Disrupting protease and deubiquitinase activities of SARS-CoV-2 papain-like protease by natural and synthetic products discovered through multiple computational and biochemical approaches.

The single-stranded RNA genome of SARS-CoV-2 encodes several structural and non-structural proteins, among which the papain-like protease (PLpro) is crucial for viral replication and immune evasion and has emerged as a promising therapeutic target. The current study aims to discover new inhibitors of PLpro that can simultaneously disrupt its protease and deubiquitinase activities. Using multiple computational approaches, six compounds (CP1-CP6) were selected from our in-house compounds database, with higher docking scores (-7.97 kcal/mol to -8.14 kcal/mol) and fitted well in the active pocket of PLpro. Furthermore, utilizing microscale molecular dynamics simulations (MD), the dynamic behavior of selected compounds was studied. Those molecules strongly binds at the PLpro active site and forms stable complexes. The dynamic motions suggest that the binding of CP1-CP6 brought the protein to a closed conformational state, thereby altering its normal function. In an in vitro evaluation, CP2 showed the most significant inhibitory potential for PLpro (protease activity = 2.71 ± 0.33 µM and deubiquitinase activity = 3.11 ± 0.75 µM), followed by CP1, CP5, CP4 and CP6. Additionally, CP1-CP6 showed no cytotoxicity at a concentration of 30 µM in the human BJ cell line.

Synthesis and Enzymatic Evaluation of a Small Library of Substituted Phenylsulfonamido-Alkyl Sulfamates towards Carbonic Anhydrase II.

A small library of 79 substituted phenylsulfonamidoalkyl sulfamates, 1b-79b, was synthesized starting from arylsulfonyl chlorides and amino alcohols with different numbers of methylene groups between the hydroxyl and amino moieties yielding intermediates 1a-79a, followed by the reaction of the latter with sulfamoyl chloride. All compounds were screened for their inhibitory activity on bovine carbonic anhydrase II. Compounds 1a-79a showed no inhibition of the enzyme, in contrast to sulfamates 1b-79b. Thus, the inhibitory potential of compounds 1b-79b towards this enzyme depends on the substituent and the substitution pattern of the phenyl group as well as the length of the spacer. Bulkier substituents in the para position proved to be better for inhibiting CAII than compounds with the same substituent in the meta or ortho position. For many substitution patterns, compounds with shorter spacer lengths were superior to those with long chain spacers. Compounds with shorter spacer lengths performed better than those with longer chain spacers for a variety of substitution patterns. The most active compound held inhibition constant as low as Ki = 0.67 µM (for 49b) and a tert-butyl substituent in para position and acted as a competitive inhibitor of the enzyme.

Dehydroabietylamine-substituted trifluorobenzene sulfonamide rhodamine B hybrids as anticancer agents overcoming drug resistance.

Attachment of a conjugate assembled from a novel fluorinated carbonic anhydrase inhibitor and rhodamine B onto dehydroabietylamine (DHA) or cyclododecylamine led to first-in-class conjugates of good cytotoxicity; thereby IC50 values (from SRB assays; employed tumor cell lines A2780, A2780Cis, A549, HT29, MCF7, and non-malignant human fibroblasts CCD18Co) between 0.2 and 0.7 µM were found. Both conjugates showed similar cytotoxic activity but the dehydroabietylamine derived conjugate outperformed its cyclododecyl analog in terms of tumor cell/non-tumor cell selectivity. Both conjugates accumulate intracellular, and the DHA conjugate was able to overcome drug resistance which is effective independent of the expression status of carbonic anhydrase IX.

Synthesis of Rhodamine-Conjugated Lupane Type Triterpenes of Enhanced Cytotoxicity.

