Anti-urease therapy: a targeted approach to mitigating antibiotic resistance in Helicobacter pylori while preserving the gut microflora

HPU consists of two structural subunits, UreA (26 kDa) and UreB (61 kDa), which assemble into a functional enzyme complex [47], forming a complex tetrahedral structure by four trimers of dimers ((αβ)₃)₄ [48], resulting in twelve active sites per functional HPU molecule. This intricate arrangement harbors a unique catalytic site featuring two Ni(II) ions bridged by a carbamylated lysine, facilitating efficient urea hydrolysis. This arrangement underscores the ability of the enzyme to catalyze the hydrolysis of urea with remarkable efficiency [30, 49]. A detailed description of ligand–receptor interactions is crucial for rational drug design targeting H. pylori urease. The enzyme’s active site contains a binuclear nickel center that is coordinated by conserved residues, including His219, His275, and Asp363, which are critical for urea hydrolysis. Importantly, the His492 residue resides within a flexible mobile flap region, which acts as a gate regulating access to the active site. Upon substrate or inhibitor binding, this flap undergoes conformational changes that modulate catalytic activity. Selective inhibitors often form hydrogen bonds or coordinate directly with the nickel ions, while also interacting with flap-region residues like His492 to stabilize binding. For instance, hydroxamic acid-based inhibitors can chelate both nickel atoms and form additional polar contacts with the surrounding amino acids, enhancing affinity and specificity. This dual mechanism—metal coordination and hydrogen bonding with the dynamic flap—has been shown to be a key strategy in designing inhibitors that are both potent and selective for H. pylori urease, minimizing off-target effects on other microbial or human metalloenzymes [50‐52]. Notably, tetrahedral assembly contributes significantly to enzyme stability and functionality, allowing for the simultaneous processing of multiple urea molecules and increasing the efficiency of ammonia and bicarbonate production [31]. This structural architecture highlights the sophistication embedded in urease design (Fig. 3). The catalytic site's involvement of nickel ions and a carbamylated lysine residue is pivotal for the enzyme's catalytic ability [47]. This unique configuration gives urease an extraordinary rate enhancement, with a half-life of 20 ms and a rate enhancement of 3 × 10¹⁵, surpassing those of other known hydrolases [53]. Such dynamics at the catalytic site contribute to the efficiency of urea hydrolysis, a process integral to bacterial survival strategies [54].

Bild vergrößern

The urease enzyme of H. pylori is composed of subunits UreA and UreB. While UreA does not participate directly in catalysis, it is essential for maintaining the structural integrity of the enzyme. UreB, on the other hand, contributes directly to the formation of the active site that houses nickel ions—crucial cofactors required for enzymatic function [59, 60].

Nickel ions are embedded within the UreB subunit and are indispensable for catalysis [52]. The maturation of urease into its active form requires the action of several accessory proteins, including UreE, UreF, UreG, UreH, and UreI. These proteins coordinate the delivery and incorporation of nickel into the urease enzyme, ensuring its catalytic competence.

UreI, a membrane protein, functions as an H⁺-gated urea channel. Under acidic stress, UreI is activated, facilitating the influx of urea into the cytoplasm. This mechanism is critical for acid tolerance, as intracellular urease hydrolyzes urea into ammonia and carbon dioxide, effectively neutralizing the acidic environment [61‐63].

Nickel acquisition in H. pylori is mediated by outer-membrane transport systems such as FrpB4 (a TonB-dependent transporter) and possibly FecA3. Intracellular nickel trafficking involves complex protein networks that manage distribution between urease and hydrogenase systems [39]. Proteins like HspA serve dual roles in nickel storage and delivery, acting as metallo-chaperones crucial for bacterial survival under acidic conditions [39].

The nickel-responsive transcriptional regulator NikR modulates gene expression related to urease activity, maintaining homeostasis in response to intracellular nickel levels. Meanwhile, the NixA transporter plays a central role in nickel uptake, supporting urease function and contributing to H. pylori virulence [38, 64‐66].

Beyond NixA, H. pylori employs additional transporters, including FrpB4 and the recently characterized NiuBDE system. FrpB4 is particularly responsive in acidic environments, while NiuBDE operates effectively across a broader pH range. The complementary pH responsiveness of these systems underlines their adaptive importance for nickel acquisition [67, 68].

Nickel-binding proteins such as HypB, a GTPase involved in urease and hydrogenase maturation, further illustrate the complexity of metal coordination. Nickel binding to HypB is regulated by nucleotide interaction, with His-107 playing a pivotal role in metal coordination. These interactions highlight the tight coupling between metal binding, GTP hydrolysis, and enzymatic biosynthesis in H. pylori [69].

Accessory proteins UreH, UreF, UreG, and UreE are indispensable for urease activation. UreF, for example, acts as a GTPase-activating protein, promoting GTP hydrolysis by UreG—a necessary step in nickel incorporation. Structural modeling studies show that UreF undergoes conformational changes upon complexing with UreH, facilitating UreG recruitment and heterotrimeric complex formation (UreG–UreF–UreH), which is critical for urease maturation [70‐72].

UreF also forms dimers with high α-helical content and binds two nickel ions per dimer. Specific residues are involved in Ni²⁺ coordination, and mutations in these residues impair binding, reinforcing UreF’s role in nickel trafficking [73]. UreG, a SIMIBI-class GTPase, remains structurally flexible even when bound to nickel and GTP. This flexibility suggests that allosteric communication is vital for effective urease activation [74].

