Protease Research Articles

Development of lipopeptide-based HIV-1/2 fusion inhibitors targeting the gp41 pocket site with a new design strategy

Emerging studies demonstrate that lipid conjugation is a vital strategy for designing peptide-based viral fusion inhibitors, and the so-called lipopeptides exhibit greatly improved antiviral activity. In the design of lipopeptides, a flexible linker between the peptide sequence and lipid molecule is generally required, mostly with a short polyethylene glycol or glycine-serine sequence. Very recently, we discovered that the helix-facilitating amino acid sequence "EAAAK" as a rigid linker is a more efficient method in the design of SARS-CoV-2 fusion inhibitory lipopeptides. In this study, we comprehensively characterized the functionalities of different linkers in HIV fusion inhibitors. A short-peptide inhibitor 2P23, which mainly targets the gp41 pocket site, was used as a design template, generating a group of cholesterol-modified lipopeptides. In the inhibition of HIV-1 infection, the lipopeptide inhibitors with a rigid linker were much superior than those with the flexible linkers, as indicated by LP-37 with the "EAAAK" linker and LP-39 with the repeated "EP" amino acid sequences. Both lipopeptides were very potent inhibitors of HIV-2 and simian immunodeficiency (SIV) either. Promisingly, LP-37 displayed high α-helicity, thermostability and binding ability to a target-mimic peptide, and it was metabolically stable when treated with temperature, proteolytic enzymes or human sera. Taken together, our studies have verified a universal strategy for designing viral fusion inhibitors and offered a novel HIV fusion inhibitor for drug development.

Molecular Characterization and Antibiotic sensitivity of Staphylococcus sp. Isolated from Animal Skin wounds

A wide variety of commensal microorganisms reside on the skin, many of which secrete proteolytic enzymes that might have detrimental effects following an injury. These bacteria are considered opportunistic pathogens. The objective of the study is to isolate Staphylococcus sp. from animal skin and conduct molecular characterization of the recovered strains in order to examine their enzymatic production capabilities. The objective is to assess the antibiotic susceptibility of Staphylococcus sp. and determine the ideal conditions for bacterial development. A total of 25 swab samples were obtained from animal skin wounds in AL-Shatrah city. Each sample was cultured on nutrient agar, blood agar, and Mannitol salt agar (with a 7.5% NaCl concentration) as a specialized medium. All isolates underwent molecular diagnosis using the PCR technique. Additionally, 10 isolates were selected for sequencing using the Sanger dideoxy 16S rRNA sequencing method. Enzyme production properties were analyzed, and the sensitivity of bacteria to six antibiotics was studied. Additionally, the researchers investigated the ideal circumstances for bacterial proliferation. Analysis of 25 swab samples taken from animal skin wounds yielded a total of 18 isolates. Based on biochemical tests, the majority of these isolates were identified as Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, and Staphylococcus borealis. Staphylococcus warneri and Staphylococcus epidermidis exhibited sensitivity to Novobiocin, with inhibition zones of 22mm and 21mm respectively. However, all other isolates that were examined showed resistance to novobiocin.

Identification of the enzyme activity of human Demodex aspartic protease and its function to hydrolyse host macromolecules and skin cell proteins

Aspartic protease (ASP), a common proteolytic enzyme, plays an important role in the pathogenesis of numerous parasites. However, its role in Demodex remains unclear. Herein, we studied the expression, purification, enzymatic activity detection, and hydrolysis function of human Demodex ASP. The findings showed that recombinant ASP (rASP) possessed aspartic protease activity, which reached optimum levels at pH 2.5–3.0 and 35 °C. Furthermore, the activity of Demodex folliculorum rASP (Df. ASP) was considerably higher than that of Demodex brevis rASP (Db.rASP). Df.rASP also exhibited a more potent hydrolytic ability than Db.rASP. Df.rASP hydrolysed IgG, IgM, and fibronectin, whereas Db.rASP only slightly hydrolysed IgG. Mass spectrometry analysis revealed that Df.rASP exerted hydrolytic effects on 38 HaCaT proteins, more than the 23 proteins hydrolysed by Db.rASP. Sequence alignment and structure modelling of the substrate binding cleft identified three distinct amino acids between Df. ASP and Db. ASP, which should be the molecular basis for their difference in enzymatic activity and hydrolytic function. These results imply that Df.rASP may play a more critical role in the pathogenesis of human Demodex, and molecular data will provide a scientific basis for future analyses of their molecular pathogenesis.