Various conjugates with rhodamines were prepared by starting with betulinic acid (BA) and platanic acid (PA). The molecules homopiperazine and piperazine, which were identified in earlier research, served as linkers between the rhodamine and the triterpene. The pentacyclic triterpene's ring A was modified with two acetyloxy groups in order to possibly boost its cytotoxic activity. The SRB assays' cytotoxicity data showed that conjugates 13-22, derived from betulinic acid, had a significantly higher cytotoxicity. Of these hybrids, derivatives 19 (containing rhodamine B) and 22 (containing rhodamine 101) showed the best values with EC50 = 0.016 and 0.019 µM for A2780 ovarian carcinoma cells. Additionally, based on the ratio of EC50 values, these two compounds demonstrated the strongest selectivity between malignant A2780 cells and non-malignant NIH 3T3 fibroblasts. A375 melanoma cells were used in cell cycle investigations, which showed that the cells were halted in the G1/G0 phase. Annexin V/FITC/PI staining demonstrated that the tumor cells were affected by both necrosis and apoptosis.

Combination of Betulinic Acid Fragments and Carbonic Anhydrase Inhibitors-A New Drug Targeting Approach.

Human carbonic anhydrase IX (hCA IX) is a zinc(II)-dependent metalloenzyme that plays a critical role in the conversion of carbon dioxide and water to protons and bicarbonate. It is a membrane-bound protein with an extracellular catalytic center that is predominantly overexpressed in solid hypoxic tumors. Sulfamates and sulfonamides, for example acetazolamide (AZA), have been used to inhibit hCA IX in order to improve the response to solid hypoxic tumors. In the present study, we propose a new drug targeting approach by attaching the natural cytotoxic substances betulin and betulinic acid (BA) via a linker to sulfonamides. The conjugate was designed with different spacer lengths to accumulate at the target site of hCA IX. Computational and cell biological studies suggest that the length of the linker may influence hCA IX inhibition. Cytotoxicity tests of the newly synthesized bifunctional conjugates 3, 5, and 9 show effective cytotoxicity in the range of 6.4 and 30.1 µM in 2D and 3D tumor models. The hCA IX inhibition constants of this conjugates, measured using an in vitro enzyme assay with p-nitrophenyl acetate, were determined in a low µM-range, and all compounds reveal a significant inhibition of hypoxia-induced CA activity in a cell-based assay using the Wilbur-Anderson method. In addition, the cells respond with G1 increase and apoptosis induction. Overall, the dual strategy to produce cytotoxic tumor therapeutics that inhibit tumor-associated hCA IX was successfully implemented.

Diethoxyphosphoryloxy-oleanolic acid is a nanomolar-inhibitor of butyrylcholinesterase.

A series of new betulin, lupeol, erythrodiol, and oleanolic acid phosphoryloxy- and furoyloxy-derivatives has been synthesized and their structure was confirmed by NMR spectroscopy. Synthesized compounds were subjected to Ellman's assays to determine their ability to inhibit the enzymes AChE and BChE. Among them, diethoxyphosphoryloxy-oleanolic acid inhibited BChE with a value of 99%, thereby acting as a mixed-type inhibitor holding very low Ki values of Ki = 6.59 nM and Ki ' = 1.97 nM, respectively.

An asiatic acid derived trisulfamate acts as a nanomolar inhibitor of human carbonic anhydrase VA.

This investigation delves into the inhibitory capabilities of a specific set of triterpenoic acids on diverse isoforms of human carbonic anhydrase (hCA). Oleanolic acid (1), maslinic acid (2), betulinic acid (3), platanic acid (4), and asiatic acid (5) were chosen as representative triterpenoids for evaluation. The synthesis involved acetylation of parent triterpenoic acids 1-5, followed by sequential reactions with oxalyl chloride and benzylamine, de-acetylation of the amides, and subsequent treatment with sodium hydride and sulfamoyl chloride, leading to the formation of final compounds 21-25. Inhibition assays against hCAs I, II, VA, and IX demonstrated noteworthy outcomes. A derivative of betulinic acid, compound 23, exhibited a Ki value of 88.1 nM for hCA VA, and a derivative of asiatic acid, compound 25, displayed an even lower Ki value of 36.2 nM for the same isoform. Notably, the latter compound displayed enhanced inhibitory activity against hCA VA when compared to the benchmark compound acetazolamide (AAZ), which had a Ki value of 63.0 nM. Thus, this compound surpasses the inhibitory potency and isoform selectivity of the standard compound acetazolamide (AAZ). In conclusion, the research offers insights into the inhibitory potential of selected triterpenoic acids across diverse hCA isoforms, emphasizing the pivotal role of structural attributes in determining isoform-specific inhibitory activity. The identification of compound 25 as a robust and selective hCA VA inhibitor prompts further exploration of its therapeutic applications.