Recent studies have identified UreG as a target for urease inhibition. Colloidal bismuth subcitrate disrupts urease maturation by interfering with UreG, suggesting a potential antimicrobial strategy [75]. Protein–protein interactions within the urease gene cluster have also been elucidated, including complexes such as UreF-UreH and UreG-UreE, which are essential for the delivery of nickel ions to the urease active site [76, 77].

UreG forms a (UreE)₂-(UreG)₂ complex that mediates nickel transfer from UreE, a critical step for enzyme activation [78]. Molecular dynamics studies have also identified nickel-transport tunnels within the HpUreDFG complex. These tunnels facilitate targeted nickel transfer, underscoring their importance in urease biosynthesis and offering new therapeutic targets for inhibiting enzyme activation [79].

Autoinducer-2 (AI-2), a quorum-sensing molecule, has been shown to upregulate urease expression via suppression of the HP1021 response regulator. This modulation enhances H. pylori survival in acidic environments and suggests novel avenues for antimicrobial intervention targeting urease regulation [80].

The histidine-rich protein Hpn plays a central role in nickel trafficking. It binds nickel with high affinity, contributing to both storage and mobilization. Comparative studies reveal differences in nickel-binding thermodynamics across Hpn variants from different H. pylori strains, highlighting the significance of histidine and glutamine residues in metal binding [64, 81, 82].

Finally, the interplay between urease and carbonic anhydrases (CAs) is crucial for H. pylori’s acid acclimatization. Urease hydrolyzes urea to ammonia (NH₃) and carbon dioxide (CO₂). In the cytoplasm, β-CA hydrates CO₂, while α-CA in the periplasm performs a similar function. These reactions produce ions that interact with ammonia to form NH₄⁺ and bicarbonate (HCO₃⁻), buffering protons and stabilizing intracellular and periplasmic pH [83‐85].

This dual-enzyme system—urease and carbonic anhydrases—demonstrates an evolved strategy by which H. pylori survives the gastric niche (Fig. 4). These mechanisms represent critical vulnerabilities that may be exploited in the development of targeted therapies aimed at disrupting bacterial acid resistance and colonization [86, 87].

Bild vergrößern

In the context of plant-based therapies for combating H. pylori, phytochemicals, which are natural compounds found in plants and have been demonstrated to have anti-H. pylori activity [100]. Among these compounds, flavonoids such as quercetin and catechins have been reported to inhibit the growth of H. pylori and its adherence to the gastric mucosa [101‐103]. Additionally, essential oils from herbs and spices such as oregano, thyme, and cinnamon are recognized for their antibacterial properties against H. pylori, potentially disrupting the bacterial cell membrane and interfering with its metabolic processes [104, 104‐106]. Ali et al. demonstrated that two bioactive compounds (Eugenol and cinnamaldehyde, both present in essential oils of plants) inhibited the growth of 30 H. pylori strains, with enhanced activity at acidic pH and no resistance developing [107]. Another promising avenue for generating H. pylori treatments is the exploration of medicinal plants, particularly herbal extracts. Extracts from certain herbs have shown potential in treating H. pylori infections. For example, liquorice root, which contains the compound glycyrrhizin, has been demonstrated to be effective in inhibiting H. pylori growth [106, 108]. Similarly, green tea, which is rich in catechins, especially epigallocatechin gallate, has notable anti-H. pylori activity [109, 110]. Despite the potential of these natural compounds, none of the plant-based compounds are currently approved by the FDA as individual standard therapies against H. pylori. In addition, a lack of standardization and quality control, insufficient clinical evidence and potential interactions with other medications can lead to undesirable side effects, leading to limitations in the use of plant-based compounds [111, 112].

Probiotic therapy has gained attracted significant attention as potential treatment for H. pylori infections. These live beneficial microorganisms offer a multifaceted approach to combating H. pylori. Probiotics target H. pylori through several mechanisms. Immunologically, they can modulate the host immune response by secreting anti-inflammatory cytokines and regulating the mucosal immune response. This modulation reduces the release of inflammatory cytokines and chemokines, thereby mitigating the gastric inflammation induced by H. pylori. Probiotics stimulate the secretion of mucins and glycoproteins, which protect the gastric mucosa by preventing H. pylori adhesion to epithelial cells [83, 113‐115]. Non-immunologically, probiotics produce substances such as short-chain fatty acids (e.g., acetic and propionic acids), lactic acid, and bacteriocins. These substances inhibit the growth of H. pylori by lowering the local pH and directly reducing bacterial viability. Probiotics also compete for adhesion sites on the gastric mucosa, effectively limiting the ability of H. pylori to colonize and establish infection.

In addition to their direct antimicrobial effects, probiotics play a crucial role in modulating the immune system, potentially enhancing the overall response of the host to infection [113, 116]. Specific strains, particularly Lactobacillus and Bifidobacterium have demonstrated effectiveness against H. pylori. These probiotic strains have been shown to inhibit H. pylori, reduce the side effects commonly associated with standard antibiotic treatments, and, importantly, enhance treatment success when used as adjuncts. This suggests a synergistic effect, where probiotics not only contribute to their anti-H. pylori activity but also enhances the efficacy of conventional treatments. This insight into probiotic therapy underscores its potential as a complementary approach to H. pylori management [117‐121].

