4-{3-[(Pyridin-4-ylmethyl)amino]-[1,2,4]triazolo[4,3-b][1,2,4]triazin-6-yl}phenol: An improved anticancer agent in hepatocellular carcinoma and a selective MDR1/MRP modulator.

Hepatocellular carcinoma is the most common type of primary liver cancer. However, multidrug resistance (MDR) is a major obstacle to the effective chemotherapy of cancer cells. This report documents the rational design, synthesis, and biological evaluation of a novel series of triazolotriazines substituted with CH2NH-linked pyridine for use as dual c-Met/MDR inhibitors. Compound 12g with IC50 of 3.06 µM on HepG2 cells showed more potency than crizotinib (IC50 = 5.15 µM) in the MTT assay. In addition, 12g inhibited c-Met kinase at a low micromolar level (IC50 = 0.052 µM). 12g significantly inhibited P-gp and MRP1/2 efflux pumps in both cancerous HepG2 and BxPC3 cells starting from the lower concentrations of 3 and 0.3 µM, respectively. 12g did not inhibit MDR1 and MRP1/2 in noncancerous H69 cholangiocytes up to the concentration of 30 and 60 µM, respectively. Current results highlighted that cancerous cells were more susceptible to the effect of 12g than normal cells, in which the inhibition occurred only at the highest concentrations, suggesting a further interest in 12g as a selective anticancer agent. Overall, 12g, as a dual c-Met and P-gp/MRP inhibitor, is a promising lead compound for developing a new generation of anticancer agents.

Synthesis and evaluation of 99mTc-labeled 1-(2-Pyridyl)piperazine derivatives as radioligands for 5HT7 receptors.

Glioblastoma multiform (GBM) is one of the most aggressive tumors of the central nervous system in humans. GBM overexpresses serotonin-7 receptors (5-HT7Rs); hence, this study aims to develop 5-HT7R targeted radiotracers. Aryl piperazine derivatives can act as ligands for 5-HT7R. Therefore, compounds 6 and 7 as 1-(3-nitropyridin-2-yl)piperazine derivatives were synthesized and radiolabeled with 99mTcN2+ core. Radiolabeled 6 and 7 (99mTcN-[6] and 99mTcN-[7]) were prepared with high radiochemical purity (RCP > 96%). They displayed high affinity toward U-87 MG cell line 5-HT7R. The calculated Ki for 99mTcN-[7] was lower than that of 99mTcN-[6] (14.85 ± 0.32 vs 22.57 ± 0.73 nM) which indicates the higher affinity of 99mTcN-[7] toward 5-HT7R. A molecular docking study also confirmed the binding of these radiotracers to 5-HT7R. The biodistribution study in normal mice revealed that 99mTcN-[7] has the highest brain accumulation at 30 min post-injection (0.54 ± 0.12 %ID/g) while the uptake of 99mTcN-[6] is much lower (0.14 ± 0.02 %ID/g). The biodistribution study in the xenograft model confirms that the radiotracers recognize the tumor site. 99mTcN-[6], and 99mTcN-[7] showed the highest tumor uptake at 1-hour post-injection (5.44 ± 0.58 vs 4.94 ± 1.65 %ID/g) and tumor-to-muscle ratios were (4.61 vs. 5.61). The injection of pimozide blocks the receptors and significantly reduces the tumor-to-muscle ratios at 1-hour post-injection to 0.81 and 0.31, respectively. In correlation with in vitro study, 99mTcN-[6] and 99mTcN-[7] visualize the tumor site in U-87 MG glioma xenografted nude mice and display the tumor-to-muscle ratios of 7.05 and 6.03.

Dual action anti-inflammatory/antiviral isoquinoline alkaloids as potent naturally occurring anti-SARS-CoV-2 agents: A combined pharmacological and medicinal chemistry perspective.

