antibiotics, commonly amoxicillin, clarithromycin, or metronidazole. Triple therapy with clarithromycin and nonbismuth and bismuth quadruple therapy are the most popular first-line of treatment. When first-line treatment fails, a second-line treatment regimen is often prescribed, which includes a levofloxacin-based triple therapy. This regimen showed an eradication rate of 69% in 7 days and 84% in 10 days of treatment. However, increased resistance to levofloxacin, particularly due to its frequent use to treat pneumonia, poses a clinical challenge. An alternative, second-line treatment option is a sequential therapy with moxifloxacin and levofloxacin. − If both first- and second-line treatment fails, then physicians recommend antibiotic susceptibility testing since options become restricted. Third-line treatment includes the use of rifabutin, rifaximin, and sitafloxacin. These treatment regimens are very intensive yet unable to eradicate the organism completely, resulting in antibiotic resistance. Several factors lead to treatment failure, including the bacterium’s ability to survive in the acidic gastric environment leading to high bacterial load, biofilm formation, and the limited penetration of antibiotics into the deeper layers of the gastric epithelium where the bacteria reside.

H. pylori has several unique adaptations to thrive in a harsh acidic environment and colonize in the stomach. The unique mechanisms including, but not limiting to, urease production, motility, and adhesion, help it to reside in the gastric environment. The urease found in the cytosol and on the surface of H. pylori, hydrolyses urea to ammonia and carbon dioxide. ,− The resulting ammonia neutralizes the gastric acidic environment around the bacteria. Once H. pylori survives the acidic environment, it penetrates the basal layer of the stomach mucosal epithelial layer using its sheathed flagella, which facilitates motility through gastric mucosa. Here, the outer membrane proteins (OMP) of H. pylori serve as adhesins, allowing it to attach to the gastric mucosa, especially mucin (MUC5AC) of the stomach. Proteins such as sialic acid-binding adhesin, blood-antigen binding protein A (BabA), lacdiNAc-binding adhesin, neutrophil-activating protein, heat shock protein 60, outer membrane protein (HopZ), and adherence-associated proteins (AlpA and AlpB), play essential roles in bacterial adhesion and pathogenesis, such as inflammation and ulceration which may further lead to cancer. , Considering these special adaptive features of the bacterium, one potential approach to prevent colonization could be by targeting H. pylori urease enzyme with specific urease inhibitors. This article explores the potential of urease inhibitors, as an adjuvant, with antibiotic therapy for eradication of the organism.

2. H. pylori Urease

2.1. H. pylori Urease Genes

H. pylori urease enzyme is a large assembly of structures with a molecular weight (300–550 kDa). , The enzyme consists of seven gene clusters, which makes it active in carrying out urea hydrolysis (Table ). These seven genes are present in chromosomes of H. pylori in a single 6.13 kb gene cluster, where all genes are transcribed in the same direction. It also has nickel ions, which is a metal cofactor. The supramolecular assembly of urease contributes to acid tolerance and H. pylori’s ability to endure in the gastric acidic environment. ,

1. Role of H. pylori Urease Genes.

urease genes of H. pylori role
Structural Genes
UreA (mol. mass 29.5 kDa) produce assembled apoenzyme ,
UreB (mol. mass 66 kDa) produce assembled apoenzyme ,
Accessory Genes
UreE (mol. mass 19.5 kDa) incorporates nickel ion to urease apoenzyme ,
UreF (mol. mass 28.6 kDa) regulates folding and function of UreG ,
UreG (mol. mass 22 kDa) couples GTP hydrolysis ,,
UreH (mol. mass 29.7 kDa) along with other accessory proteins helps in urease maturation process ,
Acid Gated Channel
UreI (mol. mass 21.7 kDa) transport of urea
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2.1.1. Catalytic Subunits: ureA/B

The ureA subunit is encoded by ureA gene whereas the ureB subunit is encoded by ureB genes. , The ureA of H. pylori urease is unusual, as a single ureA gene encodes its amino acid sequence, whereas two separate genes encode the amino acid sequence in other bacterial species. The peculiarity could be because two smaller genes from other species may have been fused and formed ureA of the H. pylori urease. The ureA/B genes can encode a fully assembled apoenzyme adequately, but the apoenzyme will be catalytically inactive.

2.1.2. Accessory Proteins: ureE, ureF, ureG, ureH

The ureE of H. pylori functions as a metallochaperone and plays a crucial role in incorporating nickel ions into the urease apoprotein. , In solution, ureE exists as a dimer and binds with 0.5 nickel ion per monomer indicating stoichiometry of 1:1 dimer to one nickel ion. UreG, another accessory protein of H. pylori urease functions as a GTPase that couples GTP hydrolysis and helps in urease maturation and activation process. ,, The GTP hydrolysis is needed for transfer of nickel. The ureG contains the metal binding motif Cys–Pro–His, and substitution of this abolishes the maturation of urease. UreE with UreH supplies the nickel and helps in maturation of urease.

UreF of urease in H. pylori regulates the folding and function of UreG which acts as a GTPase-activating protein. , Another accessory protein, UreH, helps in urease maturation. , The accessory proteins, ureE, ureF, ureG, and ureH are located downstream of ureA and ureB and are necessary for the urease enzyme to be active.

2.1.3. UreI: Acid-Gated Urea Channel

UreI of H. pylori is an internal proton-gated protein and plays a crucial role in urea transport and acid resistance. It has six membrane-spanning segments and histidine residues. , UreI plays various roles in urea transport; it acts as an uptake system that helps maintain the intracellular concentration of ammonia and exports the intracellular ammonia that is in access. , The ureI pore is regulated by external pH through a periplasmic pH shift. When the medium pH drops below pH 6.5, the ureI pore opens, allowing urea entry into the cytoplasm of the bacteria. This urease activity results in ammonia that neutralizes the acidic environment, and as the pH reaches neutrality, the urease enzyme denies the substrate urea as the ureI pore closes, avoiding the excess of ammonia. ,

