Discovery of a Novel Inhibitor Structure of Mycobacterium tuberculosis Isocitrate Lyase

Tuberculosis remains a global threat to public health, and dormant Mycobacterium tuberculosis leads to long-term medication that is harmful to the human body. M. tuberculosis isocitrate lyase (MtICL), which is absent in host cells, is a key rate-limiting enzyme of the glyoxylic acid cycle and is essential for the survival of dormant M. tuberculosis. The aim of this study was to evaluate natural compounds as potential MtICL inhibitors through docking and experimental verification. Screening of the TCMSP database library was done using Discovery Studio 2019 for molecular docking and interaction analysis, with the putative inhibitors of MtICL, 3-BP, and IA as reference ligands. Daphnetin (MOL005118), with a docking score of 94.8 and -CDOCKER interaction energy of 56 kcal/mol, was selected and verified on MtICL in vitro and M. smegmatis; daphnetin gave an IC₅₀ of 4.34 μg/mL for the MtICL enzyme and an MIC value of 128 μg/mL against M. smegmatis, showing enhanced potential in comparison with 3-BP and IA. The interactions and essential amino acid residues of the protein were analyzed. In summary, natural daphnetin may be a promising new skeleton for the design of inhibitors of MtICL to combat dormant M. tuberculosis.     

The Important Roles of Water in Protein Folding: an Approach by Single Molecule Force Spectroscopy

The single-chain elasticity of a completely unfolded protein ((I27)8,modules of human cardiac titin) is studied in different liquid environments by the atomic force microscopy (AFM)-based single molecule force spectroscopy (SMFS).The experimental results show that there is a clear deviation between the force curves obtained in the aqueous and nonaqueous environments.Such a deviation can be attributed to the additional energy consumed by the rearrangement of the bound water molecules around the chain of the completely unfolded (I27)8 chain upon stretching in aqueous solution,which is very similar to the partial dehydration process from a denatured/unfolded to a native/folded protein.Through the analysis of the free energy changes involved in protein folding,we conclude that it is due to the weak disturbance of water molecules and the special backbone structures of proteins that the self-assembly of proteins can be achieved in physiological conditions.We speculate that water is likely to be an important criterion for the selection of self-assembling macromolecules in the prebiotic chemical evolution.     

In Silico Drug Repurposing Approach: Investigation of Mycobacterium tuberculosis FadD32 Targeted by FDA-Approved Drugs

Background: Despite the enormous efforts made towards combating tuberculosis (TB), the disease remains a major global threat. Hence, new drugs with novel mechanisms against TB are urgently needed. Fatty acid degradation protein D32 (FadD32) has been identified as a promising drug target against TB, the protein is required for the biosynthesis of mycolic acids, hence, essential for the growth and multiplication of the mycobacterium. However, the FadD32 mechanism upon the binding of FDA-approved drugs is not well established. Herein, we applied virtual screening (VS), molecular docking, and molecular dynamic (MD) simulation to identify potential FDA-approved drugs against FadD32. Methodology/Results: VS technique was found promising to identify four FDA-approved drugs (accolate, sorafenib, mefloquine, and loperamide) with higher molecular docking scores, ranging from −8.0 to −10.0 kcal/mol. Post-MD analysis showed that the accolate hit displayed the highest total binding energy of −45.13 kcal/mol. Results also showed that the accolate hit formed more interactions with FadD32 active site residues and all active site residues displayed an increase in total binding contribution. RMSD, RMSF, Rg, and DCCM analysis further supported that the presence of accolate exhibited more structural stability, lower bimolecular flexibility, and more compactness into the FadD32 protein. Conclusions: Our study revealed accolate as the best potential drug against FadD32, hence a prospective anti-TB drug in TB therapy. In addition, we believe that the approach presented in the current study will serve as a cornerstone to identifying new potential inhibitors against a wide range of biological targets.     

Development of Novel Isatin-Tethered Quinolines as Anti-Tubercular Agents against Multi and Extensively Drug-Resistant Mycobacterium tuberculosis

We describe the design and synthesis of two isatin-tethered quinolines series (Q6a–h and Q8a–h), in connection with our research interest in developing novel isatin-bearing anti-tubercular candidates. In a previous study, a series of small molecules bearing a quinoline-3-carbohydrazone moiety was developed as anti-tubercular agents, and compound IV disclosed the highest potency with MIC value equal to 6.24 µg/mL. In the current work, we adopted the bioisosteric replacement approach to replace the 3,4,5-trimethoxy-benzylidene moiety in the lead compound IV with the isatin motif, a privileged scaffold in the TB drug discovery, to furnish the first series of target molecules Q6a–h. Thereafter, the isatin motif was N-substituted with either a methyl or benzyl group to furnish the second series Q8a–h. All of the designed quinoilne-isatin conjugates Q6a–h and Q8a–h were synthesized and then biologically assessed for anti-tubercular actions towards drug-susceptible, MDR, and XDR strains. Superiorly, the N-benzyl-bearing compound Q8b possessed the best activities against the examined M. tuberculosis strains with MICs equal 0.06, 0.24, and 1.95 µg/mL, respectively.     

