Ligand fishing as a tool to screen natural products with anticancer potential.

Cancer is the second leading cause of death in the world and its incidence is expected to increase with the aging of the world's population and globalization of risk factors. Natural products and their derivatives have provided a significant number of approved anticancer drugs and the development of robust and selective screening assays for the identification of lead anticancer natural products are essential in the challenge of developing personalized targeted therapies tailored to the genetic and molecular characteristics of tumors. To this end, a ligand fishing assay is a remarkable tool to rapidly and rigorously screen complex matrices, such as plant extracts, for the isolation and identification of specific ligands that bind to relevant pharmacological targets. In this paper, we review the application of ligand fishing with cancer-related targets to screen natural product extracts for the isolation and identification of selective ligands. We provide critical analysis of the system configurations, targets, and key phytochemical classes related to the field of anticancer research. Based on the data collected, ligand fishing emerges as a robust and powerful screening system for the rapid discovery of new anticancer drugs from natural resources. It is currently an underexplored strategy according to its considerable potential.

High-resolution inhibition profiling and ligand fishing for screening of nucleoside hydrolase ligands in Moringa oleifera Lamarck.

In Leishmania donovani, the causative protozoan of visceral leishmaniasis, nucleoside hydrolase enzyme (NH) is fundamental for the biosynthesis of its DNA and RNA. Therefore, LdNH is considered a potential target for the development of new leishmaniasis chemotherapy. Moringa oleifera Lamarck is a medicinal plant native to northeastern India with numerous pharmacological properties, including antileishmanial activity. Thus, this study aimed to explore the inhibitory activity of different extracts from M. oleifera leaves and flowers on LdNH. Using LdNH covalently immobilized on magnetic particles (LdNH-MPs), a novel activity assay was developed based on the direct quantification of the formed product by HPLC-DAD. This study screened 12 extracts from leaves and flowers of M. oleifera using different extraction methods. The hydroethanolic (70% ethanol) extract from flowers, obtained by infusion (FIEH) or ultrasound-assisted extraction (FUEH), exhibited respectively IC50 values of 26.2 ± 4.63 µg/mL and 4.96 ± 0.52 µg/mL. The most promising extract (FUEH) was investigated by high-resolution LdNH inhibition profiling, which showed different regions of inhibition in the biochromatogram. A ligand fishing assay was attempted to pinpoint the bioactive compounds. Experimental conditions employed in the elution step of the ligand fishing assay did not result in ligands isolation. However, the analyses of the crude extract solution and the supernatants after the incubation with the active and inactive LdNH-MPs indicated missing peaks referring to compounds selectively retained in the active LdNH-MPs incubation. The missing peaks eluted in the same region that exhibits inhibition in the high-resolution LdNH inhibition profiling. The ligands were identified by UHPLC-MS/MS as palatinose, adenosine, 3-p-coumaroylquinic acid, 4-p-coumaroylquinic acid, hyperoside, quercetin-3-O-malonyl glycoside, and kaempferol-3-O-galactoside.

Anti-tubercular profile of new selenium-menadione conjugates against Mycobacterium tuberculosis H37Rv (ATCC 27294) strain and multidrug-resistant clinical isolates.

Tuberculosis (TB) is one of the most fatal diseases and is responsible for the infection of millions of people around the world. Most recently, scientific frontiers have been engaged to develop new drugs that can overcome drug-resistant TB. Following this direction, using a designed scaffold based on the combination of two separate pharmacophoric groups, a series of menadione-derived selenoesters was developed with good yields. All products were evaluated for their in vitro activity against Mycobacterium tuberculosis H37Rv and attractive results were observed, especially for the compounds 8a, 8c and 8f (MICs 2.1, 8.0 and 8.1 µM, respectively). In addition, 8a, 8c and 8f demonstrated potent in vitro activity against multidrug-resistant clinical isolates (CDCT-16 and CDCT-27) with promising MIC values ranging from 0.8 to 3.1 µM. Importantly, compounds 8a and 8c were found to be non-toxic against the Vero cell line. The SI value of 8a (>23.8) was found to be comparable to that of isoniazid (>22.7), which suggests the possibility of carrying out advanced studies on this derivative. Therefore, these menadione-derived selenoesters obtained as hybrid compounds represent promising new anti-tubercular agents to overcome TB multidrug resistance.

Formulation and Evaluation of a Novel Itraconazole-Clotrimazole Topical Emulgel for the Treatment of Sporotrichosis.

In recent years, the development of new pharmaceutical formulations for the treatment of sporotrichosis has become a relevant research field. In this work, we aimed to develop an emulgel containing itraconazole and clotrimazole to ensure therapeutic effectiveness against Sporothrix brasiliensis. The topical use of a formulation that combines both drugs represents an interesting option for the complementary treatment of sporotrichosis. The emulgel formulation was prepared and evaluated for its zeta potential, viscosity, in vitro antifungal activity and stability at different storage conditions. The results showed that the newly developed emulgel displayed promising physicochemical characteristics, as well as a good in vitro inhibitory activity against S. brasiliensis yeasts. The results obtained in this work suggest that the emulgel containing itraconazole and clotrimazole might highly be efficient and a complementary therapy to oral administration in the treatment of sporotrichosis.