2.1.4. Nickel

Nickel is an important nutrient required for bacteria and to ensure sufficient nickel ions for urease enzyme. , H. pylori requires it as a cofactor of urease and hydrogenase. , The homeostasis of nickel in H. pylori is controlled by NikR, a ribbon-helix–helix regulatory protein that acts as an activator or repressor based on the target protein. ,, NikR has two dimeric N-terminal DNA-binding domains and a C-terminal domain required for nickel binding and tetramerization. ,− NikR regulates gene transcription in response to nickel availability. It activates genes involved in nickel metabolism (nixA, nikABCDE, hpn-like, hpn, ureA, and ureB) and also downregulates the genes involved in iron storage and uptake (fur, pfr, and exbB/exbD), motility (flaA, flab, and cheV), and stress responses involving OMP (omp31, omp32, and omp11). The nickel ions are imported to H. pylori through NixA, a high affinity metal permease located in the cytoplasmic membrane. − NixA is necessary for H. pylori to colonize the stomach mucosa. NixA has 331 amino acid residues accounting for a molecular weight of 37 kDa and consisting of eight transmembrane spinning helices. ,

2.2. Roles of Urease in Pathogenesis

2.2.1. Role of Urease in Colonization and Survival of H. pylori

Once H. pylori enter the human stomach, it uses a sophisticated mechanism to adapt to the acidic conditions, primarily through the urease enzyme. As the enzyme is activated, the ureI channel opens to allow entry of only urea in an acidic environment. Porins in the outer membrane of H. pylori is permeable to urea, and urea travels to cytoplasm through the ureI channel. This results in hydrolysis of urea to ammonia (NH3) and carbon dioxide (CO2), which diffuses into the periplasm. NH3 neutralizes the surrounding acidic environment and forms NH4 by reaction with protons. The carbonic anhydrase converts the released CO2 to HCO3 – which acts as a buffer. − This dual activity helps the bacteria maintain cellular homeostasis, ensuring that biochemical processes continue without interruption. , A study conducted by Eaton et al., in piglets revealed that the urease-negative mutant of H. pylori did not colonize in the gut, while the piglets that were inoculated with the parent strain of H. pylori had colonization ranging from 4.4 + 1.5 log10 CFU/g to 6.9 + 0.5 log10 CFU/g of gastric mucosa in piglets sacrificed after 5 days. The role of urease in the colonization and survival of H. pylori is depicted in Figure .

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Role of H. pylori urease in colonization and survival of H. pylori.

2.2.2. Role of Urease in Angiogenesis and Tumor Growth

H. pylori urease not only helps in survival but is also involved in the process of angiogenesis and tumor growth through the following processes. (A) Internalization and cellular activation: H. pylori urease is rapidly internalized into gastric epithelial cells through cholesterol-rich lipid rafts, and activates signaling events that alter cellular gene expression. (B) Direct induction of VEGF-mediated angiogenesis: H. pylori urease directly upregulates VEGF, a key mediator in angiogenesis, inducing new blood vessel formation and enhancing nutrient and oxygen supply to enable tumor growth. (C) Ammonia and chronic inflammation: ammonia released due to urea hydrolysis by urease enzyme leads to mucosal damage. It also increases gastrin levels and stimulate cell proliferation. H. pylori urease also activates neutrophils and monocytes leading to pro-inflammatory cytokines. This results in recruiting more immune cells and sustaining chronic inflammation. , (D) Antiapoptotic effect: H. pylori urease exerts antiapoptotic effects in gastric epithelial cells by increasing the expression of antiapoptotic proteins like Bcl-xL (B-cell lymphoma extra large) and decreasing the expression of pro-apoptotic proteins like BAD (Bcl-2-associated death promoter), both of which are mitochondrial proteins involved in the regulation of apoptosis. This antiapoptotic activity allows genetically damaged or mutated cells to survive, further supporting angiogenesis and facilitating tumor growth. (E) Stabilization of hypoxia-inducible factor 1-α (HIF-1α): H. pylori urease stabilizes HIF-1α under normoxic conditions, enabling angiogenesis and tumor growth. By enhancing angiogenesis, H. pylori urease increases nutrient and oxygen supply to the tumor, facilitating its growth, invasion, and metastasis. , Given its role in promoting angiogenesis, urease could be a potential therapeutic target. Inhibiting urease activity may reduce angiogenesis, thereby limiting tumor growth and spread. Role of urease in angiogenesis and tumor growth is depicted in Figure .

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Role of H. pylori urease in angiogenesis and tumor growth.

2.2.3. Role of Urease in Platelets Aggregation

Platelets, also known as thrombocytes, play a role in hemostasis, thrombosis, inflammatory responses, vascular integrity maintenance, and wound healing. In a healthy individual, platelet aggregation maintains hemostasis. When a blood vessel is injured, endothelial damage exposes subendothelial matrix components such as collagen and von Willebrand factor, enabling platelets to adhere to the exposed surface via receptors such as GP lb-IX-V and GP VI. This adhesion activates platelets, triggering the release of ADP, thromboxane A2 (TxA2), and serotonin from dense granules, amplifying platelet activation and recruiting additional platelets. Activated platelets undergo a morphological change and form a stable plug to stop bleeding. This process is localized and self-limiting to prevent excessive clot formation. However, in case of infection due to H. pylori, the urease enzyme dysregulates the platelet aggregation, leading to excessive clotting or inflammation. H. pylori urease binds to specific receptors on platelets, leading to the activation of calcium channels and subsequent release of ADP. Concurrently, platelets produce 12-lipoxygenase (12-LOX) metabolites, which further promotes platelet aggregation. Aggregated platelets release inflammatory mediators such as reactive oxygen species (ROS), interleukin-1 β (IL-1β), and tumor necrosis factor-α (TNF-α), which cause tissue damage. This aggregation leads to microthrombi, forming in the gastric mucosa, obstructing small blood vessels and reducing blood flow, thereby exacerbating ischemia and delaying the repair of gastric mucosal cells. Consequently, the prolonged healing process intensifies the severity and duration of gastric ulcers. Chronic inflammation resulting from this interaction can lead to conditions such as chronic gastritis and the risk of severe complications like gastric cancer due to ongoing cycles of tissue damage and regeneration. − These effects highlight the importance of managing H. pylori infections by targeting its urease to prevent platelet dysfunction and associated complications. The role of urease in platelet aggregation is depicted in Figure .

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Role of H. pylori urease in platelet aggregation.

