A Complex Interplay Between Melatonin and RORß: RORß is Unlikely a Putative Receptor for Melatonin as Revealed by Biophysical Assays.

A nuclear retinoic acid receptor (RAR)-related orphan receptor ß (RORß) is strictly expressed in the brain, particularly in the pineal gland where melatonin is primarily synthesized and concentrated. The controversial issues regarding the direct interaction of melatonin toward ROR receptors have prompted us to investigate the potential melatonin binding sites on different ROR isoforms. We adopted computational and biophysical approaches to investigate the potential of melatonin as the ligand for RORs, in particular RORß. Herein, possible melatonin binding sites were predicted by molecular docking on human RORs. The results showed that melatonin might be able to bind within the ligand-binding domain (LBD) of all RORs, despite their difference in sequence homology. The predicted melatonin binding scores were comparable to binding energies with respect to those of melatonin interaction to the well-characterized membrane receptors, MT1 and MT2. Although the computational analyses suggested the binding potential of melatonin to the LBD of RORß, biophysical validation failed to confirm the binding. Melatonin was unable to alter the stability of human RORß as shown by the unaltered melting temperatures upon melatonin administration in differential scanning fluorometry (DSF). A thermodynamic isothermal titration calorimetry (ITC) profile showed that melatonin did not interact with human RORß in solutions, even in the presence of SRC-1 co-activator peptide. Although the direct interaction between the LBD of RORß could not be established, RORα and RORß gene expressions were increased upon 24 h treatment with µM-range melatonin. Our data, thus, support the studies that the nuclear effects of melatonin may not be directly mediated via its interaction with the RORß. These findings warrant further investigation on how melatonin interacts with ROR signaling and urge the melatonin research community for a paradigm shift in the direct interaction of melatonin toward RORs. The quest to identify nuclear receptors for melatonin in neuronal cells remains valid for the community to achieve.

Kaemtakols A-D, highly oxidized pimarane diterpenoids with potent anti-inflammatory activity from Kaempferia takensis.

Four highly oxidized pimarane diterpenoids were isolated from Kaempferia takensis rhizomes. Kaemtakols A-C possess a tetracyclic ring with either a fused tetrahydropyran or tetrahydrofuran motif. Kaemtakol D has an unusual rearranged A/B ring spiro-bridged pimarane framework with a C-10 spirocyclic junction and an adjacent 1-methyltricyclo[3.2.1.02,7]octene ring. Structural characterization was achieved using spectroscopic analysis, DP4 + and ECD calculations, as well as X-ray crystallography, and their putative biosynthetic pathways have been proposed. Kaemtakol B showed significant potency in inhibiting nitric oxide production with an IC50 value of 0.69 µM. Molecular docking provided some perspectives on the action of kaemtakol B on iNOS protein.

ECDD-S16 targets vacuolar ATPase: A potential inhibitor compound for pyroptosis-induced inflammation.

BACKGROUND: Cleistanthin A (CA), extracted from Phyllanthus taxodiifolius Beille, was previously reported as a potential V-ATPase inhibitor relevant to cancer cell survival. In the present study, ECDD-S16, a derivative of cleistanthin A, was investigated and found to interfere with pyroptosis induction via V-ATPase inhibition. OBJECTIVE: This study examined the ability of ECDD-S16 to inhibit endolysosome acidification leading to the attenuation of pyroptosis in Raw264.7 macrophages activated by both surface and endosomal TLR ligands. METHODS: To elucidate the activity of ECDD-S16 on pyroptosis-induced inflammation, Raw264.7 cells were pretreated with the compound before stimulation with surface and endosomal TLR ligands. The release of lactate dehydrogenase (LDH) was determined by LDH assay. Additionally, the production of cytokines and the expression of pyroptosis markers were examined by ELISA and immunoblotting. Moreover, molecular docking was performed to demonstrate the binding of ECDD-S16 to the vacuolar (V-)ATPase. RESULTS: This study showed that ECDD-S16 could inhibit pyroptosis in Raw264.7 cells activated with surface and endosomal TLR ligands. The attenuation of pyroptosis by ECDD-S16 was due to the impairment of endosome acidification, which also led to decreased Reactive Oxygen Species (ROS) production. Furthermore, molecular docking also showed the possibility of inhibiting endosome acidification by the binding of ECDD-S16 to the vacuolar (V-)ATPase in the region of V0. CONCLUSION: Our findings indicate the potential of ECDD-S16 for inhibiting pyroptosis and prove that vacuolar H+ ATPase is essential for pyroptosis induced by TLR ligands.