Despite the promising potential of probiotics as alternative therapies for H. pylori, several limitations should be considered. One major challenge is the lack of standardization in probiotic formulations. The efficacy of different strains of probiotics can vary widely, and the optimal strains and dosages for H. pylori eradication are not yet well defined​​. Additionally, clinical evidence supporting the ability of probiotics to significantly reduce H. pylori colonization or improve clinical outcome remains limited [113]. While some studies show that probiotics can enhance treatment response and alleviate side effects and reduce treatment-related side effects [122], other studies report no significant benefit [123].

The use of nanoparticles (NPs) for treating H. pylori and gastric cancer shows significant promise because of their unique physicochemical properties. NPs enhance immune regulation and enable targeted drug delivery and the controlled release of drugs, antigens, and adjuvants directly to the intended target sites while avoiding pathological disorders [113, 124]. This controlled drug release property allows for sustained therapeutic effects, reducing the frequency of administration and minimizing systemic toxicity, which is particularly beneficial for treating chronic infections such as H. pylori and gastric cancer. Nanoparticles can be broadly classified into three major categories: metal-based (e.g., silver, gold, and zinc oxide), polymer-based (e.g., chitosan and other biodegradable polymers), and lipid-based (e.g., liposomes and solid lipid nanoparticles), each offering unique advantages in drug delivery and inhibit bacterial growth [125, 126].

Metal NPs, including those made from silver, gold, and zinc oxide, have demonstrated strong antibacterial properties against H. pylori. Silver NPs interact with proteins in bacterial membranes, leading to disruption of bacterial cell functions and bacterial death. Studies have shown that silver NPs synthesized through green methods, such as using plant extracts, can effectively inhibit the growth of H. pylori [127]. In addition to their direct antibacterial effects, silver NPs have been shown to inhibit H. pylori quorum sensing (QS) and biofilm formation, disrupting bacterial communication pathways that contribute to antibiotic resistance and persistence in the gastric environment [128]. Likewise, gold and zinc oxide NPs have been shown to damage bacterial cell membranes and inhibit H. pylori growth, making them potent agents against antibiotic-resistant strains [124, 129, 130]. Notably, gold NPs have been investigated as drug carriers to enhance the antibacterial effects of conventional antibiotics. A study by Fateh et al. demonstrated that conjugating gold NPs with metronidazole significantly increased its anti-H. pylori activity, overcoming metronidazole resistance and resulting in a 17-mm growth inhibition zone against resistant strains [131]. Additionally, an innovative approach using nanocluster-mediated photothermal therapy has demonstrated significant potential in inhibiting H. pylori growth. A study by Meng et al. showed that zinc ferrite nanoclusters, when exposed to near-infrared (808 nm) irradiation, effectively inhibited H. pylori growth by inducing bacterial membrane disruption and ribosome damage. Moreover, this treatment enhanced antibiotic sensitivity, reducing resistance to levofloxacin and clarithromycin while also decreasing H. pylori biofilm formation [132].

Polymeric NPs offer a biocompatible platform for targeted drug delivery in inhibiting H. pylori growth. These versatile carriers can be loaded with antibiotics or natural antimicrobials, improving their potency and efficacy. Chitosan-based NPs have been widely researched due to their mucoadhesive properties, which allow them to adhere to the gastric mucosa and deliver drugs directly to the site of infection. These NPs can encapsulate antibiotics such as amoxicillin, improving their stability and effectiveness against H. pylori in the acidic environment of the stomach [133]. Furthermore, the ability of chitosan NPs to provide controlled drug release ensures prolonged drug action at the infection site, enhancing bacterial inhibition while minimizing off-target effects. Surface modifications of chitosan NPs with targeting ligands have further enhanced their efficacy by directing them to specific sites of bacterial colonization [133‐135]. Additionally, pH-responsive coatings can be employed to ensure the controlled release of the encapsulated drug only within the acidic environment of the stomach, further enhancing the specificity and effectiveness of the therapy [136, 137]. A recent study by Grosso et al. introduced a novel approach using semi-interpenetrating polymer networks (semi-IPN) as gastroretentive drug delivery systems for H. pylori treatment. These biodegradable, super porous, and mucoadhesive matrices demonstrated sustained amoxicillin release at pH 1.2 and pH 5.0 over 24 h, ensuring prolonged drug retention in the stomach. Furthermore, formulations incorporating vonoprazan (a potassium-competitive acid blocker) alongside amoxicillin showed promise in enhancing drug stability and improving efficacy while mitigating the development of antibiotic resistance [138].

Lipid-based NPs, such as liposomes, solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) have also been explored for H. pylori treatment. These systems provide a biocompatible and biodegradable drug delivery platform with the ability to encapsulate both hydrophilic and hydrophobic drugs. A recent study by Lopes-de-Campos et al. demonstrated the potential of amoxicillin-loaded lipid NPs functionalized with dioleoylphosphatidylethanolamine (DOPE) for targeted H. pylori. These lipid NPs exhibited strong bacterial adhesion inhibition, increased gastric retention, and direct bactericidal activity, even in the absence of antibiotics. Additionally, the study introduced a novel floating system to prolong gastric retention time, ensuring sustained drug release and improved therapeutic efficacy [139]. SLNs and NLCs further offer improved drug stability, controlled release properties, and enhanced penetration of the gastric mucosal barrier, making them promising candidates for H. pylori treatment therapy [140, 141]. Despite these advancements, challenges remain, such as stability, gastrointestinal degradation, immune response, and the need for biodegradability and compatibility of NPs for clinical use. Future efforts aim to overcome these hurdles, promising wider biomedical applications and individualized precision therapy [142, 143].