2.2.4. Role of H. pylori Urease in Alzheimer’s Disease (AD)

AD is a neurodegenerative condition that causes dementia, leading to impairments in cognitive function. This results in symptoms such as aphasia (language impairment), amnesia (memory loss), agnosia (inability to recognize people, objects, or sounds despite intact sensory function), and apraxia (difficulty performing tasks or movements). AD can also cause behavioral disturbances, including personality changes, misidentifications, hallucinations, delusions, and depression. , Emerging evidence highlights the role of the H. pylori urease enzyme in AD pathogenesis as it contributes to neurodegeneration. Infection with H. pylori elevates levels of pro-inflammatory cytokines, including TNF-α and IL-1β, which can cross the blood–brain barrier (BBB). These cytokines initiate and sustain chronic neuroinflammation, a hallmark feature of AD pathology. Chronic neuroinflammation accelerates neuronal injury and functional decline, heightening the risk of neurodegeneration. The urease activity also generates excessive ammonia by catalyzing the hydrolysis of urea into ammonia and carbon dioxide. Ammonia, a highly neurotoxic molecule, can traverse BBB and contribute to neuronal damage. − Clinical studies have further highlighted the role of urea metabolism in neurological disorders, such as the association between elevated blood urea nitrogen and poor outcomes in ischemic stroke, underscoring the systemic impact of urea-derived toxicity. − Tau, a microtubule-stabilizing protein in neurons, is known to become hyperphosphorylated in AD. Hyperphosphorylation of tau involves the excessive accumulation of phosphate groups, leading to neurofibrillary tangles. These tangles disrupt cellular function and are toxic to neurons, ultimately contributing to neuronal death. Study suggested that H. pylori urease can induce tau hyperphosphorylation in neural cells, linking enzyme to this hallmark of AD pathology. H. pylori urease also produces ROS thereby affecting cellular components such as proteins, lipids, and DNA. The generation of ROS directly damages neurons, exacerbates tau hyperphosphorylation, and accelerates the aggregation of amyloid β (Aβ), a key protein involved in AD. Aβ plaques, which are abnormal clumps of protein, accumulate in the brain of AD patients, contributing to neurotoxicity and dysfunction. Additionally, H. pylori releases outer membrane vesicles (OMVs) containing urease and other bacterial components such as lipopolysaccharides, cytotoxin-associated gene A (CagA), and vacuolating cytotoxin A (VacA). These OMVs can cross the BBB and enter the brain, promoting Aβ aggregation and plaque formation. The accumulation of these plaques is toxic to neurons and further advances AD progression. These evidence underscore the potential of targeting H. pylori urease as an effective therapeutic strategy for mitigating the advancement of AD (Figure ).

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Role of H. pylori urease in AD.

3. Challenges in H. pylori Eradication and Potential of Urease Inhibitors in Overcoming Them

Eradication of H. pylori continues to be a worldwide challenge despite decades of therapeutic advancement. This is mainly attributed to increased antibiotic resistance, the coccoid form of bacteria, high bacterial load, biofilm formation, and patient-related challenges. The current treatment regimen causes side effects and also decreases the efficacy with time. , As the effectiveness of the regimen diminishes, the risk of reoccurrence and reinfection of the bacteria increases. Urease, a crucial enzyme that enables H. pylori to thrive in the stomach’s acidic environment has been identified as a promising target for therapy. Urease inhibitors offer a new strategy by breaking this survival pathway, potentially enhancing the treatment efficacy. Here, we discuss the major challenges associated with the eradication of H. pylori and how urease inhibitors could help to overcome them.

3.1. Antibiotic Resistance

A primary reason for resistance of antibiotics to H. pylori is the insufficient reach of antibiotics to the inner layer of mucosa where it resides. ,− The antibiotics show a topical effect for a shorter duration of time until they are present in the stomach. Resistance to metronidazole, clarithromycin, and levofloxacin is increasingly on the rise, particularly after failed eradication attempts. Research indicates that resistance to clarithromycin and levofloxacin is associated with a 7-fold and 8.2-fold increase in treatment failure respectively, in both treatment-naive and refractory infections. The primary resistance rates of clarithromycin, amoxicillin, tetracycline, metronidazole, and levofloxacin are 34.1–55.2%, 15.0%, 17.9%, 69.4–71.3%, and 18.4–27.9%, respectively. This increasing pattern of resistance creates a difficult cycle in which each failed treatment further reduces the effectiveness of future therapies.

3.2. The Coccoid Form of H. pylori

Most of the H. pylori bacteria are spiral in shape but under unfavorable conditions and on long-term contact with antibiotics, H. pylori convert into coccoid form. Coccoid form of H. pylori contributes to immune invasion, allowing the bacterium to persist undetected and sustain chronic infection. , These coccoid forms may continue to remain dormant for a long period of time in the gastric tissues and can retain virulence factors. This may lead to failure of treatment and reinfection of H. pylori. , Among amoxicillin, metronidazole, and clarithromycin, it is reported that amoxicillin showed pronounced effect with more than 90% conversion to coccoid from at 1/2 MIC after 72 h, and lead to treatment failure after suboptimal therapy. , It is essential to explore unconventional antimicrobial approaches, especially in the case of recurrent infection.

3.3. High Bacterial Load

Increased H. pylori density is associated with more intense inflammation and higher risk of diseases. Certain virulence factors, including the cagA gene, are associated with increased bacterial loads in the gastric mucosa. Clinical studies conducted by Lai et al., demonstrate that pretreatment density of H. pylori considerably influences eradication as well as ulcer healing rates. In patients with mild and moderate H. pylori density, eradication rate of H. pylori was 88.9%, and 94.3% respectively. While, the eradication rate was only 69.7%, in patients with high bacterial density. These results point out the direct association of the bacterial load and treatment outcome.

3.4. Biofilm Formation

H. pylori forms a biofilm, protecting it from antibiotics. Biofilms are microorganism communities attached to the surface and embedded by matrix composed of polysaccharide, lipids, DNA, and proteins that acts as shield, limiting antibiotic penetration and protecting bacteria from host immune systems. − Biofilm of H. pylori also has efflux pump genes such as HP1181, HP1165, hefA, and gluP that expels the antibiotic before reaching bacterial cells contributing to multidrug resistance. Biofilms reduce the diffusion of antibiotics, requiring 10–1000 times higher antibiotic concentration as compared to planktonic bacteria. It is reported that biofilm leads to antibiotic resistance. As a result, we should consider the impact of biofilm formation while developing a new drug against H. pylori.