Rational Design and Lead Optimisation of Potent Antimalarial Quinazolinediones and Their Cytotoxicity against MCF-7.

Quinazolinedione is one of the most outstanding heterocycles in medicinal chemistry thanks to its wide ranges of biological activities including antimalarial, anticancer, and anti-inflammatory. TCMDC-125133 containing a quinazolinedione pharmacophore displays promising antimalarial activity and low toxicity, as described in the GlaxoSmithKline (GSK) report. Herein, the design and synthesis of novel quinazolinedione derivatives is described on the basis of our previous work on the synthesis of TCMDC-125133, where low-cost chemicals and greener alternatives were used when possible. The initial SAR study focused on the replacement of the valine linker moiety; according to the in silico prediction using SwissADME, concise four-step syntheses toward compounds 4-10 were developed. The in-house synthesized compounds 4-10 were assayed for antimalarial activity against P. falciparum 3D7, and the result revealed that only the compound 2 containing a valine linker was tolerated. Another round of lead optimization focused on the replacement of the m-anisidine moiety in compound 2. A library of 12 derivatives was prepared, and the antimalarial assay showed that potent antimalarial activity could be maintained by replacing the methoxy group in the meta position of the phenyl side chain with a fluorine or chlorine atom (21: IC50 = 36 ± 5 nM, 24: IC50 = 22 ± 5 nM). Further lead optimization is underway to enhance the antimalarial activity of this class of compound. The compounds included in the study possess little to no antiproliferative activity against MCF-7 cells.

Receptor binding protein of prophage reversibly recognizes the low-molecular weight subunit of the surface-layer protein SlpA in Clostridioides difficile.

Receptor-binding proteins (RBPs) are located at the viral tail and mediate the initial recognition of phage to a specific bacterial host. Phage RBPs have co-evolved with numerous types of host receptors resulting in the formation of a diverse assortment of cognate pairs of RBP-receptors that function during the phage attachment step. Although several Clostridioides difficile bacteriophages have been discovered, their RBPs are poorly described. Using homology analysis, putative prophage-tail structure (pts) genes were identified from the prophage genome of the C. difficile HN10 strain. Competition and enzyme-linked immunosorbent assays, using recombinant PtsHN10M, demonstrated the interaction of this Pts to C. difficile cells, suggesting a role as a phage RBP. Gel filtration and cross-linking assay revealed the native form of this protein as a homotrimer. Moreover, truncated variants indicated that the C-terminal domain of PtsHN10M was important for binding to C. difficile cells. Interaction of PtsHN10M was also observed to the low-molecular weight subunit of surface-layer protein A (SlpA), located at the outermost surface of C. difficile cells. Altogether, our study highlights the function of PtsHN10M as an RBP and potentially paves the way toward phage engineering and phage therapy against C. difficile infection.

Development of Inhibitors of SAICAR Synthetase (PurC) from Mycobacterium abscessus Using a Fragment-Based Approach.

Mycobacterium abscessus (Mab) has emerged as a challenging threat to individuals with cystic fibrosis. Infections caused by this pathogen are often impossible to treat due to the intrinsic antibiotic resistance leading to lung malfunction and eventually death. Therefore, there is an urgent need to develop new drugs against novel targets in Mab to overcome drug resistance and subsequent treatment failure. In this study, SAICAR synthetase (PurC) from Mab was identified as a promising target for novel antibiotics. An in-house fragment library screen and a high-throughput X-ray crystallographic screen of diverse fragment libraries were explored to provide crucial starting points for fragment elaboration. A series of compounds developed from fragment growing and merging strategies, guided by crystallographic information and careful hit-to-lead optimization, have achieved potent nanomolar binding affinity against the enzyme. Some compounds also show a promising inhibitory effect against Mab and Mtb. This work utilizes a fragment-based design and demonstrates for the first time the potential to develop inhibitors against PurC from Mab.

A new strategy for hit generation: Novel in cellulo active inhibitors of CYP121A1 from Mycobacterium tuberculosis via a combined X-ray crystallographic and phenotypic screening approach (XP screen).