Phage therapy, which uses bacteriophages (phages) and phage lysins, offers several advantages over traditional antibiotics. Phages exhibit high target specificity, minimizing disruption of the gut microbiome [144, 145]. Additionally, phages exhibit a unique characteristic benefit: they self-replicate at the infection site once they encounter their target bacteria. This ability to self-replicate at the infection site reduces the risk of off-target effects. Furthermore, phages have been shown to rapidly enter a death phase when the target bacteria are reduced, minimizing the risk of prolonged exposure and potential development of adverse immune responses [146]. Moreover, phages mutate more frequently than bacteria do, potentially aiding in the treatment of phage-resistant H. pylori strains [147].

Despite these advantages, limited research has been conducted on the use of phages for H. pylori. Recent studies show promise for phage-based therapies, as some phages isolated from wastewater (HPE1 and HPE2) have demonstrated the ability to adapt to the acidic environment of the human stomach and exhibit high thermal stability [148]. However, the harsh physiological conditions inside the stomach can compromise the effectiveness of most phages. The acidity of gastric juice and digestive enzymes can greatly affect the biological and structural composition of phages, thus reducing their proliferation and concentration at the infected site [149, 150]. Cuomo et al. demonstrated the effectiveness of lactoferrin-hydroxyapatite complex phages (Hp + LF-HA) in specifically killing H. pylori without harming host cells. Furthermore, hydroxyapatite nanoparticles have also been shown to increase phage stability and lytic activity within harsh gastric environments [151]. These advancements highlight the potential for developing targeted and effective phage therapies for H. pylori. While the advantages of phage therapy are compelling, limitations exist. The narrow cleavage spectrum of some phages might necessitate a cocktail approach for broader efficacy. Additionally, potential drawbacks include lysogenic phage-mediated transfer of toxins and antibiotic resistance genes, as well as bacterial endotoxin release upon lysis [144, 152]. Further research is needed to address these challenges and optimize phage delivery systems. Importantly, well-designed clinical trials are crucial for assessing the safety and efficacy of phage therapy for H. pylori treatment.

Antimicrobial peptides (AMPs) represent a naturally occurring and diverse class of gene-encoded proteins produced by a wide range of organisms. AMPs function as the first line of defense in the host innate immune response and play a critical role in combating microbial invasions [153]. Unlike conventional antibiotics that target specific bacterial processes, AMPs primarily exert bactericidal effects rather than bacteriostatic, as they disrupt the microbial cell membrane [154]. This multipronged approach to microbial treatment contributes to broad-spectrum activity and significant selectivity toward bacterial cells. Most AMPs are cationic because of the presence of positively charged residues such as arginine, lysine, and histidine. This positive charge allows AMPs to interact with the negatively charged bacterial cell membrane, leading to increased permeability, pore formation, and ultimately, cell lysis [155, 156]. Additionally, AMPs can traverse the bacterial cytoplasm and disrupt essential processes such as cell wall synthesis, DNA and RNA replication, protein synthesis, and cell division [153]. Furthermore, the relatively low cost of synthesis makes AMPs a potentially cost-effective therapeutic strategy against antibiotic-resistant H. pylori strains [155]. Compared with conventional antibiotics which have a single target, the complex mode of action of AMPs makes it significantly more challenging for bacteria to develop resistance [157]. This low incidence of resistance development positions AMPs as a promising strategy to combat multidrug-resistant (MDR) bacteria.

Several AMPs have demonstrated both in vitro and in vivo activity against H. pylori. Synthetic analogs such as pexiganan (MSI-78), derived from the frog skin peptide magainin 2, exhibit rapid bactericidal activity against H. pylori and synergize with beta lactams, potentially reducing required drug dosage and mitigating associated side effects [158, 159]. Tilapia piscidin 4 (TP4) from tilapia fish mast cells shows potent anti-H. pylori activity in vitro and reduces bacterial colonization in animal models, suggesting its potential for effective treatment and prevention of bacterial spread [160, 161]. Similarly, epinecidin-1 (Epi-1) derived from grouper fish manifests dose- and time-dependent bactericidal effects against H. pylori and has synergistic effects with antibiotics [162‐164].

Cathelicidins, a class of AMPs with immunomodulatory properties, represent another promising avenue for H. pylori treatment. Other AMPs, such as human LL-37 and its murine homolog cathelin-related antimicrobial peptide (CRAMP), have shown significant anti-H. pylori activity. Notably, human LL-37 exhibits increased resistance to the acidic stomach environment, making it a potentially well-suited candidate for H. pylori treatment [165‐167]. Furthermore, a study by Jiang et al. suggested that Cbf-K16, a cathelicidin-like peptide, has bactericidal effects on H. pylori at low pH and inhibits colonization in an animal model, making it a potentially well-suited candidate for H. pylori treatment [168]. Defensins, such as human neutrophil peptide 1 (HNP-1), and plant-derived AMPs, such as SolyC, have also demonstrated efficacy against H. pylori, including antibiotic-resistant strains [169, 170]. Bicarinalin, an AMP from ant venom, has dual modes of action: directly targeting H. pylori and inhibiting bacterial adhesion to gastric cells. This dual mode of action could significantly reduce bacterial colonization and persistence within the stomach lining [171, 172]. Odorranain-HP from the odorous frog possesses anti-H. pylori activity with low hemolytic potential, minimizing potential side effects [173]. PGLa-AM1, a cationic AMP from the African clawed frog, has rapid bactericidal activity against H. pylori [174‐176]. Finally, bacteriocins produced by certain probiotic bacteria, such as nisin A, pediocin BA28, and bulgaricus BB18, have been shown to have inhibitory effects on H. pylori strains [177‐180].