3.5. Complexity of Therapy and Patient Noncompliance

Standard triple or quadruple therapies demand patients to take multiple medications several times in a day for up to 2 weeks or beyond. The complexity and pill burden overwhelm patients leading to missed doses or early discontinuation. Eradication of H. pylori depends strongly on patients receiving at least 90% of the prescribed medication. Noncompliance is greater with a more prolonged or complicated regimen, with rescue treatment, and when the patient experiences adverse effects. Evidence indicates that approximately 1.7% to 10% of patients fail to complete treatment as directed, with less compliance in regimens involving multiple doses or large pill burdens. Adverse effects related to the prolonged use of antibiotics also lead to discontinuation of treatment. Common noncompliance includes the severe diarrhea abdominal pain associated with amoxicillin and levofloxacin, appearance of metallic taste associated with metronidazole and clarithromycin, mineralization and bone calcification of tetracycline and the bitter taste, nausea, and dark stools associated with bismuth. −

3.6. Targeting Urease as a Promising Approach to Overcome the Challenges

Targeting the urease enzyme offers a promising strategy to overcome the above challenges. Since urease is pivotal for gastric survival, inclusion of a urease inhibitor as an adjuvant with antibiotics can provide an additive antimicrobial effect, resulting in eradication of the organism and prevent the development of resistance. This adjuvant approach does not interfere with the mechanism of antibiotic action and thus provides an additive effect.

The coccoid form of H. pylori is a dormant, nonreplicative state with lowered metabolism than the spiral form. , By directly inhibiting urease, these dormant forms can be deprived of their primary acid-neutralizing mechanism, impairing their capacity to survive in the stomach leading to their eradication where antibiotics are not effective.

High bacterial loads overwhelm the host’s defenses and diminish antibiotic effectiveness, particularly when there is resistance. Some urease inhibitors like acetohydroxamic acid and baicalin have been reported to profoundly decrease the viability of H. pylori and ATP synthesis irrespective of bacterial load. By inhibition of a key survival process, urease inhibition can reduce the bacterial burden and augment the efficacy of antibiotics.

Biofilms shield H. pylori from antibiotics and immune responses of the host, allowing for chronic infection and recurrence. The activity of the urease is responsible for biofilm development by establishing a localized pH-neutral environment. Inhibition of urease dismantles this protective niche, making bacteria within biofilms more susceptible to immune clearance and antimicrobials.

Today’s standard regimen tend to involve several antibiotics and proton pump inhibitors which results in a high pill burden and complicated dosing schedule, both of which can have negative effects on adherence. , Urease inhibitors, specifically attacking one of the most vital survival mechanism of bacteria, is expected to decrease the number of antibiotics needed, making the regimen simpler and can help in reducing the adverse effects. , Less complicated regimen are highly correlated with improved adherence. , Role of urease inhibitors in overcoming the challenges of H. pylori eradication is depicted in Table .

2. Potential Role of Urease Inhibitors to Overcome the Challenges Associated with H. pylori Eradication.

sr. no. challenges overcoming the challenges by urease inhibitors
1 antibiotic resistance bypasses antibiotic resistance mechanism
2 bacterial coccoid form reduces acid resistance, targets dormant cells
3 high bacterial burden reduces viability and ATP production
4 biofilm formation disrupts biofilm stability and persistence
5 poor adherence to therapy simplify the regimen and reduce the pill burden
6 complexity and duration of treatment fewer drugs and shorter courses
7 adverse effects lower doses, improved tolerability
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4. Three-Dimensional Structure of H. pylori Urease

Understanding the structure of H. pylori urease is vital in designing targeted urease inhibitors, as it reveals the enzyme’s active site architecture, enabling the design of compounds that can block its function. The two subunits, α and β, make up the urease. These two subunits combine to form heterodimer αβ. The heterodimeric unit can then form a trimeric structure (αβ)3, which later forms tetrahedral structure ((αβ)3)4 or a dodecameric assembly having 12 active sites. These active sites have two nickel ions, and this region is also denoted as “flap” region since it has the flexibility to change from the closed state to the open state to provide access to urea at this two-nickel region. Figure depicts the formation of the structure of the urease and the flap region in the urease’s active site.

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Three-dimensional structure of urease enzyme (a) formation of dodecameric assembly of H. pylori urease (b) flap region of active site of urease in closed and open state.

The study on H. pylori urease has advanced through homology modeling, structural experimental studies, and computational drug design to identify novel inhibitors. Homology modeling was the first tool to understand the urease of H. pylori. The crystal structure of H. pylori urease (Protein Databank Bank (PDB) 1E9Z) was modeled from Klebsiella aerogenes (PDB 1KAU) as it is homologous to K. aerogenes. This structure helped the researchers understand the interaction of the known urease inhibitor, acetohydroxamic acid, in the H. pylori urease active site. The residues present in H. pylori urease active sites have four histidines (His138, His136, His274, and His248), one aspartic acid (Asp362), one carbamylated lysine (KCX219), and two nickel ions. The H. pylori urease in complex with ligand acetohydroxamic acid (HAE) was determined by X-ray diffraction technique at 3.00 Å and it is deposited in the PDB with the ID 1E9Y. , Similarly, the cryo-EM structures of H. pylori urease with ligands 2-{[1-(3,5-dimethylphenyl)-1H-imidazol-2-yl]­sulfanyl}-N-hydroxyacetamide (DJM) and β-mercaptoethanol (BME) determined at 2.00 Å and 2.40 Å resolution are also deposited in PDB under the accession ID 6ZJA and 6QSU, respectively. The active site residues of H. pylori urease bound to ligands HAE, DJM, and BME are shown in Figure . The PDB IDs of H. pylori urease bound to ligands along with details on mutation, their activity, and active site residues are given in Table .

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Ligplot diagram of (a) acetohydroxamic acid (HAE), (b) 2-{[1-(3,5-dimethylphenyl)-1H-imidazol-2-yl]­sulfanyl}-N-hydroxyacetamide (DJM), and (c) β-mercaptoethanol (BME) in the H. pylori urease active site.