Degradation of gluten using plant proteases

The paper describes the fact of the presence of a proteolytic enzyme in the leaves of the natural hybrid variety of grape - Isabella (Vitis Labrusca L.) and the perspective of its practical application for breaking down wheat gluten. As our research has shown, this enzyme effectively degradates the constituent components of the protein fraction of wheat flour-the gluten fraction. It is known that the wheat gluten is a strong allergen for many people, which can initiate a disease such as celiac disease. Protease from Isabella leaves carries out deep hydrolysis of gluten to low molecular weight soluble peptides and amino acids. This effect can be successfully used in the production of gluten-free bread products.

Effects of microplastics on the physiology of living organisms on the example of laboratory reared bloodsucking mosquitoes Aedes aegypti L.

AbstractThe presence of environmental microplastics (MPs) poses a significant threat to terrestrial and aquatic animals, including insects such as blood‐sucking mosquitoes. The paper reports on the laboratory study of the effect of three different types of MPs, including fragmented high‐density polyethylene (HDPE), polypropylene (PP) and polystyrene (PS), on the viability, innate immune responses, activity of detoxifying enzymes and malondialdehyde (MDA) concentration in Aedes aegypti (Linnaeus, 1762). The results showed that dietary administration of microfragments of PP, PS and HDPE at low concentrations (4 mg/L) had no effect on the survival rate of mosquito larvae, but was observed to suppress the larval immune response. The addition of MPs to the diet resulted in a significant suppression of phenoloxidase activity compared to the control. A decrease in the activity of the detoxifying enzymes glutathione‐S‐transferase and non‐specific esterase was observed. Dietary administration of MPs did not cause any significant change in alkaline proteolytic enzyme activity in larvae compared to the control. However, we observed a twofold increase in the activity of acid proteolytic enzymes in all experiments compared to the control (p < 0.05). MDA levels in larval homogenates remained unchanged, while lysozyme‐like activity showed a slight decrease compared to the control. The observed processes may be a consequence of intestinal obstruction by MPs, which may cause microtraumas to intestinal tissues and changes in the structure and composition of the microbiota. These changes may have a profound effect on the resistance of mosquito larvae to insecticides and pathogens.

Unveiling the potential of a protease inhibitor from Pleiogynium cerasiferum to combat Helicoverpa armigera by employing sequential purification and multivariate statistical analysis

Crop protection is heavily dependent on the use of chemical pesticides that are not only toxic but also detrimental to the environment. To minimize the chemical approaches of crop protection, this study investigates a natural protease inhibitor PCPI (Pleiogynium cerasiferum protease inhibitor) extracted from the seeds of Pleiogynium cerasiferum as a viable bioresource with biopesticidal properties. PCPI demonstrated inhibitory activity of 92% against bovine trypsin and 89% against Helicoverpa armigera's midgut proteolytic enzymes. Characterization of PCPI revealed a 24 kDa protein with critical inhibitory concentrations of 1.53 µg/ml for H. armigera midgut proteases and 1.31 µg/ml for bovine trypsin. At the concentration of 0.5% (w/w) of PCPI, the deformities in pupae were ~ 82%, adult insects ~ 87%, and egg-laying as well as egg hatching capacity dropped by ~ 81% and 17% respectively. Principal component analysis (PCA), complemented by hierarchical clustering, was applied to assess the nuanced effects of PCPI across the pest's lifecycle. The analysis identified deformities in pupae, adult, adult emergence, and notably, egg laying reduction as variables most responsive to PCPI concentrations. Hierarchical clustering analysis corroborated these PCA findings indicating a concentrated influence on developmental stages and reproduction metrics, signifying the multifaceted impact of PCPI. These insights affirm PCPI's promise as a cornerstone of eco-friendly pest management strategies.

One-step ultra-rapid immunoassay of calcitonin gene-related peptide for migraine diagnosis

Migraine is known to be caused by calcitonin gene-related peptide (CGRP), prompting the need for quantitative analysis of CGRP for the clinical treatment of monoclonal antibodies targeting CGRP. Since CGRP is cleaved by proteolytic enzymes post-blood collection, rapid analysis methods are required. In this study, a one-step immunoassay for CGRP was developed using chemically mimicking peptides (mimotopes) with an analysis time of 32 min. Four clones from an Fv-antibody library were screened using two types of monoclonal antibodies against CGRP. Mimotopes for each monoclonal antibody were synthesized into peptides of 15 residues. The binding affinity (KD) was estimated, and the interaction with monoclonal antibodies was analyzed using docking simulations. Finally, a one-step immunoassay for CGRP was demonstrated using migraine patient samples (n = 57) and healthy volunteer controls (n = 18). The limit of detection (LOD) of one-step immunoassay based on Fremanezumab (mimotope F1) was estimated to be 8.8 pg/mL with the limit of quantification (LOQ) of 125.9 pg/mL. And, the one-step immunoassay based on Galcanezumab (mimotope G7) showed the LOD of 9.4 pg/mL and the LOQ of 84.7 pg/mL. The total analysis time was estimated to be approximately 32 min and the assay results were estimated to be statistically consistent with conventional CGRP assay.

