Design of a potent anticancer lead inspired by natural products from traditional Indian medicine

Abstract

Among the plant constituents of Clerodendrum colebrookianum Walp., acteoside, martinoside, and osmanthuside β6 interact with ROCK, a drug target for cancer. In this study, aglycone fragments of these plant constituents (caffeic acid, ferulic acid, and p-coumaric acid) along with the homopiperazine ring of fasudil (standard ROCK inhibitor) were used to design hybrid molecules. The designed molecules interact with the key hinge region residue Met156/Met157 of ROCK I/II in a stable manner according to our docking and molecular dynamics simulations. These compounds were synthesized and tested in vitro in SW480, MDA-MB-231, and A-549 cancer cell lines. The most promising compound was chemically optimized to obtain a thiourea analog, 6a (IC₅₀ = 25?µM), which has >3-fold higher antiproliferative activity than fasudil (IC₅₀ = 87?µM) in SW480 cells. Treatment with this molecule also inhibits the migration of colon cancer cells and induces cell apoptosis. Further, SPR experiments suggests that the binding affinity of 6a with ROCK I protein is better than that of fasudil. Hence, the drug-like natural product analog 6a constitutes a highly promising new anticancer lead.

Communicated by Ramaswamy H. Sarma     

Simple plant-based production and purification of the assembled human ferritin heavy chain as a nanocarrier for tumor-targeted drug delivery and bioimaging in cancer therapy

Nanoparticles are used as carriers for the delivery of drugs and imaging agents. Proteins are safer than synthetic nanocarriers due to their greater biocompatibility and the absence of toxic degradation products. In this context, ferritin has the additional benefit of inherently targeting the membrane receptor transferrin 1, which is overexpressed by most cancer cells. Furthermore, this self-assembling multimeric protein can be loaded with more than 2000 iron atoms, as well as drugs, contrast agents, and other cargos. However, recombinant ferritin currently costs ~3.5 million € g⁻¹, presumably because the limited number of producers cannot meet demand, making it generally unaffordable as a nanocarrier. Because plants can produce proteins at very-large-scale, we developed a simple, proof-of-concept process for the production of the human ferritin heavy chain by transient expression in Nicotiana benthamiana. We optimized the protein yields by screening different compartments and 5′-untranslated regions in PCPs, and selected the best-performing construct for production in differentiated plants. We then established a rapid and scalable purification protocol by combining pH and heat treatment before extraction, followed by an ultrafiltration/diafiltration size-based separation process. The optimized process achieved ferritin levels of ~40 mg kg⁻¹ fresh biomass although depth filtration limited product recovery to ~7%. The purity of the recombinant product was >90% at costs ~3% of the current sales price. Our method therefore allows the production of affordable ferritin heavy chain as a carrier for therapeutic and diagnostic agents, which is suitable for further stability and functionality testing in vitro and in vivo.     

Effect of different cooking methods on loss of iron and zinc micronutrients in fortified and non-fortified rice

Malnutrition is considered as major public health concern and is emerging challenge to food and nutrition security particularly in developing countries. Rice is the staple food and consumed by the half of the world's population which is the source of daily requirement of the nutrients. Attempts are being made to fortify rice with micronutrients, but the loss or retention of these micronutrients in different cooking methods is not well studied and documented especially in fortified rice. In the present study, paddy seeds of six Indian varieties were fortified with iron and zinc by parboiling process. Consequently, fortified polished rice had higher micronutrient contents (Fe, 106.31 ± 12.56; Zn, 97.72 ± 9.75) than non-fortified polished rice (Fe, 7.44 ± 1.05; Zn, 14.74 ± 2.94) expressed in ppm. Polished rice of both fortified and non-fortified were cooked under five different cooking conditions and analyzed for remaining iron and zinc content. Cooking rice in rice cooker without prior washing (NRC) retained highest concentration of Fe and Zinc in both fortified and non-fortified rice varieties. It also showed that fortified rice suffered higher percentage loss of micronutrient, than the non-fortified rice. But the average retained micronutrient amount measured in ppm, was higher in fortified rice (Fe, 43.54 ± 6.88; Zn, 36.7 ± 3.12) than in non-fortified rice (Fe, 4.24 ± 0.87; Zn, 9.3 ± 2.11). Hence, adopting appropriate cooking method, higher amount of micronutrients will be retained in the cooked food which will in turn help in combating the malnutrition and improve health.     

CULTURE AND BIOCHEMICAL ANALYSIS OF A TEA ALGAL PATHOGEN,CEPHALEUROS PARASITICUS1

To assess the current situation regarding the incidence of red rust disease in tea plants, an extensive survey was conducted in southern Indian tea plantations covering different tea cultivars and agroclimatic zones. The results indicated that the incidence of disease was more severe in tea seedlings than clones in all the agroclimatic zones. On the other hand, a simple, reliable, and reproducible technique was standardized for culturing Cephaleuros parasiticus G. Karst. isolated from infected tea leaves. Ten isolates were obtained, of which two were screened based on growth rate and culture characters for further studies. Ten culture media were tested for the culturing of C. parasiticus in which Trebouxia and Bristol media were the best followed by George, Go algal, and tea leaf extract media. Variations between isolates (Valparai C. parasiticus field number 27 [VCP27], Munnar C. parasiticus field number 11 [MCP11], and University of Texas culture number 2412 [UTEX2412]) of C. parasiticus were studied based on the growth pattern, protein expression profile, and cellular constituents in the filaments. The quantitative estimation of cellular constituents showed that there was no significant difference in these constituents among isolates. The detection of amino acids in the filaments of C. parasiticus isolates showed 16 free forms and 11 bound forms. Amino acids in bound form were higher in all the isolates than in free form of amino acids. The three isolates of C. parasiticus were closely related, with bands lying between the molecular weight of 116 and 35 kDa.     

De novo 3D models of SARS-CoV-2 RNA elements from consensus experimental secondary structures

The rapid spread of COVID-19 is motivating development of antivirals targeting conserved SARS-CoV-2 molecular machinery. The SARS-CoV-2 genome includes conserved RNA elements that offer potential small-molecule drug targets, but most of their 3D structures have not been experimentally characterized. Here, we provide a compilation of chemical mapping data from our and other labs, secondary structure models, and 3D model ensembles based on Rosetta''s FARFAR2 algorithm for SARS-CoV-2 RNA regions including the individual stems SL1-8 in the extended 5′ UTR; the reverse complement of the 5′ UTR SL1-4; the frameshift stimulating element (FSE); and the extended pseudoknot, hypervariable region, and s2m of the 3′ UTR. For eleven of these elements (the stems in SL1–8, reverse complement of SL1–4, FSE, s2m and 3′ UTR pseudoknot), modeling convergence supports the accuracy of predicted low energy states; subsequent cryo-EM characterization of the FSE confirms modeling accuracy. To aid efforts to discover small molecule RNA binders guided by computational models, we provide a second set of similarly prepared models for RNA riboswitches that bind small molecules. Both datasets (‘FARFAR2-SARS-CoV-2’, https://github.com/DasLab/FARFAR2-SARS-CoV-2; and ‘FARFAR2-Apo-Riboswitch’, at https://github.com/DasLab/FARFAR2-Apo-Riboswitch’) include up to 400 models for each RNA element, which may facilitate drug discovery approaches targeting dynamic ensembles of RNA molecules.