3. List of PDB ID of H. pylori Urease Bound to the Ligand with Details of Mutation and Active Site Residues.

PDB ID method used for determination of protein structure resolution (Å) pH mutation ligand ligand activity active site residues (as per PDBsum)
1E9Y X-ray diffraction 3.00 6.5 N6-carboxylysine is modified residue at position 219 of ureB acetohydroxamic acid Ki: 96,000 ± 17,000 nM, IC50: 156 ± 32 μM histidines (His221, His274, His248), aspartic acid (Asp362, Asp223), alanine (Ala169, Ala365) and glycine (Gly279)
6ZJA electron microscopy 2.00   no 2-{[1-(3,5-dimethylphenyl)-1H-imidazol-2-yl]sulfanyl}-N-hydroxyacetamide Ki: 0.630 μM, IC50: 19.6 μM methionine (Met366, Met317), isoleucine (Ile467), aspartic acid (Asp223), histidine (His322, His248, His221), carbamylated lysine (KCX219), cysteine (Cys321), alanine (Ala278), and glycine (Gly279)
6QSU electron microscopy 2.40   no β-mercaptoethanol Ki: 435 μM, IC50: 13.5 mM histidines (His221, His138, His274, His248), aspartic acid (Asp362, Asp223), alanine (Ala169, Ala365) and glycine (Gly279)
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5. Urease Inhibitors for Treatment of H. pylori Infection

Inhibiting urease enzyme activity has been explored by many researchers to prevent pathogenesis and make the organism less adaptable to the gastric acidic environment. − In this section, we discuss the potent urease inhibitors based on their structural class. The reported half-maximal inhibitory concentration (IC50) values, type of inhibition, the key pharmacophoric features and its significance are highlighted in Table . Compounds showing urease inhibitory activity belonging to the classes imidazole, hydroxamic acid, flavonoid, barbituric acid, thiourea, coumarin, catechol, and chlorogenic acid derivatives are discussed.

4. Urease Inhibitors with Their IUPAC Name, IC50 Value, Type of Inhibition, Pharmacophoric Features with Key Interactions and Their Significance .

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5.1. Imidazole and Its Derivatives

Imidazole derivatives, characterized by their nitrogen-containing heterocyclic ring, have gained attention in research due to wide range of biological activities like antifungal, anticancer, anti-HIV, antitubercular, antibacterial, and analgesic effects. Notably, imidazole as a scaffold has been used for the design and synthesis of H. pylori urease inhibitors. Among these derivatives, five compounds showed promising activity (Table ). A benzimidazole-2-thione derivative (Compound 1), showed a high binding free energy of −17.20 kcal/mol as compared to standard acetohydroxamic acid (binding free energy = −7.78 kcal/mol) in docking studies (PDB: 1E9Y). Docking interactions suggest a structural requirement of thiocarbonyl, benzimidazole, and ethoxymethyl groups as essential for activity in Compound 1. The in silico ADMET (adsorption, distribution, metabolism, excretion, and toxicity) and cytotoxicity study confirmed the safety. A nitroimidazole derivative, (Compound 2), demonstrated a 15-fold and 70-fold increase in urease inhibition compared to thiourea (IC50 of 22.01 μM) and hydroxyurea (IC50 of 100 μM), respectively. The docking study (PDB: 3LA4) showed binding energy of −9.54 kcal/mol revealing the presence of indole ring as a structural requirement for target affinity. Furthermore, the ADME studies also highlight its drug likeness. A metronidazole salicylate derivative (Compound 3), showed a 1.4-fold increase in urease inhibition as compared to standard acetohydroxamic acid (IC50 of 17 ± 2 μM). The docking studies (PDB: 1E9Z) supported the role of halogenated aromatic system and ether linkages essential for activity. A secnidazole salicylate conjugate (Compound 4), showed 16-fold increase in activity compared to acetohydroxamic acid (IC50 of 16 ± 2 μM). Docking studies (PDB ID: 1E9Z) established hydroxyl-substituted benzene rings key for activity. An imidazole­[2,1-b]­thiazole-sulfonate derivative, (Compound 5), was found to increase urease inhibition by 8-fold than standard thiourea (IC50 of 22.3 ± 0.031 μM). The docking study (PDB: 3LA4) of this compound revealed N-propyl and mesyl phenyl groups of the compound important for enzyme binding. Molecular dynamics also confirmed the stability of protein and its complexes with the compound.

5.2. Hydroxamic Acid Derivatives

Hydroxamic acids are a class of compounds with a wide range of biological activities. Derivatives belonging to this class exhibit remarkable inhibitory properties targeting peroxidase, matrix metalloproteinase, and urease. They also function as siderophores, competing for iron­(III). Hydroxamic acid represents a promising avenue in managing H. pylori infection by targeting the urease enzyme. Acetohydroxamic acid from this class is used as a reference compound in studies to target the urease enzyme. Researchers have developed urease inhibitors based on the hydroxamic acid scaffold. In this discussion, we highlight five compounds exhibiting potent urease inhibitory activity (Table ). An N-arylamino hydroxamic acid derivative, (Compound 6), exhibited 100% eradication rate showing 1500-fold more efficacy than standard acetohydroxamic (44.4% eradication rate). Docking studies (PDB: 1E9Y) revealed 2,4-dichloro substitution on benzene is essential for urease inhibition. The acute toxicity studies in mice showed LD50 of 3126.9 mg/kg supporting its potential for clinical development. A 3-arylpropionylhydroxamic acid derivative (Compound 7), demonstrated 20-fold increase in activity than standard acetohydroxamic acid (IC50 of 23.8 ± 1.5 μM). Docking studies (PDB: 1E9Y) of this compound revealed 5-chlorosalicylic acid and benzyl group as an essential structural feature for urease inhibition. A phenoxyacylhydroxamic acid derivative (Compound 8), showed 457-fold increase in the urease inhibitory activity than the standard acetohydroxamic acid (IC50 of 27.9 ± 1.5 μM). The docking studies (PDB: 1E9Y) showed the acylhydroxamic moiety, ether oxygen atom, and 3,4-dichlorophenyl group essential for potent inhibitory activity. A hydroxamic acid derivative containing a chromone ring system, (Compound 9), exhibited a 332-fold increase in activity as compared to acetohydroxamic acid (IC50 value of 27.6 ± 2.5 μM). Docking studies (PDB: 1E9Y) revealed that the compound can act in both, anionic and neutral forms, targeting the active site and the flap region, leading to potent inhibition. A dihydropyrimidine hydroxamic acid derivative, (Compound 10), demonstrated ∼2000-fold higher urease inhibitory activity than the commercially available acetohydroxamic acid (IC50 value of 27.4 ± 1.2 μM). Docking studies revealed that hydroxamic acid moiety, NH linker, carbonyl of dihydropyrimidine, and phenyl ring are essential for enhancing the activity.