In the search for compounds that inhibit the SARS-CoV-2 after the onset of the COVID-19 pandemic, isoquinoline-containing alkaloids have been identified as compounds with high potential to fight the disease. In addition to having strong antiviral activities, most of these alkaloids have significant anti-inflammatory effects which are often manifested through the inhibition of a promising host-based anti-COVID-19 target, the p38 MAPK signaling pathway. In the present review, our pharmacological and medicinal chemistry evaluation resulted in highlighting the potential of anti-SARS-CoV-2 isoquinoline-based alkaloids for the treatment of COVID-19 patients. Considering critical parameters of the antiviral and anti-inflammatory activities, mechanism of action, as well as toxicity/safety profile, we introduce the alkaloids emetine, cephaeline, and papaverine as high-potential therapeutic agents for use in the treatment of COVID-19. Although preclinical studies confirm that some isoquinoline-based alkaloids reviewed in this study have a high potential to inhibit the SARS-CoV-2, their entry into drug regimens of COVID-19 patients requires further clinical trial studies and toxicity evaluation.

Polyethyleneglycol-serine nanoparticles as a novel antidote for organophosphate poisoning: synthesis, characterization, in vitro and in vivo studies.

Acute organophosphate pesticide poisoning causes considerable worldwide mortality and morbidity. In this study, serine was attached to the polyethylene glycol-bisaldehyde (PEG) as a novel antidote for diazinon (DZ) poisoning. Serine and PEG were conjugated with a reductive amination reaction. PEG-serine NPs (PEG-NPs) were purified and their structure was analyzed by 1H NMR, 13 C NMR, IR, and particle size was determined via dynamic light scattering. In vitro studies, including hemolysis assay and cytotoxicity on SK-BR-3 and HFFF2 cell lines, were performed. In vivo studies of PEG-NPs were evaluated on DZ-exposed mice. PEG-NPs were administered (i.p.) 20 min after a single dose of DZ (LD50; 166 mg/kg). Atropine (20 mg/kg, i.p.) with pralidoxime (20 mg/kg, i.p.) was used as the standard therapy compared to PEG-NPs. NMR and IR data confirmed that the conjugation of PEG to serine occurred successfully. The average NP size was 22.1 ± 1.8 nm. The hemolysis of the PEG-NPs was calculated at 0.867%, 50% inhibitory concentration (IC50) was calculated 36 ± 4.5, and 41 ± 3.4 mg/mL on SK-BR-3 and HFFF2 cell lines, respectively. Percentage of surviving significantly improved by 12.5, 25, and 25% through the usage of PEG-NPs at doses of 100, 200, and 400 mg/kg, respectively, when compared with the DZ group. Cholinesterase enzyme activity, lipid peroxidation, and mitochondrial function significantly improved through PEG-NPs when compared with the DZ group. PEG conjugated serine is very biocompatible with low toxicity and can reduce the acute toxicity of DZ as a new combination therapy.

Discovery of Chalcone-Based Hybrid Structures as High Affinity and Site-Specific Inhibitors against SARS-CoV-2: A Comprehensive Structural Analysis Based on Various Host-Based and Viral Targets.

Previous studies indicated that natural-based chalcones have significant inhibitory effects on the coronavirus enzymes 3CLpro and PLpro as well as modulation of some host-based antiviral targets (HBATs). In this study, a comprehensive computational and structural study was performed to investigate the affinity of our compound library consisting of 757 chalcone-based structures (CHA-1 to CHA-757) for inhibiting the 3CLpro and PLpro enzymes and against twelve selected host-based targets. Our results indicated that CHA-12 (VUF 4819) is the most potent and multi-target inhibitor in our chemical library over all viral and host-based targets. Correspondingly, CHA-384 and its congeners containing ureide moieties were found to be potent and selective 3CLpro inhibitors, and benzotriazole moiety in CHA-37 was found to be a main fragment for inhibiting the 3CLpro and PLpro. Surprisingly, our results indicate that the ureide and sulfonamide moieties are integral fragments for the optimum 3CLpro inhibition while occupying the S1 and S3 subsites, which is fully consistent with recent reports on the site-specific 3CLpro inhibitors. Finding the multi-target inhibitor CHA-12, previously reported as an LTD4 antagonist for the treatment of inflammatory pulmonary diseases, prompted us to suggest it as a concomitant agent for relieving respiratory symptoms and suppressing COVID-19 infection.