While these AMPs hold immense promise, certain limitations need to be addressed. Bacterial resistance to AMPs, although less common than resistance to conventional antibiotics, can occur through mechanisms such as cell envelope modification or AMP degradation [155]. Additionally, natural AMPs often suffer from proteolytic degradation, poor bioavailability, and short half-lives, necessitating the development of more stable analogs or delivery systems [113, 181]. Further research on their mechanisms of action, overcoming limitations, and optimizing delivery methods is crucial to fully understand their therapeutic potential in the fight against H. pylori infection.

Vaccination offers a compelling approach to prevent H. pylori infection and reduce the associated disease burden. While no licensed H. pylori vaccine exists yet, recent advancements in vaccine development technologies and a deeper understanding of H. pylori pathogenesis hold promise for overcoming past challenges. Preclinical studies have yielded encouraging results with novel vaccine technologies such as vector-based vaccines using bacteria and multiepitope vaccines carrying specific T and B cells. Vector-based vaccines, particularly those utilizing live bacteria, have demonstrated the ability to elicit a robust immune response due to their efficient delivery and recognition by the immune system [182]. Multiple epitope vaccines, which are designed to target a broader range of H. pylori antigens, offer an advantage in combating strains with diverse antigenic profiles [183]. Additionally, vaccines targeting BabA, an adhesin molecule crucial for H. pylori adherence to the gastric epithelium, have shown promise in animal models. The BabA vaccine has the ability to offer protection against severe gastric disease even without complete H. pylori suppression, suggesting its potential to reduce disease burden independent of bacterial clearance [184].

Despite these advancements, several challenges remain in H. pylori vaccine development. Elucidating the optimal T-cell polarization (Th1 vs. Th2) required for effective H. pylori clearance is crucial for vaccine design [185, 186]. Additionally, the increased cost of clinical trials, particularly in developing countries with limited resources and expertise, presents a significant hurdle to progress. Continued research on novel vaccine technologies, a deeper understanding of the immune response to H. pylori and increased global collaboration in clinical trials are essential steps toward the development of safe and effective H. pylori vaccines. Despite the promising potential of alternative antibiotic-free therapies for combating H. pylori, significant limitations hinder their effectiveness and reliability. Plant-based compounds, while showing anti-H. pylori activity in vitro lacks FDA approval due to insufficient clinical evidence and standardization. Probiotics, although beneficial, face challenges such as variable efficacy and undefined optimal strains and dosages. Nanoparticle-based treatments encounter issues of stability, gastrointestinal degradation, and immune response. Although highly specific, phage therapy requires overcoming the harsh gastric environment and potential safety concerns. Antimicrobial peptides, though potent, suffer from proteolytic degradation and limited bioavailability. Vaccines for H. pylori are still in developmental stages, and no licensed options are available. These limitations underscore the critical need for effective anti-urease compounds, which offer a targeted, scientifically validated approach to H. pylori inhibition, ensuring better clinical outcomes and patient safety (Fig. 5) (see Table 1).

Bild vergrößern

Natural compounds found in plants, animals, fungi, and microorganisms possess diverse biological activities, making them important in fields such as medicine, where they are often used as the basis for drug development owing to their therapeutic properties. Owing to their bioactive compounds, such as phenolics, terpenoids, and alkaloids, plants are a significant source of natural urease inhibitors [194]. These inhibitors are not only environmentally benign but also potentially more effective because of their biodegradability and synergistic actions. Studies have investigated various plant extracts for their anti-H. pylori and urease inhibitory activities. Amin et al. evaluated the effects of extracts of Acacia nilotica, Calotropis procera, Adhatoda vasica, Fagonia arabica, and Casuarina equisetifolia against H. pylori [195]. These findings which suggest competitive inhibition of A. nilotica and mixed-type C. procera, support the traditional use of these plants for stomach ailments and highlight their potential as natural anti-H. pylori agents [195].

Another study aimed to identify the antibacterial and urease inhibitory effects of Oliveria decumbens extracts and fractions against H. pylori and other bacteria. Researchers isolated three new kaempferol derivatives and two thymol derivatives from plants and reported that the n-hexane fraction exhibited significant anti-H. pylori activity. Stigmasterol, tiliroside, and carvacrol were the most potent urease inhibitors identified. This work aligns with the traditional use of Oliveria decumbens to treat gastrointestinal infections [196]. Coptisine, the main alkaloid in Rhizoma Coptidis, was investigated for its urease inhibitory effects in an interesting study [197]. This study demonstrated the antibacterial and bactericidal effects of coptisine against H. pylori strains, including those resistant to antibiotics. Notably, the minimum inhibitory concentration (MIC) of Coptisine against H. pylori ranged from 25 to 50 μg/mL. Interestingly, coptisine disrupts urease maturation by inhibiting the activity of UreG, a key accessory protein required for urease assembly. It specifically hinders urease’s ability to form dimers and bind nickel, effectively preventing the formation of functional urease enzymes. This multifaceted action suggests a comprehensive inhibitory effect on both urease activity and its maturation process [197].