Reconstitution of a new cysteine biosynthetic pathway in Mycobacterium tuberculosis

A new pathway for cysteine biosynthesis has been elucidated in Mycobacterium tuberculosis. This pathway involves a protein-bound thiocarboxylate (CysO-SH) as the sulfide donor, similar to thiamin biosynthesis. Cysteine synthase M (CysM) catalyzes the addition of cysteine to the carboxy terminus of the protein-bound thiocarboxylate to generate a CysO-cysteine adduct. A protease, Mec+, hydrolyzes the CysO-cysteine adduct to release cysteine and regenerate CysO. Mec+ contains a JAMM motif, and this work provides the first functional characterization of the JAMM motif in prokaryotes. MoeZ, a paralogue of ThiF, has been shown to transfer sulfur onto CysO.     

Free Radicals as a Double-Edged Sword: The Cancer Preventive and Therapeutic Roles of Curcumin

Free radicals, generally composed of reactive oxygen species (ROS) and reactive nitrogen species (RNS), are generated in the body by various endogenous and exogenous systems. The overproduction of free radicals is known to cause several chronic diseases including cancer. However, increased production of free radicals by chemotherapeutic drugs is also associated with apoptosis in cancer cells, indicating the dual nature of free radicals. Among various natural compounds, curcumin manifests as an antioxidant in normal cells that helps in the prevention of carcinogenesis. It also acts as a prooxidant in cancer cells and is associated with inducing apoptosis. Curcumin quenches free radicals, induces antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase), and upregulates antioxidative protein markers–Nrf2 and HO-1 that lead to the suppression of cellular oxidative stress. In cancer cells, curcumin aggressively increases ROS that results in DNA damage and subsequently cancer cell death. It also sensitizes drug-resistant cancer cells and increases the anticancer effects of chemotherapeutic drugs. Thus, curcumin shows beneficial effects in prevention, treatment and chemosensitization of cancer cells. In this review, we will discuss the dual role of free radicals as well as the chemopreventive and chemotherapeutic effects of curcumin and its analogues against cancer.     

Pharmacological and Therapeutic Properties of Punica granatum Phytochemicals: Possible Roles in Breast Cancer

Background: Pomgranate (Punica granatum) represents a high source of polyphenols with great bioavailability. The role of this fruit in the prevention and treatment of various malignant pathologies has been long time cited in both scientific and non-scientific literature, making thus important to identify its involvement in the pathophysiological processes. The treatment for breast cancer had focused on the inhibition of the mechanisms that governs the estrogen activity. These mechanisms are covered either by the antagonism of the estrogen receptor (ER) or by the inhibition of the estrogen synthesis. Our interest in identifying a bioactive compound rich in polyphenols, which induces both the antagonism of the estrogen receptor, and the inhibition of the estrogen synthesis, revealed us the pomegranate fruit and its derivatives: peel and seeds. Pomegranates’ chemical composition include many biological active substances such as flavonols, flavanols, anthocyanins, proanthocyanidins, ellagitannins and gallotannins. Materials and Methods: We performed a review of the scientific literature by using the following keywords: “pomegranate”, “breast cancer”, “Punica granatum”, “pomegranate polyphenols”. Our search was performed in the PubMed and Google Scholar databases, and it included only original research written in English from the last 20 years. None of the articles were excluded due to affiliation. A total number of 28 original papers, which mentioned the beneficial activity of pomegranate against breast cancer, were selected. Both clinical and preclinical studies were considered for this review. Results: Recent discoveries pointed out that polyphenols from Punica granatum possess strong anti-cancer activity, exhibited by a variety of mechanisms, such as anti-estrogenic, anti-proliferative, anti-angiogenetic, anti-inflammatory, and anti-metastatic. Pomegranate extracts induced cell cycle arrest in the G0/G1 phase, and induced cytotoxicity in a dose- and time-dependent manner. Moreover, several polyphenols extracted from pomegranate inhibited the invasion potential, migration and viability of breast cancer cells. The effects of pomegranate juice on serum estrogens and other sexual hormones levels were also investigated on two human cohorts. Conclusions: Punica granatum represents a promising area in oncology. The large availability and low cost, associated with the lack of side effects, made from this natural product a great strategy for the management of breast cancer. There are several mechanistic studies in mouse models and in breast cancer cell lines, suggesting the possible pathways through which polyphenols from pomegranate extracts act, but larger and better-controlled studies are necessary in the future. Only two small clinical trials were conducted on humans until now, but their results are contradictory and should be considered preliminary.     