Papaverine, a promising therapeutic agent for the treatment of COVID-19 patients with underlying cardiovascular diseases (CVDs).

The causative agent of coronavirus disease-2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), enters the host cells via an angiotensin-converting enzyme 2 (ACE2)-mediated endocytosis-dependent manner. Because ACE2 is highly expressed in the heart, SARS-CoV-2 can severely infect heart tissue and arteries, causing acute and chronic damage to the cardiovascular system. Therefore, special attention should be paid to finding appropriate agents to protect this vital system during COVID-19 treatment. Papaverine is a unique vasodilator alkaloid that is clinically used in the treatment of vasospasm. Interestingly, this compound has potent and direct effects on a wide range of viruses, and could also prevent viral exploitation mechanisms of the host cell facilities by inhibiting some cellular signaling pathways such as p38 MAPK. This pathway was recently introduced as a promising target for the treatment of COVID-19. Papaverine also has anti-inflammatory effects which is useful in combating the hyper-inflammatory phase of the COVID-19. Unlike some medications that have severe dosage-restrictions in the treatment of COVID-19 due to cardiac side effects, papaverine is recommended for use in many heart disorders. The ability of papaverine to treat COVID-19 has become more promising when the results of some extensive screenings showed the strong ability of this compound to inhibit the cytopathic effects of SARS-CoV-2 with EC50 of 1.1 µM. Having several therapeutic effects along with desired safety profile raises this hypothesis that papaverine could be a promising compound for the suppression of SARS-CoV-2 and prevention of ischemia/vasoconstriction-related complications in COVID-19 disease, especially in patients with underlying cardiovascular diseases (CVDs).

Synthesis, in silico, in vitro and in vivo evaluations of isatin aroylhydrazones as highly potent anticonvulsant agents.

In this study, a series of new isatin aroylhydrazones (5a-e and 6a-e) was synthesized and evaluated for their anticonvulsant activities. The (Z)-configuration of compounds was confirmed by 1H NMR. In vivo studies using maximal electroshock (MES) and pentylenetetrazole (PTZ) models of epilepsy in mice revealed that while most of compounds had no effect on chemically-induced seizures at the higher dose of 100 mg/kg but showed significant protection against electrically-induced seizures at the lower dose of 5 mg/kg. Certainly, N-methyl analogs 6a and 6e were found to be the most effective compounds, displaying 100% protection at the dose of 5 mg/kg. Protein binding and lipophilicity(logP) of the selected compounds (6a and 6e) were also determined experimentally. In silico evaluations of title compounds showed acceptable ADME parameters, and drug-likeness properties. Distance mapping and docking of the selected compounds with different targets proposed the possible action of them on VGSCs and GABAA receptors. The cytotoxicity evaluation of 6a and 6e against SH-SY5Y and Hep-G2 cell lines indicated safety profile of compounds on the neuronal and hepatic cells.

Potentiation of Low-Dose Doxorubicin Cytotoxicity by Affecting P-Glycoprotein through Caryophyllane Sesquiterpenes in HepG2 Cells: an in Vitro and in Silico Study.