Another natural protoberberine alkaloid, epiberberine, has also been investigated for its urease inhibitory properties. Epiberberine has been identified as the most potent urease inhibitor, exhibiting significant inhibition at low micromolar concentrations. Research indicates that its mechanism of action involves slow-binding, competitive inhibition by directly targeting the enzyme’s active site [198]. Further studies have investigated the inhibitory effects of evodiamine, a compound derived from Evodia rutaecarpa, on H. pylori growth and inflammation. Evodiamine effectively suppresses H. pylori by downregulating genes essential for its replication, transcription, and urease production. Additionally, it diminishes the translocation of cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA) into gastric cells, contributing to reduced inflammation by inhibiting NF-κB and MAPK pathway activation [199]. Another example of a natural compound with anti-urease activity is hespertin, which is a flavonoid found in citrus fruits. Hesperetin significantly inhibits H. pylori growth by downregulating genes involved in bacterial replication, transcription, motility, and adherence [187]. It reduces urease activity, H. pylori colonization and survival in the gastric mucosa. Additionally, hesperetin was also found to inhibit, similar to evodiamine, the translocation of the virulence factors cagA and vacA into gastric cells, which are key to H. pylori pathogenicity [200]. On the other hand, Canarium album fruits have emerged as promising natural sources for the development of new treatments for H. pylori infection. A study by Jiahui Yan et al. revealed that an ethyl acetate extract notably inhibited H. pylori growth and urease activity while downregulating key virulence genes such as vacA and cagA [201]. Another natural compound, zerumbone, extracted from Zingiber zerumbet, has unique effects on HPU activity through dimerization, trimerization, or tetramerization with urease molecules without affecting any urease gene transcription or urease protein expression [202]. Among the natural anti-urease compounds, flavonoids have shown promising results through molecular docking and dynamics simulations alongside analyses of their physicochemical properties and toxicity. For example, flavonoids, specifically chrysin, galangin, kaempferol, luteolin, morin, and quercetin, demonstrate a stronger binding affinity to urease than traditional inhibitors, such as acetohydroxamic acid (AHA) [100]. Motivated by the therapeutic potential of quercetin for the treatment of gastric ulcers, Zhu-Ping Xiao et al. investigated its urease inhibitory activity against H. pylori, along with its analogs. The findings revealed the remarkable inhibitory potency of quercetin, with molecular docking and kinetics suggesting a noncompetitive inhibition mechanism [203]. To further research flavonoids, Izabela Korona-Glowniak et al. investigated the efficacy of 26 different commercial essential oils for their MICs against the H. pylori ATCC 43504 strain. The thyme, lemongrass, cedarwood, and lemon balm oils presented the most potent anti-H. pylori activities, with MIC values as low as 15.6 µg/ml. This study not only expands the series of natural agents used against H. pylori but also underscores the potential of essential oils, particularly cedarwood and oregano oils, to inhibit urease activity at sub-MIC concentrations [204]. Another study investigated the effects of acetohydroxamic acid (AHA), baicalin, and ebselen on H. pylori urease activity, bacterial survival, and gene expression. AHA and ebselen strongly inhibited urease activity at low doses, reducing it by 84% and 71%, respectively, whereas baicalin requires relatively high doses to achieve similar effects. All three compounds significantly reduced bacterial viability, with AHA being the most effective. Interestingly, the inhibitors caused an increase in ureA and ureB gene expression, suggesting a survival response by the bacteria. Baicalin also reduced ATP production, whereas AHA and ebselen did not. An analysis of clinical isolates revealed differences in urease activity but no link to infection severity [205]. An innovative strategy for managing H. pylori involves the use of nanoparticles. A previous study demonstrated this by synthesizing silver nanoparticles (AgNPs) from Ficus carica leaf extract. These AgNPs manifested significant inhibitory activity against the urease enzyme, suggesting promising potential for H. pylori treatment via the integration of traditional plant extracts with nanotechnology [206]. Another study employed green synthesis methods to create silver nanoparticles from Solanum xanthocarpum berry extract. These nanoparticles exhibited potent anti-H. pylori activities and significant urease inhibitory effects. This innovative approach aligns with green chemistry principles and supports the potential of silver nanoparticles as antibacterial agents, particularly against H. pylori infections [207] (Among anti-urease compounds, Acetohydroxamic Acid (AHA) is the only agent that reached clinical use, primarily for urease-positive urinary tract infections. However, due to its toxicity profile and limited gastric applicability, it was not advanced further for H. pylori therapy) (Table 2).