5.3. Barbituric Acid Derivatives

Barbituric acid is a versatile heterocyclic compound with a pyrimidine ring with three carboxyl groups and diverse pharmacological and biological applications. It serves as a core scaffold for synthesizing barbiturates, a class of drugs historically utilized as hypnotics, anticonvulsants, and sedatives. Barbituric acid derivatives exhibit activities such as antileprotic, antihistamine, antiurease, anti-inflammatory, antiviral, antioxidant, anti-AIDS, anesthetic, anticonvulsant, sedative-hypnotic, antimicrobial, anticancer, and antitumor properties. Barbituric acid has also been explored for H. pylori urease inhibitory activity. Among the barbituric acid derivatives, four compounds showed potent urease inhibition (Table ). A 5-benzylidene barbiturate (Compound 11), exhibited an increase in activity of 2.4-fold compared to standard hydroxyurea (IC50 = 100 ± 3.03 μM). The docking study (PDB: 1E9Y) showed docking score of −9.47 kcal/mol revealing para-substitution important for enhancing the urease inhibition than ortho or meta substitution. An N,N-dimethylbarbituric-pyridinium derivative (Compound 12) showed an increase in urease inhibition of 2.2-fold and 10-fold in comparison to standard thiourea (IC50 value of 22.0 ± 0.03 μM) and hydroxyurea (IC50 of 100.0 ± 0.2 μM), respectively. Docking studies revealed that N,N dimethyl substitution, pyridinium ring, and 2-methylphenyl moieties are involved in potent urease inhibition. A thio-barbiturate, (Compound 13) exhibited 144-fold and 33-fold increase in urease inhibition as compared to standard urease inhibitors, such as hydroxyurea (IC50 of 100 ± 1.7 μM) and thiourea (IC50 of 23 ± 0.73 μM). Docking studies (PDB: 4H9M) showed that the benzylidene group, amide linker, and 2,3-dichlorophenyl have enhanced the urease inhibition. The in silico pharmacokinetic study demonstrated that the compound has a drug-like PK profile and good oral bioavailability. A 1,2,3-triazole–(thio)­barbituric acid derivative, (Compound 14), showed a 12.5-fold and 2.75-fold urease inhibition compared to hydroxyurea (IC50 of 100 ± 0.20 μM) and thiourea (IC50 of 22.0 ± 0.03 μM), respectively. Docking studies (PDB: 4H9M) revealed that a reduction in the flexibility of the flap region at the entrance of active site is essential for inhibiting the ureolytic activity. The in silico ADME profile showed that this compound is orally active and has no toxicity effect.

5.4. Thiourea Derivatives

Thiourea (SC­(NH2)2), also known as thiocarbamide, is a multifunctional organosulfur compound, with its derivatives exhibiting various biological applications like antiviral, antibacterial, antifungal, anticonvulsant, anti-inflammatory, antitubercular, antithyroid, and anticancer properties. Derivatives from this class have been explored for their urease inhibition activity against H. pylori in which five compounds have shown potent urease inhibition (Table ). The N-monosubstituted thiourea derivative (Compound 15), exhibited 170-fold more potency than standard acetohydroxamic acid (IC50 of 27.2 ± 0.7 μM). The docking studies (PDB: 1E9Y) revealed monosubstituted thiourea, benzene ring, and aceto and para positions of chlorine enhances the urease inhibition. The compound showed lower cell toxicity. An N-monosubstituted aroylthiourea (Compound 16), exhibited 450-fold and 543-fold more potency than standard acetohydroxamic acid (IC50 of 27.2 ± 0.7 μM) and thiourea (IC50 of 32.6 ± 0.9 μM), respectively. The compound was bound to the urea binding site with an exceptionally low K D value of 0.420 ± 0.003 nM and a prolonged residence time of 6.7 min. The docking studies revealed that the thiourea moiety, ortho-nitro substituted benzene ring, and aceto moiety enhances the urease inhibition. An acetylphenol-based acyl thiourea derivative, (Compound 17), exhibited ∼413-fold more potent urease inhibition than thiourea (IC50 of 22.3 ± 0.031 μM). The docking studies (PDB: 3LA4) showed carbamothioyl, hydroxybenzene, 2-bromobenzamide, phenyl ring of bromobenzamide, and thiourea nitrogen are essential for activity. The in silico pharmacokinetic study revealed that this compound is safe. A thiourea moiety with lipophilic chain and phenyl residue derivative (Compound 18), showed potent urease inhibition of 1.7-fold than standard thiourea (IC50 value of 18.61 ± 0.11 μM). The docking studies (PDB: 4H9M) revealed that the aliphatic tail of this compound orienting toward the solvent and the bromo group at para position on benzene ring enhances the urease inhibition. The in silico ADME showed compound is safe to administer.

5.5. Chromone Derivatives

5.5.1. Flavonoid Derivatives

Flavonoids, a wide group of polyphenolic compounds with a benzo-γ-pyrone ring, are found in plants. They are known for their renowned health benefits, which include anti-inflammatory, antiviral, hepatoprotective, and antioxidant properties. Their biological activity depends on the structure, degree of hydroxylation, and functional group substitutions. In plants, flavonoids function as growth regulators and secondary antioxidants during biotic and abiotic stress. Flavonoid derivatives have gained attention as natural urease inhibitors for H. pylori. Here, we discuss seven flavonoid-based urease inhibitors (Table ). A 4′,7,8-trihydroxyl-2-isoflavene (Compound 19), has shown 20-fold more potent urease inhibition than acetohydroxamic acid (IC50 value of 18.2 ± 1.6 μM). The docking study (PDB: 1E9Y) showed the isoflavene core, phenyl ring attached to position 2 of isoflavene, 7-hydroxyl, 8-hydroxyl moiety, and 4′-hydroxyl moiety essential for the urease inhibition. Quercetin (Compound 20), showed 1.76-fold increased urease inhibition than acetohydroxamic acid (IC50 value of 19.4 ± 2.0 μM). The docking studies (PDB: 1E9Y) showed 3-OH, 5-OH, and 3′,4′-dihydroxyl groups play a major role in the inhibition of H. pylori urease. It has low side effects and can be taken orally up to 1000 mg/d. , Baicalin (Compound 21) and scutellarin (Compound 22), derived from Scutellaria baicalensis, have shown 0.17-fold and 0.30-fold increased urease inhibitory activity, respectively, than acetohydroxamic acid (IC50 value of 0.147 ± 0.05 mM). The docking study (PDB: 1E9Y) showed that these two compounds tightly anchored the flap region, preventing the flap from backing into its closed position. The 4′-hydroxyl group provides high binding affinity toward H. pylori urease. Baicalin and scutellarin have been reported to be safe with high LD50 values. , Hesperetin-7-rhamnoglucoside (Compound 23), a bioflavonoid isolated from peel of Citrus uranium fruit has shown 2000-fold increase in urease inhibition compared to thiourea (IC50 value of 23.4 μM). The docking studies (PDB: 4HI0) revealed that hydroxyl group, aromatic ring, and alkyl substituents as essential for the activity. A hybrid of diosmin and thiourea (Compound 24), exhibited 1.8-fold increased urease inhibitory activity compared to standard thiourea which has IC50 value of 22.80 ± 0.011 μM. The substitution of nitro group at 3-position on aryl thiourea was responsible for potent urease inhibition. The docking study (PDB: 3LA4), showed good binding to the urease enzyme. The in silico ADMET study revealed the compound has drug-like profile and has good oral bioavailability. A hybrid of morin and thiourea (Compound 25), exhibited 2-fold increase in the urease inhibition compared to the control thiourea (IC50 value of 22.80 ± 0.011 μM). The anti-H. pylori activity showed a minimum inhibitory concentration (MIC) of 500 μg/mL with a zone of inhibition of 15 mm. The substitution of the bromo group and aryl thiourea enhanced the effectiveness of the compound in inhibiting the urease. The docking studies showed that the hydroxyl moiety, NH, and aromatic ring firmly fix the compound in the active site of the urease enzyme. The in silico ADMET study showed that this compound has favorable PK profile.