Modification of adipogenesis and oxidative stress by quercetin: positive or negative impact on adipose tissue metabolism of obese diabetic Zucker rats?

Reactive oxygen species (ROS) play a key role in the regulation of adipogenesis. The aim of our study was to investigate the effect of quercetin (QCT) supplement on obese adipose tissue metabolism of 30-week-old diabetic Zucker rats (ZDF), not well examined yet. QCT was administered orally at dose of 20mg/kg body weight/day for 6 weeks. Adipocytes from subcutaneous adipose tissue (ScWAT) were isolated and their size was evaluated by light microscopy. Gene expression of adipogenic markers in subcutaneous and visceral adipose tissue was determined by real-time PCR and expression of proteins involved in lipid and glucose metabolism was determined in ScWAT by immunoblotting. Obese ZDF rats suffered from diabetes, hyperinsulinemia and had higher index HOMA-IR (Homeostatic Model Assessment for Insulin Resistance). Treatment with QCT had no significant impact on these metabolic disorders in genetic model of obesity and type 2 diabetes used in our study. Nevertheless, QCT reduced expression of inflammatory cytokine tumour necrosis factor alpha in ScWAT and also visceral adipose tissue and up-regulated expression of anti-inflammatory adiponectin in ScWAT. A shift in redox equilibrium was detected via inhibition of pro-oxidant genes by QCT. Furthermore, QCT reduced adipocyte size in ScWAT, down-regulated expression of fatty acid synthase and adipogenic markers, and moreover stimulated expression of proteolytic enzymes. These changes likely resulted in reduced fat deposition in ScWAT, which was reflected in the elevated circulated levels of free fatty acids in QCT-treated obese ZDF rats compared with obese untreated controls. This increase could, at least in part, explain why we did not observe an improvement in systemic metabolic health by QCT in our model. In conclusion, our study suggests that preventive treatment with QCT might be more effective than its administration in the stage of fully developed diabetes, and further research in this area is needed.

Role of Metalloproteinases in Adhesion to Radicular Dentin: A Literature Review.

Root dentin is a porous and complex dental surface that may have irregularities and deposits of organic material. To achieve an effective bond between restorative materials and root dentin, it is necessary that the restorative materials adhere intimately to the dentin surface. Metalloproteinases (MMPs) are a group of proteolytic enzymes that perform an important role in degrading the extracellular matrix and remodeling connective tissue. The aim of this research was to determine the scientific evidence available on the role played by MMPs in adhesion to root dentin and their putative inhibitors. Several techniques have been used to evaluate the presence of MMPs in the root dentin of human and bovine teeth, such as Western blot, immunohistochemistry, immunofluorescence, and zymography, the latter also being used together with the EnzCheck assay to evaluate the inhibitory effect of adhesion protocols on the activity of root MMPs in vitro. When analyzing the databases, 236 articles were found, 12 of which met the selection criteria. The variables analyzed were articles that evaluated different MMP inhibitors in root dentin. In the adhesion to radicular dentin, MMPs have a crucial role in the degradation of the extracellular matrix of dentin and the remodeling of the dentin surface because excessive MMP activity can be harmful to dental health, since excessive degradation of the extracellular matrix of dentin can weaken the tooth structure and decrease fracture resistance. Therefore, it is important to monitor MMP activity during root dentin bonding procedures.

Changes in Protease Activity of Ginger Rhizome during Postharvest Storage

It has been reported that ripe ginger rhizomes contain proteolytic enzymes. In this study, we investigated the fluctuations in protease activity from ginger rhizomes stored after harvest for the utilization of proteases. The juice from ginger rhizomes that had been stored for 4 months after harvest exhibited the highest specific protease activity. Additionally, juices with high specific protease activity could form soymilk and cow milk gels. Electrophoresis experiments revealed that the protein bands in ginger rhizome juice were degraded by the proteases contained in the juice. This degradation was prevented by adding the cysteine protease inhibitor E-64.