Semisynthetic substances derived from naturally occurring compounds through chemical modifications can enhance or modify the properties of the original compound, thus increasing its suitability for pharmaceutical development. Compared to their natural counterparts, semisynthetic derivatives can offer improved efficacy, stability, or safety profiles. Sulforaphane (SF), a compound derived from broccoli and other crucifers, is an example of this approach. Fahey et al. reported that SF inactivation of urease follows first-order kinetics, which are dependent on the enzyme and SF concentrations. Their study also provided evidence of dithiocarbamate formation between SF and the cysteine thiols of urease [213]. Another study explored the synthesis and molecular docking of morin analogs to identify potent urease inhibitors and antioxidants. This research identified N-(2-chlorophenyl)-N-((4E)-2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-ylidene) thiourea (M2b) and N-(4-bromophenyl)-N-((4E)-2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-ylidene) thiourea (M2i) as potent urease inhibitors and antioxidants [214]. Xiao et al. presented a study on the synthesis and antibacterial properties of palmatine (PMT) derivatives against H. pylori, with a focus on strains resistant to metronidazole. This finding reveals that introducing a secondary amine at the 9-position of PMT derivatives could increase their anti-H. pylori potency. These derivatives are exceptionally effective against metronidazole-resistant H. pylori strains, with MICs ranging from 4 to 16 µg/mL [215]. Among the reductive derivatives of compound W, the 4-deoxy analogs exhibit the most potent inhibitory effects. Specifically, the compound 4′7,8-trihydroxyl-2-isoflavene emerged as the most active, with an IC₅₀ value of 0.85 mM, indicating greater than 20-fold greater potency than AHA, a commercial urease inhibitor [216] (Table 3).

Synthetic compounds are artificially created chemicals or substances rather than being naturally occurring. They are designed and manufactured through specific chemical processes in laboratories or industrial settings. A literature review of anti-urease compounds revealed that many of them are synthetic, as shown in Table 4. The synthesis and evaluation of a series of novel amantadine-thiourea conjugates (3a–j) as inhibitors of jack bean urease identified N-(adamantan-1-ylcarbamothioyl) octanamide (3j), with a 7-carbon alkyl chain, as the most promising urease inhibitor candidate, with an IC₅₀ of 0.0085 µM. This finding highlights the importance of the alkyl chain length within this series for enzyme inhibition. Molecular docking analysis supported these findings by showing good binding affinity with the target protein. The study concluded that this compound, owing to its noncompetitive mode of inhibition and strong binding affinity, could serve as a lead structure for designing more potent urease inhibitors [217] (Table 4).

Richa Arora et al. leveraged computational tools to discover potential urease inhibitors against H. pylori. Their approach utilized pharmacophore modeling, virtual screening, and molecular docking to identify four promising compounds from known inhibitors. This research represents an advancement in combating H. pylori infection by demonstrating the power of computational chemistry in drug discovery and opening new avenues for therapeutic intervention [218] (Table 4). This study navigates through the chemical space of N-acylglycino- and hippurohydroxamic acid derivatives, known urease inhibitors, to construct a robust three-featured pharmacophore model. Among their notable discoveries, miglitol and marimastat have emerged as the most potent compounds, exemplifying the power of targeted chemical investigations in unveiling potential therapeutic agents. This work sets a new standard in the quest for effective treatments against H. pylori [218].

A new class of urease inhibitors combines nitrogen-containing heterocycles. This study demonstrated the synthesis of carbazole-triazine hybrids, with compounds with bulky iodo- and strong electron-withdrawing nitro groups exhibiting the highest activity and potent urease inhibition [219]. Another study highlighted the potential of virtual screening and molecular dynamics simulations for discovering new anti-urease therapeutic agents. They employed rapid overlay of chemical structures (ROCS) shape-based screening and molecular docking software to identify several potential urease inhibitors from over 1.83 million compounds, specifically those that target antibiotic-resistant H. pylori strains [220]. In another study, the synthesis and evaluation of arylmethylene hydrazine derivatives for urease inhibition were investigated. These compounds exhibited significant activity, with IC₅₀ values ranging from 0.61 to 4.56 µM, surpassing those of standard inhibitors such as hydroxyurea and thiourea. Notably, a compound featuring a 2-nitrobenzylidene group displayed the highest potency [221]. Another study investigated the role of specific functional groups in urease inhibition by evaluating series containing 4-dihydropyrimidine-2-thiones, N,S-dimethyl-dihydropyrimidines, hydrazine derivatives of dihydropyrimidine, and tetrazolo dihydropyrimidine derivatives. This research suggests that the free sulfur atom and hydrazine moiety might be key pharmacophores for inhibiting the H. pylori urease enzyme [222]. By utilizing large-scale virtual screening of the ZINC database, Azizian et al. selected compounds on the basis of their docking energy calculations for further in vitro screening against H. pylori urease. Their findings revealed that barbituric acid and certain derivatives, particularly those with para substitution on the benzylidene moiety, exhibited greater urease inhibitory activity than did the standard inhibitor hydroxyurea. This study aimed to identify new scaffolds for H. pylori urease inhibition, potentially leading to novel therapies for managing H. pylori infections. Barbituric acid derivatives have emerged as promising candidates because of their superior potency and potential ability to chelate with the enzyme’s active site [223]. Building upon previous research, Biglar et al. synthesized a series of NN-dimethylbarbituric-pyridinium derivatives and evaluated their potency as H. pylori urease inhibitors. The results demonstrated that these compounds were more effective than the standard inhibitors hydroxyurea and thiourea. This work offers promising new candidates for H. pylori urease inhibition [224]. Recent studies have explored various metals for their ability to inhibit urease enzyme activity. Among these complexes, copper (II) complexes have emerged as promising candidates. Investigations by Cui et al. into two such complexes revealed their substantial inhibitory action against H. pylori urease, surpassing the standard inhibitor in effectiveness. This highlights the potential of metal complexes for the development of new H. pylori treatments [225]. Another study synthesized six copper complexes with IC₅₀ values ranging from 0.20 to over 100 μM. The study also incorporated molecular docking studies to elucidate the mechanism of inhibition, concluding that copper complexes with square planar coordination are more effective urease inhibitors [226]. To expand the exploration of metal-based urease inhibitors, another study investigated four new cobalt (III) complexes with Schiff bases. This work contributes to ongoing research on metal complexes as potential urease inhibitors [227].