Doxorubicin represents a valuable choice for different cancers, although the severe side effects occurring at the high effective dose limits its clinical use. In the present study, potential strategies to potentiate low-dose doxorubicin efficacy, including a metronomic schedule, characterized by a short and repeated exposure to the anticancer drug, and the combination with the natural chemosensitizing sesquiterpenes ß-caryophyllene and ß-caryophyllene oxide, were assessed in human hepatoma HepG2 cells. The involvement of P-glycoprotein (P-gp) in the HepG2-chemosensitization to doxorubicin was evaluated. Also, the direct interaction of caryophyllene sesquiterpenes with P-gp was characterized by molecular docking and dynamic simulation studies. A metronomic schedule allowed us to enhance the low-dose doxorubicin cytotoxicity and the combination with caryophyllane sesquiterpenes further potentiated this effect. Also, an increased intracellular accumulation of doxorubicin and rhodamine 123 induced by caryophyllane sesquiterpenes was found, thus suggesting their interference with P-gp function. A lowered expression of P-gp induced by the combinations, with respect to doxorubicin alone, was observed too. Docking studies found that the binding site of caryophyllane sesquiterpene was next to the ATP binding domain of P-gp and that ß-caryophyllene possessed the stronger binding affinity and higher inhibition potential calculated by MM-PBSA. Present findings strengthen our hypothesis about the potential chemosensitizing power of caryophyllane sesquiterpenes and suggest that combining a chemosensitizer and a metronomic schedule can represent a suitable strategy to overcome drawbacks of doxorubicin chemotherapy while exploiting its powerful activity.

Design, synthesis, in vivo and in silico evaluation of phenacyl triazole hydrazones as new anticonvulsant agents.

A series of phenacyl triazole hydrazones 3 have been designed based on the hybridization of (arylalkly)triazole and aroyl hydrazone scaffolds as new anticonvulsant agents. The target compounds 3 were easily synthesized from appropriate phenacyl triazoles and aryl acid hydrazides and characterized by IR, NMR and Mass spectroscopy. The in vivo anticonvulsant evaluation of synthesized compounds by using MES and PTZ tests revealed that they are more effective in MES model respect to PTZ test. All compounds showed 33-100% protection against MES-induced seizures at the dose of 100 mg/kg. However, the isonicotinic acid hydrazide derivative 3h showed the best profile of activity in both models. Molecular docking studies of compound 3h with different targets (NMDA, AMPA, GABAA and sodium channel), postulated that the compound acts mainly via GABAA receptors. In silico molecular properties predictions indicated that all compounds have favourable oral bioavailability and BBB permeability.

New 7-piperazinylquinolones containing (benzo[d]imidazol-2-yl)methyl moiety as potent antibacterial agents.

A series of 7-piperazinylquinolones containing a (benzo[d]imidazol-2-yl)methyl moiety were designed and synthesized as new antibacterial agents. The antibacterial activity of title compounds was evaluated against Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis and Bacillus subtilis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumonia) microorganisms. Among the tested compounds, the N1-cyclopropyl derivative 4a showed the highest activity against S. aureus, S. epidermidis, B. subtilis and E. coli ([Formula: see text] [Formula: see text]g/mL), being 2-4 times more potent than reference drug norfloxacin. A structure-activity relationship study demonstrated that the effect of the nitro group on the benzimidazole ring depends on the pattern of substitutions on the piperazinylquinolone.

Synthesis and biological evaluation of fluconazole analogs with triazole-modified scaffold as potent antifungal agents.

In order to find new azole antifungals, we have recently designed a series of triazole alcohols in which one of the 1,2,4-triazol-1-yl group in fluconazole structure has been replaced with 4-amino-5-aryl-3-mercapto-1,2,4-triazole motif. In this paper, we focused on the structural refinement of the primary lead, by removing the amino group from the structure to achieve 5-aryl-3-mercapto-1,2,4-triazole derivatives 10a-i and 11a-i. The in vitro antifungal susceptibility testing of title compounds demonstrated that most compounds had potent inhibitory activity against Candida species. Among them, 5-(2,4-dichlorophenyl)triazole analogs 10h and 11h with MIC values of <0.01 to 0.5µg/mL were 4-256 times more potent than fluconazole against Candida species.

Novel triazole alcohol antifungals derived from fluconazole: design, synthesis, and biological activity.