5.5.2. Catechol Derivatives

Catechol, are phenolic compounds that have shown antibacterial and antifungal activities. Researchers have used these as scaffolds to check their activity against the H. pylori urease enzyme (Table ). A pyrogallol and catechol derivative (Compound 26), exhibited 11.4-fold increased urease inhibitory activity than acetohydroxamic acid (IC50 value of 17.2 ± 0.9 μM). The docking study (PDB: 1E9Y) showed a binding free energy of −11.48 kcal/mol revealing 2-hydroxyl moiety in A-ring and 4-hydroxyl moiety in B ring as responsible for inhibiting the urease activity. A catechol derivative (Compound 27), showed 0.398 probability of activity from prediction of activity spectra for substances test. The docking studies (PDB: 1E9Y) showed a docking score of −11.02 kcal/mol and revealed that catechol hydroxyls, carboxylic acid group, and aromatic C6 carbon are essential moieties for enhanced activity. The compound binds at the mouth of active site and also deep inside the cavity, thus inhibiting the enzyme.

5.5.3. Coumarin Derivatives

Coumarins (benzopyrones) and their derivatives are a diverse group of compounds found predominantly in plants, though they are present in animals and microorganisms. The study of coumarins dates back more than 200 years, with the first isolation of coumarin (simplest member), from Coumarouna odorata (Dipteryx odorata). Due to their structural versatility, they have gained significant attention for their diverse range of biological activities such as antimicrobial, anticancer, antiviral, anticoagulant, antioxidant, cardiovascular, anti-inflammatory, and CNS effects. Coumarins and their derivatives have also demonstrated H. pylori urease inhibitory activity. Jadhav et al., synthesized a few coumarin derivatives, among which Compounds 28, 29, 30, 31, and 32 (Table ) showed 0.8-fold, 0.9-fold, 0.82-fold, 0.8-fold, and -0.9-fold potent urease inhibition, respectively, than standard acetohydroxamic acid (IC50 value of 44.64 ± 0.36 μM). The experimental and docking data (PDB: 1E9Y) of H. pylori antiurease activity highlights that the presence of 4-, 5-, 7-, and/or 8-hydroxyl substitution and 4-phenyl group in benzenoid ring is essential to abolish the H. pylori urease activity. Coumarins has reported to be a safer molecule.

5.5.4. Chlorogenic Acid Derivatives

Chlorogenic acid, a naturally occurring polyphenolic compound, has garnered significant attention for its diverse biological activity such as anti-inflammatory, antioxidant, antimicrobial, anticarcinogenic, antihypertensive, antiviral, antihypercholesterolemia actions. Its role as a urease inhibitor has also been explored by researchers. A chlorogenic acid derivative (Compound 33) exhibited 2-fold increased urease inhibitory activity than standard thiourea (IC50 value of 22.80 ± 0.011 μM). The anti-H. pylori activity showed a zone of inhibition of 10.00 mm at an MIC of 500 μg/mL. The docking study (PDB: 3LA4) showed a docking score of −10.091 and a binding energy of −62.674 kcal/mol. It also showed that carbonyl, hydroxyl, and nitro group of the compound enhances the inhibition of urease (Table ).

From the above discussion, we can say that pharmacophoric features play an important role in identifying potent urease inhibitors. Pharmacophoric features define the key structural elements required for molecules to bind with the enzyme and enhance the inhibition. In the imidazole class of compounds, the linking of chloro substituted salicylic acid enhances the urease inhibition. In the hydroxamic class, compounds linking to the salicylic acid derivative, NH linkage, and carbon linkage and the substitution with a chloro or hydroxyl group increase the urease inhibition. Barbituric acids are also potent urease inhibitors. Substitution of a chloro or methyl group enhances the urease inhibitory activity. Aryolthiourea also plays a role in urease inhibition. Substituting electron-withdrawing groups such as chlorine or bromine on aryolthiourea enhances urease inhibition. The substitution of the hydroxyl moiety on catechol and coumarins will make the molecule effective in inhibiting the enzyme. Chlorogenic acid substituted with a chloro group also acts as urease inhibitors. Figure depicts the pharmacophoric features responsible for urease inhibitory activity from various chemical classes.

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Pharmacophoric features of potent urease inhibitors of imidazole, hydroxamic acid, barbituric acid, thiourea, flavonoid, catechol, coumarin, and chlorogenic acid class.