Prohibitin Ligands in Cell Death and Survival: Mode of Action and Therapeutic Potential

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Highlights? Prohibitins regulate cell survival, metabolism, and inflammation ? Prohibitins act as a hub for many signaling pathways ? Prohibitin ligands are examined in cancer, Parkinson’s disease, and obesity     

Arylquinolinecarboxamides: Synthesis,in vitro and in silico studies against Mycobacterium tuberculosis

A series of fourteen 6-substituted-2-(methoxyquinolin-3-yl) methyl)-N-(pyridin-3-ylmethyl) benzamides was prepared from commercially available anilines in five simple and convenient synthetic steps. The structures of all new products were confirmed by routine spectroscopic methods: IR, ¹H and ¹³C NMR, and HRMS (electrospray ionization). The resulting arylquinolinecarboxamides were subjected to biological screening assay for in vitro inhibitory activity against Mycobacterium tuberculosis (Mtb) H37Rv strain. Several compounds exhibited modest antitubercular activity with compounds 8–11 , 15 and 19 exhibiting MIC₉₀ values in the range of 32–85 μM. The antitubercular data suggested that inhibition of Mtb can be imparted by the introduction of a non-polar substituent on C-6 of the quinoline scaffold. Further, to understand the possible mode of action of the series, the reported compounds and bedaquiline were subjected to in silico docking studies against MtbATPase to determine their potential to interfere with the mycobacterial adenosine triphosphate (ATP) synthase. The results showed that these compounds have the potential to serve as antimycobacterial agents. In silico ADME pharmacokinetic prediction results showed the ability of these arylquinolinecarcboxamides to be absorbed, distributed, metabolized and excreted efficiently.     

Antioxidant Cardioprotection against Reperfusion Injury: Potential Therapeutic Roles of Resveratrol and Quercetin

Ischemia-reperfusion myocardial damage is a paradoxical tissue injury occurring during percutaneous coronary intervention (PCI) in acute myocardial infarction (AMI) patients. Although this damage could account for up to 50% of the final infarct size, there has been no available pharmacological treatment until now. Oxidative stress contributes to the underlying production mechanism, exerting the most marked injury during the early onset of reperfusion. So far, antioxidants have been shown to protect the AMI patients undergoing PCI to mitigate these detrimental effects; however, no clinical trials to date have shown any significant infarct size reduction. Therefore, it is worthwhile to consider multitarget antioxidant therapies targeting multifactorial AMI. Indeed, this clinical setting involves injurious effects derived from oxygen deprivation, intracellular pH changes and increased concentration of cytosolic Ca²⁺ and reactive oxygen species, among others. Thus, we will review a brief overview of the pathological cascades involved in ischemia-reperfusion injury and the potential therapeutic effects based on preclinical studies involving a combination of antioxidants, with particular reference to resveratrol and quercetin, which could contribute to cardioprotection against ischemia-reperfusion injury in myocardial tissue. We will also highlight the upcoming perspectives of these antioxidants for designing future studies.     

Pathogenesis of Keratinocyte Carcinomas and the Therapeutic Potential of Medicinal Plants and Phytochemicals

Keratinocyte carcinoma (KC) is a form of skin cancer that develops in keratinocytes, which are the predominant cells present in the epidermis layer of the skin. Keratinocyte carcinoma comprises two sub-types, namely basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). This review provides a holistic literature assessment of the origin, diagnosis methods, contributing factors, and current topical treatments of KC. Additionally, it explores the increase in KC cases that occurred globally over the past ten years. One of the principal concepts highlighted in this article is the adverse effects linked to conventional treatment methods of KC and how novel treatment strategies that combine phytochemistry and transdermal drug delivery systems offer an alternative approach for treatment. However, more in vitro and in vivo studies are required to fully assess the efficacy, mechanism of action, and safety profile of these phytochemical based transdermal chemotherapeutics.     