Identification of new pharmacophore against SARS-CoV-2 spike protein by multi-fold computational and biochemical techniques.

COVID-19 appeared as a highly contagious disease after its outbreak in December 2019 by the virus, named SARS-CoV-2. The threat, which originated in Wuhan, China, swiftly became an international emergency. Among different genomic products, spike protein of virus plays a crucial role in the initiation of the infection by binding to the human lung cells, therefore, SARS-CoV-2's spike protein is a promising therapeutic target. Using a combination of a structure-based virtual screening and biochemical assay, this study seeks possible therapeutic candidates that specifically target the viral spike protein. A database of ~ 850 naturally derived compounds was screened against SARS-CoV-2 spike protein to find natural inhibitors. Using virtual screening and inhibitory experiments, we identified acetyl 11-keto-boswellic acid (AKBA) as a promising molecule for spike protein, which encouraged us to scan the rest of AKBA derivatives in our in-house database via 2D-similarity searching. Later 19 compounds with > 85% similarity with AKBA were selected and docked with receptor binding domain (RBD) of spike protein. Those hits declared significant interactions at the RBD interface, best possess and excellent drug-likeness and pharmacokinetics properties with high gastrointestinal absorption (GIA) without toxicity and allergenicity. Our in-silico observations were eventually validated by in vitro bioassay, interestingly, 10 compounds (A3, A4, C3, C6A, C6B, C6C, C6E, C6H, C6I, and C6J) displayed significant inhibitory ability with good percent inhibition (range: > 72-90). The compounds C3 (90.00%), C6E (91.00%), C6C (87.20%), and C6D (86.23%) demonstrated excellent anti-SARS CoV-2 spike protein activities. The docking interaction of high percent inhibition of inhibitor compounds C3 and C6E was confirmed by MD Simulation. In the molecular dynamics simulation, we observed the stable dynamics of spike protein inhibitor complexes and the influence of inhibitor binding on the protein's conformational arrangements. The binding free energy ΔGTOTAL of C3 (-38.0 ± 0.08 kcal/mol) and C6E (-41.98 ± 0.08 kcal/mol) respectively indicate a strong binding affinity to Spike protein active pocket. These findings demonstrate that these molecules particularly inhibit the function of spike protein and, therefore have the potential to be evaluated as drug candidates against SARS-CoV-2.

F16 Hybrids Derived from Steviol or Isosteviol Are Accumulated in the Mitochondria of Tumor Cells and Overcome Drug Resistance.

Steviol and isosteviol were prepared from the commercially available sweetener stevioside and converted into lipophilic F16 hybrids. Their cytotoxicity was determined in SRB assays and showed to depend on both the substitution pattern of the aromatic substituent as well as on the spacer length. Therefore, compound 25 held an IC50 (A2780) of 180 nM, thus surpassing the activity of comparable rhodamine hybrids. Several of the compounds were also able to overcome drug resistance in the A2780/A2780cis model. Extra staining experiments showed a similar subcellular accumulation pattern of the F16 hybrids as a well-established mitocan, hence proving preferential mitochondrial accumulation but also some other accumulation in other cellular areas.

TLC and HPTLC-APCI-MS for the rapid discrimination of plant resins frequently used for lacquers and varnishes by artists and conservators.