Trypsin from digestive tract of harpiosquillid mantis shrimp: Molecular characteristics and the inhibition by chitooligosaccharide and its catechin conjugate.

Trypsin from the digestive tract of harpiosquillid mantis shrimp (HMS) was purified using ammonium sulfate precipitation and a soybean trypsin inhibitor-CNBr-activated Sepharose 4B affinity column. The purified trypsin (PTRP-HMS) had a purity of 30.4-fold, and a yield of 14.5% was obtained. PTRP-HMS had the molecular weight of 23.0kDa as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and only one isoform was detected by native-PAGE. Its optimal temperature and pH were 55°C and 8.5, respectively. TLCK almost completely inhibited the activity of trypsin. The PTRP-HMS had a Michaelis-Menten constant (Km) and catalytic constant (Kcat) of 0.87mM and 13.04s-1, respectively, toward Nα-benzoyl-l-arginine 4-nitroanilide hydrochloride. When chitooligosaccharide (COS) and COS-catechin (COS-CAT) conjugates were examined for inhibition toward the PTRP, the latter exhibited higher efficacy in inhibiting the trypsin. Both COS and COS-CAT conjugate showed mixed-type inhibition kinetics. As a consequence, COS and COS-CAT conjugate could be used as natural additives for inhibiting trypsin in whole HMS, thus retarding the softening and lengthening the shelf-life of HMS during the iced storage. PRACTICAL APPLICATION: Harpiosquillid mantis shrimp (HMS) is of high demand due to its delicacy. However, its meat undergoes rapid softening within 2-3 days when stored in ice. Understanding causative proteolytic enzymes, especially trypsin from digestive tract, paves a way for preventing their negative impact on HMS eating quality. Employment of safe inhibitors, for example, chitooligosaccharide (COS) or COS conjugated with catechin, could inhibit HMS trypsin. Overall, softening of whole HMS containing trypsin in its digestive tract can be impeded, especially when treated with COS-CAT. This finding is beneficial for the HMS local vendor or exporter, in which HMS quality can be maintained.

From Beer to Cheese: Characterization of Caseinolytic and Milk-Clotting Activities of Proteases Derived from Brewer's Spent Grain (BSG).

This study explores the extraction and characterization of proteolytic enzymes from brewer's spent grain (BSG) and their potential as sustainable coagulants in the dairy industry. BSG samples from various beer types (Blonde Ale, IPA, Kölsch, Honey, and Porter) were obtained from two artisanal breweries in Mar del Plata, Argentina. Optimization of caseinolytic activity (CA) and protein extraction was conducted using a Plackett-Burman design, followed by a Box-Behnken design. Optimal protein concentration was achieved at intermediate pH and high temperature, while CA peaked at pH 8.0. The specific caseinolytic activity (SCA) varied among the extracts, with BSG3 showing the highest activity (99.6 U mg-1) and BSG1 the lowest (60.4 U mg-1). Protease inhibitor assays suggested the presence of aspartic, serine, metallo, and cysteine proteases. BSG3 and BSG4 showed the highest hydrolysis rates for α-casein (70% and 78%). For κ-casein, BSG1, BSG2, and BSG3 demonstrated moderate activity (56.5%, 49%, and 55.8), while BSG4 and BSG5 exhibited the lowest activity. Additionally, the milk-clotting activity (MCA) of BSG extracts was comparable to plant-based coagulants like Cynara cardunculus and Ficus carica. These findings highlight the potential of BSG-derived proteases as alternative coagulants for cheese production, offering a sustainable link between the brewing and dairy industries.

Identification and characterization of cysteine-rich polycomb-like protein (CPP) gene family in rice (Oryza sativa L.) in response to phytohormones and Xanthomonas oryzae pv. oryzae stress