A series of new triazole alcohol antifungals were designed by replacing one of the triazolyl moiety from fluconazole with a distinct 4-amino-3-mercapto-1,2,4-triazole motif, which is found in some antimicrobial agents. The antimicrobial susceptibility testing of target compounds demonstrated that the direct analogs of fluconazole (difluorophenethyl-triazoles) were less active against fungi, while compound 10h containing dichloro substitutions on both phenyl rings of the molecule had potent activity against yeasts including Candida albicans (four strains) and Cryptococcus neoformans (MICs = 2-8 µg/mL). Also, compound 10h was active against Candida parapsilosis, Epidermophyton floccosum, and Trichophyton mentagrophytes, while it showed no activity against Gram-positive and Gram-negative bacteria. Finally, a molecular docking study suggested that compound 10h interacts suitably with lanosterol 14α-demethylase, which is the key enzyme in ergosterol biosynthesis.

Design, synthesis and in vitro study of 5,6-diaryl-1,2,4-triazine-3-ylthioacetate derivatives as COX-2 and ß-amyloid aggregation inhibitors.

In order to find novel cyclooxygenase (COX)-2 inhibitors for treating inflammatory-based diseases such as Alzheimer's disease (AD), an ethyl carboxylate side chain was added to 5-(4-chlorophenyl)-6-(4-(methylsulfonyl)phenyl)-3-(methylthio)-1,2,4-triazine (lead compound II) to maintain residual inhibition of COX-1 through interacting with Arg120. A preliminary molecular docking study on both the COX-1/COX-2 active sites truly confirmed our hypothesis. Accordingly, a series of ethyl 5,6-diaryl-1,2,4-triazine-3-ylthioacetate derivatives were synthesized and their chemical structures were confirmed by NMR, IR and MS spectra. Further in vitro COX-1/COX-2 evaluations revealed that compound 6c (COX-2 IC50 = 10.1 µM, COX-1 IC50 = 88.8 µM) is the most selective COX-2 inhibitor while maintaining residual inhibition of COX-1. In order to evaluate their potential use against AD, an in vitro evaluation of ß-amyloid fibril formation was performed. The results indicated that the prototype compounds 6 are effective ß-amyloid destabilizing agents while compound 6c could inhibit 94% of the ß-amyloid fibril formation after 48 h. Finally, the in silico assessment results of their blood-brain barrier permeability were satisfactory.

Synthesis, docking simulation, biological evaluations and 3D-QSAR study of 5-Aryl-6-(4-methylsulfonyl)-3-(metylthio)-1,2,4-triazine as selective cyclooxygenase-2 inhibitors.

A series of 5-Aryl-6-(4-methylsulfonyl)-3-(metylthio)-1,2,4-triazine derivatives were synthesized and their COX-1/COX-2 inhibitory activity as well as in vivo anti-inflammatory and analgesic effects were evaluated. All of compounds showed strong inhibition of COX-2 with IC50 values in the range of 0.1-0.2µM and in most cases had stronger anti-inflammatory and analgesic effects than indomethacin at doses 3 and 6mg/kg. Among them, 5-(4-chlorophenyl)-6-(4-(methylsulfonyl) phenyl)-3-(methylthio)-1,2,4-triazine (9c) was the most potent and selective COX-2 compound; its selectivity index of 395 was comparable to celecoxib (SI=405). Evaluation of anti-inflammatory and analgesic effects of 9c showed its higher potency than indomethacin and hence could be considered as a promising lead candidate for further drug development. Furthermore, the affinity data of these compounds were rationalized through enzyme docking simulation and 3D-QSAR study by k-Nearest Neighbour Molecular Field Analysis.

Imidazolylchromanones containing alkyl side chain as lanosterol 14α-demethylase inhibitors: synthesis, antifungal activity and docking study.