5.5.4.1. In Silico Evaluation of Promising Urease Inhibitors

Different PDB IDs can show variations in docking with a ligand due to differences in the resolution. These variations influence the binding affinity of the compound during molecular docking. Innovative approaches, such as reversible peptide tagging systems, have also been explored to regulate enzyme activity, offering alternative strategies for targeting urease. Therefore, to identify the best binding pose and interaction pattern, we performed molecular docking with the available PDB IDs of H. pylori urease on the most potent urease inhibitors identified from the available reports discussed in this manuscript. Accordingly, a docking study was performed on the available H. pylori urease PDB IDs such as 1E9Y, 6ZJA, and 6QSU using the Schrodinger suite (Maestro). We used Glide XP to identify compounds showing the best interaction. Among the selected eight compounds, Compound 33, Compound 26, and Compound 10 from chlorogenic acid, catechol, and hydroxamic acid class, respectively, showed better docking score as compared to the other class of compounds (Figures , and ). The docking score and interactions of all the selected eight urease inhibitors in the active site residues are shown in Supporting Information Table S1. The interactions of these compounds in the active site of urease are given in Supporting Information Figures S21–S24. The docking study of Compound 33 showed docking scores of −6.926, −8.145, and −8.767 against H. pylori urease PDB ID 1E9Y, 6ZJA, and 6QSU respectively. Compound 26 belonging to pyrogallol and catechol showed docking scores of −6.578, −8.042, and −7.129 against H. pylori urease PDB ID 1E9Y, 6ZJA, and 6QSU, respectively. Compound 10 belonging to the class hydroxamic acid showed docking scores of −7.228, −7.521, and −7.564 against H. pylori urease PDB ID 1E9Y, 6ZJA, and 6QSU respectively. The results show that difference in resolution affects the binding affinity of the compounds. These findings emphasize the importance of considering multiple PDB structures to ensure a more reliable and comprehensive assessment of inhibitor binding.

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Binding mode of Compound 33 in the active site of H. pylori urease: (1a) interactions of Compound 33 with amino acid residues at the urease active site (PDB ID 1E9Y), (1b) 3D representation of Compound 33 in the pocket of H. pylori urease (PDB ID 1E9Y), (2a) interactions of Compound 33 with amino acid residues at the urease active site (PDB ID 6ZJA), (2b) 3D representation of Compound 33 in the pocket of H. pylori urease (PDB ID 6ZJA), (3a) interactions of Compound 33 with amino acid residues at the urease active site (PDB ID 6QSU), (3b) 3D representation of Compound 33 in the pocket of H. pylori urease (PDB ID 6QSU).

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Binding mode of Compound 26 in the active site of H. pylori urease: (1a) interactions of Compound 26 with amino acid residues at urease active site (PDB ID 1E9Y), (1b) 3D representation of Compound 26 in the pocket of H. pylori urease (PDB ID 1E9Y), (2a) interactions of Compound 26 with amino acid residues at urease active site (PDB ID 6ZJA), (2b) 3D representation of Compound 26 in the pocket of H. pylori urease (PDB ID 6ZJA), (3a) interactions of Compound 26 with amino acid residues at urease active site (PDB ID 6QSU), (3b) 3D representation of Compound 26 in the pocket of H. pylori urease (PDB ID 6QSU).

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Binding mode of Compound 10 in the active site of H. pylori urease: (1a) interactions of Compound 10 with amino acid residues at the urease active site (PDB ID 1E9Y), (1b) 3D representation of Compound 10 in the pocket of H. pylori urease (PDB ID 1E9Y), (2a) interactions of Compound 10 with amino acid residues at the urease active site (PDB ID 6ZJA), (2b) 3D representation of Compound 10 in the pocket of H. pylori urease (PDB ID 6ZJA), (3a) interactions of Compound 10 with amino acid residues at the urease active site (PDB ID 6QSU), (3b) 3D representation of Compound 10 in the pocket of H. pylori urease (PDB ID 6QSU).

6. Conclusions

The supramolecular assembly of H. pylori urease is crucial in enabling the bacteria to survive the gastric acidic condition. This enzyme is also involved in the processes of angiogenesis and platelet aggregation, leading to pathogenesis. A thorough analysis of urease enzyme as discussed in this review article, clearly points out that the urease enzyme could be a potential target for effectively eradicating H. pylori. Synthetic and natural compounds such as imidazole, hydroxamic acids, barbituric acids, thiourea, flavonoids, coumarins, catechol, and chlorogenic acid have shown potent urease inhibitory activity. A detailed review of the chemistry of these compounds showed that the pharmacophoric features play an important role in identifying potent urease inhibitors. By using pharmacophore modeling, virtual screening, and molecular docking, extensive libraries of chemicals can be effectively screened to find candidates with a good optimal binding affinity and good interaction patterns with H. pylori urease. Optimizing pharmacokinetic characteristics such as half-life, gastric stability, and oral bioavailability is also necessary, especially for drugs that act in the acidic environment of stomach. The molecular docking study that we performed showed that compounds belonging to the classes of chlorogenic acid, catechol, and hydroxamic acid have better docking scores than the other classes of reported compounds. It is essential to perform pharmacophore-based screening focusing specifically on the above three scaffolds to identify a promising urease inhibitor that is safe and effective and possesses necessary pharmacokinetic features. Considering the multifaceted role of the urease enzyme in the survival and pathogenesis of H. pylori, it could be concluded that the discovery of a promising urease inhibitor will revolutionize the H. pylori treatment. The inclusion of urease inhibitors as an adjuvant with existing antibiotic therapies could help in overcoming many of the existing treatment challenges in eradicating the organism from the affected individual. It can contribute to overcoming antibiotic resistance, a growing challenge worldwide. Furthermore, it may reduce the treatment duration and improve patient compliance, contributing to better clinical outcomes. There is also a future scope in discovering molecules that can inhibit urease genes discussed in this perspective and prevent their expression.

Supplementary Material

ao5c02725_si_001.pdf (1.3MB, pdf)

Acknowledgments

We extend our gratitude to the Manipal Academy of Higher Education for providing the necessary facilities to conduct molecular docking study using Schrödinger modelling package, and BioRender license for creating illustrations. We also acknowledge the use of ChemDraw Ultra 12.0.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c02725.

  • Docking score and interactions of potent compounds; and interactions of Compounds 4, 10, 13, 17, 19, 26, 32, and 33 with amino acid residues at urease active site (PDF)

Shivani Kunkalienkar: writing original draft, conceptualization, data curation, methodology, preparation of all graphics and TOC image. Neha S. Gandhi: methodology, data curation, supervision. Ashutosh Gupta and Moumita Saha: conceptualization, writing, review & editing. Abhishek Gupta: methodology, review & editing. Namdhev Dhas, Raghu Chandrashekar Hariharapura, K. Nandakumar and Nagalakshmi Narasimhaswamy: conceptualization, review & editing. Sudheer Moorkoth: conceptualization, review & editing, data curation, methodology, and supervision.

The work is funded by the Indian Council of Medical Research (ICMR), New Delhi, Government of India under the extramural Ad-hoc program with file no. OMI/10/2022/ECD dated 10.02.2023. ICMR has also granted fellowship to Mr. Ashutosh Gupta under the ICMR SRF program with File no. 3/2/2/16/2022-NCD-III.

The authors declare no competing financial interest.

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