Cysteine-rich polycomb-like proteins (CPPs) are found in both monocotyledonous and dicotyledonous plants. These CPPs are part of nine various families of proteolytic enzymes, and they play a crucial role in various physiological, especially defence mechanisms. Nevertheless, there is a lack of findings regarding the function of CPPs in disease resistance in rice (Oryza sativa). We recently discovered eleven CPPs in the rice genome. They can be divided into three clades: I, II, and III along with CPPs from other plants like Arabidopsis thaliana (eight members), Cucumis sativus (five members), and Glycine max (thirteen members) based on their phylogenetic relationships. It is interesting to observe how segmental duplication (SD) has assisted the emergence of the OsCPP genes in rice, whereas no evidence of tandem duplication (TD) has been detected. Synteny analyses revealed three pairs of orthologous CPP genes amongst O. sativa and A. thaliana, as well as three pairs between O. sativa and C. sativus. These OsCPPs possess ten conserved amino acid motifs and several exons and introns, as determined by their gene structures. Additionally, they possess distinctive domains such as TCR and TCR superfamily domains. Cis-regulatory elements identified in the OsCPP promoter sequences were shown to be sensitive to auxin, abscisic acid, gibberellins, salicylic acid, methyl jasmonate, and light. Furthermore, stress-responsive elements and tissue-specific expression elements were identified. A large number of OsCPP genes are expressed in different kinds of tissues and organs. Several of these genes are induced by abscisic acid and jasmonic acid. Moreover, specific OsCPP genes are stimulated by Xanthomonas oryzae pv. oryzae (Xoo), the pathogen responsible for bacterial leaf blight in rice. In particular, the pathogen causes a notable overexpression of OsCPP9, suggesting its potential involvement in the plant's defense system. These results highlight the possible contributions of OsCPP genes to rice resistance against Xoo disease.

Experience of Surgical Treatment of Patients with Acute Necrotizing Pancreatitis

Acute pancreatitis occurs as a result of autolysis of pancreatic tissue by lipolytic and activated proteolytic enzymes, which leads to a wide range of changes from edema to focal and extensive necrosis and ranks third in the development of the surgical structure of the abdominal cavity. Destructive forms occur in 30-60% of cases, and mortality fluctuates between 30-85%. The frequency of purulent results reaches 21%. The article presents a technique for the surgical treatment of patients with acute necrotizing pancreatitis.

Vascular Extracellular Matrix in Atherosclerosis.

The extracellular matrix (ECM) plays a central role in the structural integrity and functionality of the cardiovascular system. Moreover, the ECM is involved in atherosclerotic plaque formation and stability. In fact, ECM remodeling affects plaque stability, cellular migration, and inflammatory responses. Collagens, fibronectin, laminin, elastin, and proteoglycans are crucial proteins during atherosclerosis development. This dynamic remodeling is driven by proteolytic enzymes such as matrix metalloproteinases (MMPs), cathepsins, and serine proteases. Exploring and investigating ECM dynamics is an important step to designing innovative therapeutic strategies targeting ECM remodeling mechanisms, thus offering significant advantages in the management of cardiovascular diseases. This review illustrates the structure and role of vascular ECM, presenting a new perspective on ECM remodeling and its potential as a therapeutic target in atherosclerosis treatments.

Optimization of Bromelain extraction from green papaya (Carica papaya) peel waste and its application in the recovery of silver from waste of X-ray photographic films.

As bromelain is a proteolytic enzyme present in many fruits, this study utilises the bromelain content present in unripe papaya (Carica papaya L.) by extraction and characterisation of bromelain extract from the unripe papaya peel waste. The extracted bromelain was utilised in the recovery of silver from waste of X-ray films, since the toxicity of silver directly impacts on the environment to treat and recover silver bromelain enzyme was used as a biocatalyst in this study. Response surface methodology with Box-Behnken design was used to optimise both the parameters included in the recovery of bromelain from papaya waste and silver from X-ray waste. Parameters involved in the recovery of bromelain include incubation time, pH of solution, temperature of solution and concentration of solution; for silver recovery along with the parameters selected for bromelain, recovery weight of X-rays is also considered in the study. Characterisation techniques like XRD, SEM, TGA, BET, FTIR and GC/MS were performed for the samples of bromelain and recovered silver from X-rays. At 30min of incubation time, pH of 8, with 15mL of bromelain solution maintained at 80 ℃ with 0.6g of X-rays gives optimum silver recovery. The optimum recovery of silver was observed to be 96.6%.

Use of temporary immersion systems for the micropropagation of Puya alpestris (Poepp.) Gay as a source to produce proteolytic enzymes

Use of temporary immersion systems for the micropropagation of Puya alpestris (Poepp.) Gay as a source to produce proteolytic enzymes

Immunology of Schizophrenia: A Modern View on Inflammatory Hypotheses of the Disease