Investigation of a Microemulsion Containing Clotrimazole and Itraconazole for Transdermal Delivery for the Treatment of Sporotrichosis.

The aim of this study was to develop a microemulsion (ME) formulation containing an association of itraconazole (ITC) and clotrimazole (CLT) as a transdermal delivery system for the treatment of sporotrichosis. Pseudoternary phase diagrams were constructed to optimize the ME formulation. The ME formulation selected contained 1% (w/w) ITC and 1% (w/w) CLT and was composed of 23.07% Tween® 60 (surfactant), 23.07% propylene glycol (cosurfactant/cosolvent), 30.77% benzyl alcohol (oil), and 21.09% water. The ITC/CLT-loaded ME (ITC/CLT-ME) had a droplet size value of 217 ± 0.9 nm, with a polydispersity index of 0.5 ± 0.1. Permeation experiments on pig ear skin were conducted for ITC/CLT-ME, and the results indicated that the drug permeation performance was influenced by CLT, indicating that CLT acts as a promoter enhancer. In the in vitro antifungal activity assay using Sporothrix brasiliensis yeast, the inhibition halo produced by ITC/CLT-ME exhibited a mean diameter of 43.67 ± 2.31 mm. The ITC/CLT-ME formulation did not cause skin irritation in mice. The results suggest that ITC/CLT-ME is a promising tool for the transdermal treatment of sporotrichosis.

Development of a Method for the Quantification of Clotrimazole and Itraconazole and Study of Their Stability in a New Microemulsion for the Treatment of Sporotrichosis.

Sporotrichosis occurs worldwide and is caused by the fungus Sporothrix brasiliensis. This agent has a high zoonotic potential and is transmitted mainly by bites and scratches from infected felines. A new association between the drugs clotrimazole and itraconazole is shown to be effective against S. brasiliensis yeasts. This association was formulated as a microemulsion containing benzyl alcohol as oil, Tween® 60 and propylene glycol as surfactant and cosurfactant, respectively, and water. Initially, the compatibility between clotrimazole and itraconazole was studied using differential scanning calorimetry (DSC), thermogravimetric analysis (TG), Fourier transform infrared spectroscopy (FTIR), and X-ray powder diffraction (PXRD). Additionally, a simple and efficient analytical HPLC method was developed to simultaneously determine the concentration of clotrimazole and itraconazole in the novel microemulsion. The developed method proved to be efficient, robust, and reproducible for both components of the microemulsion. We also performed an accelerated stability study of this formulation, and the developed analytical method was applied to monitor the content of active ingredients. Interestingly, these investigations led to the detection of a known clotrimazole degradation product whose structure was confirmed using NMR and HRMS, as well as a possible interaction between itraconazole and benzyl alcohol.

Cathepsin D immobilized capillary reactors for on-flow screening assays.

The treatment of diseases using enzymes as targets has called for the development of new and reliable methods for screening. The protease cathepsin D is one such target involved in several diseases such as tumors, degenerative processes, and vital processes of parasites causing schistosomiasis. Herein, we describe the preparation of a fused silica capillary, cathepsin D (CatD)-immobilized enzyme reactor (IMER) using in a multidimensional High Performance Liquid Chromatography-based method (2D-HPLC) and zonal affinity chromatography as an alternative in the search for new ligands. The activity and kinetic parameters of CatD-IMER were evaluated by monitoring the product MOCAc-Gly-Lys-Pro-Ile-Leu-Phe (P-MOCAc) (KM = 81.9 ±â€¯7.49 µmol/L) generated by cleavage of the fluorogenic substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-d-Arg-NH2 (S-MOCAc). Stability studies have indicated that CatD-IMER retained 20% of activity after 5 months, a relevant result, because proteases are susceptible to autoproteolysis in solution assays with free enzyme. In the search for inhibitors, 12 crude natural product extracts were analyzed using CatD-IMER as the target, resulting in the isolation of different classes of natural products. In addition, 26 compounds obtained from different species of plants were also screened, demonstrating the efficiency and reproducibility of the herein reported assay even in the case of complex matrices such as plant crude extracts.

Immobilized purine nucleoside phosphorylase from Schistosoma mansoni for specific inhibition studies.

The parasite Schistosoma mansoni (Sm) depends exclusively on the salvage pathway for its purine requirements. The enzyme purine nucleoside phosphorylase (PNP) is, therefore, a promising target for development of antischistosomal agents and an assay for screening of inhibitors. To enable this, immobilized SmPNP reactors were produced. By quantification of hypoxanthine by liquid chromatography, kinetic constants (K M) for the substrate inosine were determined for the free and immobilized enzyme as 110 ± 6.90 µmol L (-1) and 164 ± 13.4 µmol L (-1), respectively, indicating that immobilization did not affect enzyme activity. Furthermore, the enzyme retained 25 % of its activity after four months. Non-Michaelis kinetics for the phosphate substrate, and capacity for Pi-independent hydrolysis were also demonstrated, despite the low rate of enzymatic catalysis. Use of an SmPNP immobilized enzyme reactor (IMER) for inhibitor-screening assays was demonstrated with a small library of 9-deazaguanine analogues. The method had high selectivity and specificity compared with screening by use of the free enzyme by the Kalckar method, and furnished results without the need for verification of the absence of false positives.