Previously, 2-alkylchromans have been introduced as non-azole inhibitors of 14α-demethylase. Accordingly, we incorporated imidazole ring on the 3-position of 2-alkylchromanones to design new inhibitors of 14α-demethylase and potential antifungal agents. Thus, a series of 2-alkyl-3-imidazolylchromanones were synthesized starting from 2-hydroxyphenacyl bromide. The trans-configuration of compounds was confirmed by NMR-spectroscopy. The antifungal activity of title compounds were evaluated against different fungi in comparison with fluconazole and miconazole. trans-2-(1-Pentyl)-3-imidazolylchroman-4-one (4d) showed the most potent activity against yeasts comparable to fluconazole. The experimental data based on (1)H NMR spectroscopy revealed that 2-alkyl side chain and 3-imidazolyl moiety in compound 4d exist predominantly in the di-equatorial conformation. While docking study with 14α-demethylase demonstrated that the di-axial form of compound 4d can be considered as active conformation.

Development of New PCR Protocols to Detect Genetic Diversity in the Metronidazole Metabolism Genes in Susceptible and Refractory Clinical Samples of Giardia duodenalis.

PURPOSE: Investigating the genetic variation in thioredoxin reductase (TrxR) and nitroreductase (NR) genes in both treatment-resistant and -sensitive Giardia duodenalis isolates can provide valuable information in identifying potential markers of resistance to metronidazole. The rapid increase in metronidazole treatment failures suggests the presence of genetic resistance mechanisms. By analyzing these genes, researchers can gain insights into the efficacy of metronidazole against G. duodenalis and potentially develop alternative treatment strategies. In this regard, four G. duodenalis isolates (two clinically sensitive and two clinically resistant to metronidazole) were collected from various hospitals of Shiraz, southwestern Iran. METHODS: Parasitological methods including sucrose flotation and microscopy were employed for the primary confirmation of G. duodenalis cysts in stool samples. Microscopy-positive samples were approved by SSU-PCR amplification of the parasite DNA. All four positive G. duodenalis specimens at SSU-PCR were afterward analyzed utilizing designed primers based on important metronidazole metabolism genes including TrxR, NR1, and NR2. RESULTS: Unlike TrxR gene, the results of NR1 and NR2 genes showed that there are non-synonymous variations between sequences of treatment-sensitive and -resistant samples compared to reference sequences. Furthermore, the outcomes of molecular docking revealed that there is an interaction between the protein sequence and spatial shape of treatment-resistant samples and metronidazole in the position of serine amino acid based on the NR1 gene. CONCLUSION: This issue can be one of the possible factors involved in the resistance of Giardia parasites to metronidazole. To reach more accurate results, a large sample size along with simulation and advanced molecular dynamics investigations are needed.

An Overview on Anti-COVID-19 Drug Achievements and Challenges Ahead.

The appearance of several coronavirus pandemics/epidemics during the last two decades (SARS-CoV-1 in 2002, MERS-CoV in 2012, and SARS-CoV-2 in 2019) indicates that humanity will face increasing challenges from coronaviruses in the future. The emergence of new strains with similar transmission characteristics as SARS-CoV-2 and mortality rates similar to SARS-CoV-1 (∼10% mortality) or MERS-CoV (∼35% mortality) in the future is a terrifying possibility. Therefore, getting enough preparations to face such risks is an inevitable necessity. The present study aims to review the drug achievements and challenges in the fight against SARS-CoV-2 with a combined perspective derived from pharmacology, pharmacotherapy, and medicinal chemistry insights. Appreciating all the efforts made during the past few years, there is strong evidence that the desired results have not yet been achieved and research in this area should still be pursued seriously. By expressing some pessimistic possibilities and concluding that the drug discovery and pharmacotherapy of COVID-19 have not been successful so far, this short essay tries to draw the attention of responsible authorities to be more prepared against future coronavirus epidemics/pandemics.

Benzoindolizidine Alkaloids Tylophorine and Lycorine and Their Analogues with Antiviral, Anti-Inflammatory, and Anticancer Properties: Promises and Challenges.