INTRODUCTION: Depending on their terpenoid and phenolic constituents plant resins can be classified as diterpenoid, triterpenoid or phenolic resins; thereby the profile of diterpenes and triterpenes is considered as genus- or even species-specific. OBJECTIVES: We aimed to develop a simple, rapid, inexpensive, sensitive and specific method for the identification of resin-specific triterpenoid and phenolic compounds in plant resins using (HP)TLC [(high-performance) thin-layer chromatography] combined with APCI-MS (atmospheric pressure chemical ionisation mass spectrometry) and post-chromatographic detection reactions. METHODS: Twenty resin samples from different plant species were analysed. Different extraction procedures, post-chromatographic detection reagents as well as various sorbents and solvents for planar chromatography were tested. To evaluate the potential of the optimised (HP)TLC-APCI-MS methods, parameter such as limit of detection (LOD) was determined for selected marker compounds. RESULTS: Our protocol enabled qualitative analyses of chemotaxonomic molecular markers in natural resins such as dammar, mastic, olibanum and benzoin. For the first time, the application of thionyl chloride-stannic chloride reagent for a specific post-chromatographic detection of triterpenes is reported, sometimes even allowing discrimination between isomers based on their characteristic colour sequences. For triterpene acids, triterpene alcohols and phenolic compounds, detection limits of 2-20 ng/TLC zone and a system precision with a relative standard deviation (RSD) in the range of 3.9%-7.0% were achieved by (HP)TLC-APCI-MS. The applicability of the method for the analysis of resin-based varnishes was successfully tested on a mastic-based varnish. Thus, the method we propose is a helpful tool for the discrimination of resins and resin-based varnishes with respect to their botanical origin.

Ready-to-use nanopore platform for label-free small molecule quantification: Ethanolamine as first example.

In recent decades, nanopores have become a promising diagnostic tool. Protein and solid-state nanopores are increasingly used for both RNA/DNA sequencing and small molecule detection. The latter is of great importance, as their detection is difficult or expensive using available methods such as HPLC or LC-MS. DNA aptamers are an excellent detection element for sensitive and specific detection of small molecules. Herein, a method for quantifying small molecules using a ready-to-use sequencing platform is described. Taking ethanolamine as an example, a strand displacement assay is developed in which the target-binding aptamer is displaced from the surface of magnetic particles by ethanolamine. Non-displaced aptamer and thus the ethanolamine concentration are detected by the nanopore system and can be quantified in the micromolar range using our in-house developed analysis software. This method is thus the first to describe a label-free approach for the detection of small molecules in a protein nanopore system.

Ureidobenzenesulfonamides as Selective Carbonic Anhydrase I, IX, and XII Inhibitors.

Sulfonamides remain an important class of drugs, especially because of their inhibitory effects on carbonic anhydrases. Herein, we have synthesized several sulfonamides and tested them for their inhibitory activity against carbonic anhydrases hCA I, hCA II, hCA IX, and hCA XII, respectively. Thereby, biphenyl- and benzylphenyl-substituted sulfonamides showed high selectivity against hCA IX and hCA XII; these enzymes are common targets in the treatment of hypoxic cancers, and noteworthy inhibitory activity was observed for several compounds toward hCA I that might be of interest for future applications to treat cerebral edema. Compound 3 (4-[3-(2-benzylphenyl)ureido]benzenesulfonamide) held an exceptionally low Ki value of 1.0 nM for hCA XII.

Inhibitory properties of quinopimaric acid derivatives towards cholinesterases.

A series of new diterpene quinopimaric acid derivatives modified at the hydroxyl group with different pharmacophore fragments has been synthesised and their (along with previously obtained compounds) inhibitory properties towards cholinesterases were studied. Thereby an indole-3-acetyl derivative 7 and a propargyl substituted compound 28 were shown to be excellent and acetylcholinesterase-selective inhibitors. Both compounds inhibited the enzyme as a mixed type inhibitor, and Ki values of 0.41 and 0.44 µM and Ki' values of 0.98 and 2.26 µM were determined. The binding interactions between all active compounds and ligands protein were confirmed through molecular docking study.