Expression, purification, and characterization of a galactofuranosyltransferase involved in Mycobacterium tuberculosis arabinogalactan biosynthesis

The major structural component of the cell wall of Mycobacterium tuberculosis is a lipidated polysaccharide, the mycoyl-arabinogalactan-peptidoglycan (mAGP) complex. This glycoconjugate plays a key role in the survival of the organism, and thus, enzymes involved in its biosynthesis have attracted attention as sites for drug action. At the core of the mAGP is a galactan composed of D-galactofuranose residues attached via alternating beta-(1-->5) and beta-(1-->6) linkages. A single enzyme, glfT, has been shown to synthesize both glycosidic linkages. We report here the first high-level expression and purification of glfT by expression of the Rv3808c gene in Escherichia coli C41(DE3). Following a three-step purification procedure, 3-7 mg of protein of >95% purity was isolated from each liter of culture. We subsequently probed the substrate specificity of glfT by evaluating a panel of potential mono- and oligosaccharide substrates and demonstrated, for the first time, that trisaccharides are better substrates than disaccharides and that one disaccharide, in which the terminal D-galactofuranose residue is replaced with an L-arabinofuranose moiety, is a weak substrate. Kinetic characterization of the enzyme using four of the oligosaccharide acceptors gave K(m) values ranging from 204 microM to 1.7 mM. Through the use of NMR spectroscopy and mass spectrometry, we demonstrated that this recombinant enzyme, like the wild-type protein, is bifunctional and can synthesize both beta-(1-->6) and beta-(1-->5)-linkages in an alternating fashion. Access to purified glfT is expected to facilitate the development of high-throughput assays for the identification of inhibitors of the enzyme, which are potential antituberculosis agents.     

The detection of Mycobacterium tuberculosis in sputum sample based on a wireless magnetoelastic-sensing device

This paper presents a real-time detection of Mycobacterium tuberculosis (M. TB) using a wireless magnetoelastic sensor. The sensor is fabricated by coating a magnetoelastic ribbon (Metglas 2826MB) with a polyurethane protecting film. M. TB consumes the nutrients of a liquid culture medium in growing and reproducing process, which results in properties changes (viscosity, density, elasticity, ion concentration, etc.) of the culture medium, and consequently changes in the resonance frequency of the magnetoelastic sensor. Using the described technique M. TB is quantified and sensor response is proportional to logarithmic values of the M. TB concentration from 10(4) to 10(9)cells ml(-1), with a detection limit of 10(4)cells ml(-1) at a noise level of approximately 10 Hz. The sensor can be used effectively for monitoring the bacterial growth and good results were obtained when used in sputum sample. The drug-resistance of isoniazid (INH) and rifampin (RFP) on M. TB growth in culture medium was evaluated based on this proposed method. The wireless nature of the presented device facilitates the aseptic operations.     

The absence of proximal strain in the truncated hemoglobins from Mycobacterium tuberculosis

HbN and HbO are two truncated hemoglobins from Mycobacterium tuberculosis. Resonance Raman spectra of the deoxy derivatives of these two homodimeric hemoglobins indicate that there is no proximal strain imposed by intersubunit interactions on the proximal iron-histidine bond as that observed in the tetrameric human hemoglobin. In addition, with nanosecond laser flash photolysis, it was concluded that movement along the Fe-His bond following the dissociation of CO does not trigger a quaternary structural transition in these two hemoglobins.     

Alkylation of Ethanolamines: An Approach to a Novel Class of Functional Surfactants

Some monomeric and dimeric surfactants with functional head groups have been synthesized from di- and triethanolamine synthons. The treatment of alkyl bromide with triethanolamine resulted in simultaneous N-alkylation and O-alkylation products. However, with diethanolamine, N-alkylated products were obtained, which were further used to synthesize various double-tailed surfactants and gemini surfactants.     

Fullerene quinazolinone conjugates targeting Mycobacterium tuberculosis: a combined molecular docking,QSAR, and ONIOM approach

Fullerene and its derivatives may bind to biological molecules, causing inhibitory effects. In this context, investigations of interactions of fullerene-based conjugates with proteins are of general interest. Particularly, fullerene and its derivatives demonstrate antibacterial properties; and one of the potential targets for drug design and health therapy is the inhibition of 6-oxopurine phosphoribosyltransferase in Mycobacterium tuberculosis (PDB code: 4RHY). In this article, the binding interactions between a series of quinazoline-4(3H)-ones and their fullerene derivatives with the target transferase were computationally investigated. Initially, we developed predictive quantitative structure-activity relationships (QSAR) models. Next, we introduced a simplified calculation schema that allows to evaluate relative binding affinities and to reveal specific mechanisms of action. For this purpose, the molecular docking approach was utilized to identify the native poses of the 18 transferase inhibitors. The binding pocket of the target protein was isolated and semi-empirical, and hybrid ONIOM scoring functions at different levels of theory were used to treat the ligands and the isolated binding pocket. The agreement within the calculated binding-free energies trends, as well as the agreement with the experimental data, suggests that the developed calculation schema can be used to estimate relative binding affinities towards 4RH. The combination of quantum-chemical models and QSAR models could be applied for future design of new selective inhibitors.