Ongoing viral research, essential for public health due to evolving viruses, gains significance owing to emerging viral infections such as the SARS-CoV-2 pandemic. Marine and plant alkaloids show promise as novel potential pharmacological strategies. In this narrative review, we elucidated the potential of tylophorine and lycorine, two naturally occurring plant-derived alkaloids with a shared benzoindolizidine scaffold, as antiviral agents to be potentially harnessed against respiratory viral infections. Possible structure-activity relationships have also been highlighted. The substances and their derivatives were found to be endowed with powerful and broad-spectrum antiviral properties; moreover, they were able to counteract inflammation, which often underpins the complications of viral diseases. At last, their anticancer properties hold promise not only for advancing cancer research but also for mitigating the oncogenic effects of viruses. This evidence suggests that tylophorine and lycorine could effectively counteract the pathogenesis of respiratory viral disease and its harmful effects. Although common issues about the pharmacologic development of natural substances remain to be addressed, the collected evidence highlights a possible interest in tylophorine and lycorine as antiviral and/or adjuvant strategies and encourages future more in-depth pre-clinical and clinical investigations to overcome their drawbacks and harness their power for therapeutic purposes.

In silico design of a novel peptide-based vaccine against the ubiquitous apicomplexan Toxoplasma gondii using surface antigens.

Human toxoplasmosis is a global public health concern and a commercial vaccine is still lacking. The present in silico study was done to design a novel vaccine candidate using tachyzoite-specific SAG1-realted sequence (SRS) proteins. Overlapping B-cell and strictly-chosen human MHC-I binding epitopes were predicted and connected together using appropriate spacers. Moreover, a TLR4 agonist, human high mobility group box protein 1 (HMGB1), and His-tag were added to the N- and C-terminus of the vaccine sequence. The final vaccine had 442 residues and a molecular weight of 47.71 kDa. Physico-chemical evaluation showed a soluble, highly antigenic and non-allergen protein, with coils and helices as secondary structures. The vaccine 3D model was predicted by ITASSER server, subsequently refined and was shown to possess significant interactions with human TLR4. As well, potent stimulation of cellular and humoral immunity was demonstrated upon chimeric vaccine injection. Finally, the outputs showed that this vaccine model possesses top antigenicity, which could provoke significant cell-mediated immune profile including IFN-γ, and can be utilized towards prophylactic purposes. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00140-w.

Engineering and design of promising T-cell-based multi-epitope vaccine candidates against leishmaniasis.

Cutaneous leishmaniasis (CL) is a very common parasitic infection in subtropical areas worldwide. Throughout decades, there have been challenges in vaccine design and vaccination against CL. The present study introduced novel T-cell-based vaccine candidates containing IFN-γ Inducing epitopic fragments from Leishmania major (L. major) glycoprotein 46 (gp46), cathepsin L-like and B-like proteases, histone H2A, glucose-regulated protein 78 (grp78) and stress-inducible protein 1 (STI-1). For this aim, top-ranked human leukocyte antigen (HLA)-specific, IFN-γ Inducing, antigenic, CD4+ and CD8+ binders were highlighted. Four vaccine candidates were generated using different spacers (AAY, GPGPG, GDGDG) and adjuvants (RS-09 peptide, human IFN-γ, a combination of both, Mycobacterium tuberculosis Resuscitation promoting factor E (RpfE)). Based on the immune simulation profile, those with RS-09 peptide (Leish-App) and RpfE (Leish-Rpf) elicited robust immune responses and their tertiary structure were further refined. Also, molecular docking of the selected vaccine models with the human toll-like receptor 4 showed proper interactions, particularly for Leish-App, for which molecular dynamics simulations showed a stable connection with TLR-4. Upon codon optimization, both models were finally ligated into the pET28a( +) vector. In conclusion, two potent multi-epitope vaccine candidates were designed against CL and evaluated using comprehensive in silico methods, while further wet experiments are, also, recommended.