Developing an Amide-Spacered Triterpenoid Rhodamine Hybrid of Nano-Molar Cytotoxicity Combined with Excellent Tumor Cell/Non-Tumor Cell Selectivity.

Asiatic acid, a pentacyclic triterpene, was converted into a series of piperazinyl, homopiperazinyl, and 1,5-diazocinyl spacered rhodamine conjugates, differing in the type of spacer and the substitution pattern on the rhodamine moiety of the hybrids. The compounds were tested for cytotoxic activity in SRB assays and compound 12, holding an EC50 of 0.8 nM, was the most cytotoxic compound of this series, but compound 18 (containing a ring expanded 1,5-diazocinyl moiety and n-propyl substituents on the rhodamine) was the most selective compound exhibiting a selectivity factor of almost 190 while retaining high cytotoxicity (EC50 = 1.9 nM, for A2780 ovarian carcinoma).

Substrate-like novel inhibitors of prolyl specific oligo peptidase for neurodegenerative disorders.

Prolyl specific oligopeptidase (POP), is one of the highly expressed enzymes in the brain and is a prime target to treat disorders related to the central nervous system. Here, we describe the structure-based design of the tacrine derivatives, selective, and brain-permeable POP inhibitors. These compounds inactivate POP in-vitro specifically and sustainably at very low concentrations (nano molar). Among this series, compound 6b (IC50 = 0.81 ± 0.04 µM) exhibited most potent inhibition. Furthermore, kinetic study revealed that these molecules target active site of POP which is further confirmed by in-silico molecular interaction analysis. The computational docking results indicates that the compounds are well fitted in the active site with high binding score (i.e., > -7 to > -4 kcal/mol) where Trp595, Arg643, Tyr473, and Ser554 plays important role in binding with the active compounds. The molecular dynamic simulation of most active compounds (6a, 6b, 6d, and 6f) displayed higher free energy binding, when compared to the standard drug in MM-PBSA based binding free energy calculation. In addition, the predicted pharmacokinetic profile suggests that these compounds can serve as excellent inhibitors upon additional optimization which makes them prime choice for further investigation.Communicated by Ramaswamy H. Sarma.

Mitochondria-Targeting 1,5-Diazacyclooctane-Spacered Triterpene Rhodamine Conjugates Exhibit Cytotoxicity at Sub-Nanomolar Concentration against Breast Cancer Cells.

1,5-Diazacyclooctane was prepared by a simple synthetic sequence and coupled to pentacyclic triterpenoic acids oleanolic acid, ursolic acid, betulinic acid, platanic acid, and asiatic acid; these amides were activated with oxalyl chloride and reacted with rhodamine B or rhodamine 101 to yield conjugates. The conjugates were screened in SRB assays with various human breast cancer cell lines (MDA-MB-231, HS578T, MCF-7, and T47D) and found to exert cytotoxic activity even at a low concentration. Therefore, for an asiatic acid rhodamine 101 conjugate (28), an IC50 = 0.60 nM was determined and found to induce apoptosis in MDA-MB-231 and HS578T cells. Extra experiments showed the compound to act as a mitocan and to induce inhibition of proliferation or growth arrest in MDA-MB-231 cells at lower doses followed by an induction of apoptosis at higher doses. Furthermore, differential responses to proliferation inhibition and apoptosis induction may explain differential sensitivity of mammary cell lines to compound 28.

The Finally Rewarding Search for A Cytotoxic Isosteviol Derivative.

Acid hydrolysis of stevioside resulted in a 63% yield of isosteviol (1), which served as a starting material for the preparation of numerous amides. These compounds were tested for cytotoxic activity, employing a panel of human tumor cell lines, and almost all amides were found to be non-cytotoxic. Only the combination of isosteviol, a (homo)-piperazinyl spacer and rhodamine B or rhodamine 101 unit proved to be particularly suitable. These spacered rhodamine conjugates exhibited cytotoxic activity in the sub-micromolar concentration range. In this regard, the homopiperazinyl-spacered derivatives were found to be better than those compounds with piperazinyl spacers, and rhodamine 101 conjugates were more cytotoxic than rhodamine B hybrids.