Among the limited in vivo studies conducted on urease inhibitors against H. pylori, Liu et al.’s research stands out. They analyzed aniline-containing hydroxamic acids and identified three potent compounds: 3-(3,5-dichlorophenylamino)-N-hydroxypropanamide, 3-(2-chlorophenylamino)-N-hydroxypropanamide, and 3-(2,4-dichlorophenylamino)-N-hydroxypropanamide. These compounds have demonstrated remarkable efficacy in both in vitro and in vivo studies, with complete eradication of H. pylori in mice models [228]. Researchers synthesized novel imidazothiazole derivatives containing sulfonate and sulfamate groups, which significantly inhibited urease activity. The most potent compound demonstrated an IC₅₀ of 2.94 ± 0.05 μM, outperforming the control by eightfold. An enzyme kinetics study revealed it to be a competitive inhibitor. Phenotypic screening against H. pylori identified several high-potency molecules, with compound 1d emerging as a lead candidate due to its promising inhibition profile, minimal activity against urease-negative E. coli, and low permeability in Caco-2 cells, suggesting its suitability for gastrointestinal infections without systemic effects [229]. Thirteen novel alkylated benzimidazole 2-thione derivatives were synthesized because of their urease activity toward H. pylori. The most potent compounds have potential as lead candidates for developing future anti-urease agents [230]. A novel high-throughput screening assay was developed for detecting submicromolar urease inhibitors among clinically used drugs. This assay identified panobinostat, dacinostat, ebselen, and disulfiram as effective inhibitors against plant or bacterial urease, suggesting their potential for treating H. pylori infections, particularly those resistant to antibiotics [231]. In another study, 33 (N-aryl-N-arylsulfonyl)aminoacetohydroxamic acids were synthesized to target urease inhibition in H. pylori. One compound, 2-(N-(3-nitrophenyl)-N-(4-tert-butylphenylsulfonyl)) aminoacetohydroxamic acid, displayed exceptional activity with minimal cytotoxic effects on mammalian cells. Its effectiveness, exceeding 690-fold that of AHA, is attributed to its reversible mixed-mode inhibition of urease, as demonstrated by molecular modeling and docking studies [232]. Another investigation of N-monoarylacetothioureas as anti-urease agents targeting H. pylori has demonstrated significant in vitro urease inhibition with low cytotoxicity, showing promise for further development in treating H. pylori-related diseases. The efficacy of this compound is notably greater than that of AHA, making it a potential candidate for H. pylori treatments [233]. Researchers have identified numerous potential targets for anti-urease agents by delving into the molecular intricacies of urease. This innovative approach led to the identification of the metallochaperone UreG as a novel antimicrobial target against H. pylori. This study utilized colloidal bismuth subcitrate to inhibit urease activity by disrupting the GTPase activity of UreG, thereby halting urease maturation and offering a fresh perspective on antimicrobial development [75]. A novel class of bismuth hydroxamate complexes was specifically designed to inhibit urease activity. By synthesizing and testing these complexes, their potential in combating H. pylori infections was demonstrated, leveraging both the antibacterial properties of bismuth and the urease-inhibitory capabilities of hydroxamic acids. This approach is promising for the development of effective treatments against H. pylori, particularly in light of increasing antibiotic resistance [234]. Furthermore, the synthesis of piperazine derivatives was evaluated for their potential as urease inhibitors. The synthesized derivatives exhibited excellent inhibitory potential, with IC₅₀ values ranging from 1.1 to 33.40 µM, surpassing those of the standard inhibitor thiourea. This research established a structure‒activity relationship on the basis of the substitution pattern on the phenyl ring and utilized molecular docking to understand the binding interactions within the enzyme's active site [235]. In another study, thiosemicarbazide derivatives of isoniazid were also investigated, and the results revealed a dual inhibitory effect on both inflammation and urease activity. Among these derivatives, 2- isonicotinoyl-N-(perfluorophenyl) hydrazinecarbothioamide has emerged as the most effective dual inhibitor, suggesting a new approach for developing treatments for H. pylori–related conditions [236]. Another study focused on synthesizing twenty 4-thiazolidinone analogs and examining their urease inhibitory activity. These analogs exhibited a broad range of inhibitory effects, with IC₅₀ values between 1.73 and 69.65 µM, indicating varying levels of effectiveness against urease compared with the standard inhibitor thiourea (IC₅₀ value of 21.25 µM). Research has further explored the structure‒activity relationship, highlighting the influence of substituent patterns on phenyl rings on urease inhibition efficacy [237]. Moreover, researchers have investigated barbituric acid derivatives as potential urease inhibitors and synthesized thirty-two zwitterionic adducts of diethyl ammonium salts. In vitro tests against jack bean urease identified two compounds (4i and 5 l) as the most effective, surpassing the standard inhibitor thiourea [238].