Background: the immunological direction has always been a significant part of biological studies of schizophrenia and in different years has been based on the relevant fundamental ideas about the functions of the immune system and neuroimmune relationships. Objective: to conduct a brief historical analysis of immune hypotheses of schizophrenia, reflecting the vector of research of fundamental immunology, and also to present the results of our own research, confirming the key role of chronic inflammation in the pathogenesis of schizophrenia and the possibility of using immunological indicators for diagnosis and prognosis of the course of the disease. Materials and Method: using the keywords “schizophrenia”, “immune hypotheses of schizophrenia”, “neuroinflammation”, “neuroimmune relationships” we analyzed publications from PubMed/MEDLINE, RSCI databases and other sources of the last decades in comparison with the results of clinical and biological studies of schizophrenia at the Mental Health Research Centre (MHRC). Conclusion: based on the analysis of publications, it is shown that the development of scientfic ideas about the relationship between the immune system and schizophrenia has led to the understanding of the key role of chronic inflammation in the pathogenesis of this disease. Based on comparative studies of a number of immune markers related to cytokine system, acute phase proteins, proteolytic enzymes, etc., a laboratory test system “Neuroimmuno-test”, which includes complex determination of iflammatory and autoimmune markers in blood plasma, was created at the MHRC. It is shown that the level of immune system activation correlates with the features of psychopathological symptoms of patients. Identification of the immune profiles of patients is important to differentiate disease subtypes for the purpose of diagnosis and personalized therapy.

Enhancing the quality of wholemeal bread with chia, sesame, and rosehip: mathematical modelling and organoleptic analysis

In this paper, the research was conducted using mathematical modelling methods to improve the quality of the product. This study aimed to determine the optimum composite mixture for producing whole wheat flour by adding sesame seeds, chia seeds, and crushed rosehip. Following the mathematical matrix, 20 different samples have been baked. The basic criteria were porosity and specific volume. The results were entered into Exel to draw up a graph. According to the graphic analysis, the most optimal mixture in terms of the dry matter mass in the dough was as follows in %: rosehip - 1.1%, chia seeds - 1.5%, and sesame - 2.2%. The organoleptic and physicochemical properties of the resulting samples were later analysed according to the recipes based on the selected composition of seeds. By swelling the protein shells of chia and sesame in a humid environment, amino acids in the flour combine into a chain to form a skeleton. At the same time, the ascorbic acid in the rosehip binds with the carbon atoms in the chain, strengthening the framework. As a result, large amounts of gases formed in whole grain flours are trapped in these frames, increasing the porosity of bread by 21.8%, increasing the volume of production by 29.5%, absorbing proteolytic enzymes under the influence of globulins in chia grain, slowing down amino acid degradation, reducing moisture content by 3%.

Effective Treatment of Basal Cell Carcinoma with a Topical Enzymatic Mixture Enriched in Bromelain: Summary of Proof-Of Concept Clinical Studies on the First 22 Tumors.

Background/Objectives: Basal cell carcinoma (BCC), the most prevalent form of human cancer, is traditionally treated by surgical and alternative destructive or topical chemical means, each with its advantages, challenges, and drawbacks. We describe our experience treating BCCs with a topical concentrate of proteolytic enzymes enriched in bromelain (CPEEB) sourced from pineapple stems. CPEEB has strong proteolytic, antitumor-proapoptotic, and inflammation modulation activities, and is approved for debridement of deep burns and starting phase 3 trials for chronic wounds. Methods: In the first proof-of-concept (POC) study, six BCCs on three individuals were treated with five to six daily CPEEB 10% topical applications under a zinc oxide-based occlusive dressing for 9-12 h each during a period of up to 10 days. These patients were followed for up to 4 years. In an additional two POC studies, 16 patients with one BCC each were treated every other day for a total of seven applications of topical CPEEB 5% under a variety of occlusive dressings. The wounds were followed for up to 2 months before undergoing diagnostic excisional biopsy. Results: In the first study, clinical assessment of the BCCs and two excisional biopsies after 6 months suggested that all lesions were eradicated with spontaneous healing within ~2 weeks without clinical or histological recurrence for over 4 years. In the two subsequent studies, 16 histologically diagnosed superficial and nodular BCCs were treated using four application techniques. Excisional histology after 2 months confirmed BCC eradication in seven of the patients. In nine patients, with compromised occlusive dressings, histological eradication was incomplete. Treatment was well tolerated by all patients with the expected local skin reactions, which completely healed within 2-3 weeks. Conclusions: These are POC preliminary studies aimed at indicating the potential efficacy and feasibility of topical CPEEB in eradicating BCC. In these studies, topical CPEEB 10% and 5% resulted in complete eradication of the BCC when appropriately applied. CPEEB was well tolerated in all patients, and all treated sites' erosions healed without scars in <3 weeks. Further research is necessary to corroborate the results, refine the application technique, and complete the regulatory process.