Meldrum-Based-1H-1,2,3-Triazoles as Antidiabetic Agents: Synthesis, In Vitro α-Glucosidase Inhibition Activity, Molecular Docking Studies, and In Silico Approach.

A series of novel alkyl derivatives (2-5a,b) and 1H-1,2,3-triazole analogues (7a-k) of Meldrum's acid were synthesized in a highly effective way by using "click" chemistry and screened for in vitro α-glucosidase inhibitory activity to examine their antidiabetic potential. 1H NMR, 13C-NMR, and high-resolution electrospray ionization mass spectra (HR-ESI-MS) were used to analyze each of the newly synthesized compounds. Interestingly, these compounds demonstrated high to moderate α-glucosidase inhibitory potency having an IC50 range of 4.63-80.21 µM. Among these derivatives, compound 7i showed extraordinary inhibitory activity and was discovered to be several times more potent than the parent compound Meldrum (1) and the standard drug acarbose. Later, molecular docking was performed to understand the binding mode and the binding strength of all the compounds with the target enzyme, which revealed that all compounds are well fitted in the active site of α-glucosidase. To further ascertain the structure of compounds, suitable X-ray single crystals of compounds 5a, 7a, and 7h were developed and studied. The current investigation has shown that combining 1H-1,2,3-triazole with the Meldrum moiety is beneficial. Furthermore, this is the first time that the aforementioned activity of these compounds has been reported.

Targeted theranostics: Near-infrared triterpenoic acid-rhodamine conjugates as prerequisites for precise cancer diagnosis and therapy.

Pentacyclic triterpenoic acids have shown excellent potential as starting materials for the synthesis of highly cytotoxic agents with significantly reduced toxicity for non-malignant cells. This study focuses on the development of triterpenoic acid-rhodamine conjugates with fluorescence shifted to the near-infrared (NIR) region for theranostic applications in cancer research. Spectral analysis revealed emission wavelengths around λ = 760 nm, enabling stronger signals and deeper tissue penetration. The conjugates were evaluated using SRB assays on tumor cell lines and non-malignant fibroblasts, demonstrating low nanomolar activity and high selectivity, similarly to their known rhodamine B counterparts. Additional staining experiments proved their mode of action as mitocans.

Bioactivity-Guided Subtraction of MIQOX for Easily Available Isoquinoline Hydrazides as Novel Antifungal Candidates.

The discovery of novel and easily available leads provides a convincing solution to agrochemical innovation. A bioassay-guided scaffold subtraction of the previous "Chem-Bio Model" isoquinoline-3-oxazoline MIQOX was conducted for identifying the easily available isoquinoline-3-hydrazide as a novel antifungal scaffold. The special and practical potential of this model was demonstrated by a phenotypic antifungal bioassay, molecular docking, and cross-resistance evaluation. A panel of antifungal leads (LW2, LW3, and LW11) was acquired, showing much better antifungal performance than the positive controls. Specifically, compound LW3 exhibited a broad antifungal spectrum holding EC50 values as low as 0.54, 0.09, 1.52, and 2.65 mg/L against B. cinerea, R. solani, S. sclerotiorum , and F. graminearum, respectively. It demonstrated a curative efficacy better than that of boscalid in controlling the plant disease caused by B. cinerea. The candidate LW3 did not show cross-resistance to the extensively used succinate dehydrogenase inhibitor (SDHI) fungicides and can efficiently inhibit resistant B. cinerea strains. The molecular docking of compound LW3 is quite different from that of the positive controls boscalid and fluopyram. This progress highlights the practicality of isoquinoline hydrazide as a novel model in fungicide innovation.