There is a pressing need for new drugs against tuberculosis (TB) to combat the growing resistance to current antituberculars. Herein a novel strategy is described for hit generation against promising TB targets involving X-ray crystallographic screening in combination with phenotypic screening. This combined approach (XP Screen) affords both a validation of target engagement as well as determination of in cellulo activity. The utility of this method is illustrated by way of an XP Screen against CYP121A1, a cytochrome P450 enzyme from Mycobacterium tuberculosis (Mtb) championed as a validated drug discovery target. A focused screening set was synthesized and tested by such means, with several members of the set showing promising activity against Mtb strain H37Rv. One compound was observed as an X-ray hit against CYP121A1 and showed improved activity against Mtb strain H37Rv under multiple assay conditions (pan-assay activity). Data obtained during X-ray crystallographic screening were utilized in a structure-based campaign to design a limited number of analogues (less than twenty), many of which also showed pan-assay activity against Mtb strain H37Rv. These included the benzo[b][1,4]oxazine derivative (MIC90 6.25 µM), a novel hit compound suitable as a starting point for a more involved hit to lead candidate medicinal chemistry campaign.

Frontiers in antibiotic alternatives for Clostridioides difficile infection.

Clostridioides difficile (C. difficile) is a gram-positive, anaerobic spore-forming bacterium and a major cause of antibiotic-associated diarrhea. Humans are naturally resistant to C. difficile infection (CDI) owing to the protection provided by healthy gut microbiota. When the gut microbiota is disturbed, C. difficile can colonize, produce toxins, and manifest clinical symptoms, ranging from asymptomatic diarrhea and colitis to death. Despite the steady-if not rising-prevalence of CDI, it will certainly become more problematic in a world of antibiotic overuse and the post-antibiotic era. C. difficile is naturally resistant to most of the currently used antibiotics as it uses multiple resistance mechanisms. Therefore, current CDI treatment regimens are extremely limited to only a few antibiotics, which include vancomycin, fidaxomicin, and metronidazole. Therefore, one of the main challenges experienced by the scientific community is the development of alternative approaches to control and treat CDI. In this Frontier article, we collectively summarize recent advances in alternative treatment approaches for CDI. Over the past few years, several studies have reported on natural product-derived compounds, drug repurposing, high-throughput library screening, phage therapy, and fecal microbiota transplantation. We also include an update on vaccine development, pre- and pro-biotics for CDI, and toxin antidote approaches. These measures tackle CDI at every stage of disease pathology via multiple mechanisms. We also discuss the gaps and concerns in these developments. The next epidemic of CDI is not a matter of if but a matter of when. Therefore, being well-equipped with a collection of alternative therapeutics is necessary and should be prioritized.

Anti-SARS-CoV-2 Activity of Andrographis paniculata Extract and Its Major Component Andrographolide in Human Lung Epithelial Cells and Cytotoxicity Evaluation in Major Organ Cell Representatives.

The coronaviruses disease 2019 (COVID-19) caused by a novel coronavirus (SARS-CoV-2) has become a major health problem, affecting more than 50 million people with over one million deaths globally. Effective antivirals are still lacking. Here, we optimized a high-content imaging platform and the plaque assay for viral output study using the legitimate model of human lung epithelial cells, Calu-3, to determine the anti-SARS-CoV-2 activity of Andrographis paniculata extract and its major component, andrographolide. SARS-CoV-2 at 25TCID50 was able to reach the maximal infectivity of 95% in Calu-3 cells. Postinfection treatment of A. paniculata and andrographolide in SARS-CoV-2-infected Calu-3 cells significantly inhibited the production of infectious virions with an IC50 of 0.036 µg/mL and 0.034 µM, respectively, as determined by the plaque assay. The cytotoxicity profile developed over the cell line representatives of major organs, including liver (HepG2 and imHC), kidney (HK-2), intestine (Caco-2), lung (Calu-3), and brain (SH-SY5Y), showed a CC50 of >100 µg/mL for A. paniculata extract and 13.2-81.5 µM for andrographolide, respectively, corresponding to a selectivity index of over 380. In conclusion, this study provided experimental evidence in favor of A. paniculata and andrographolide for further development as a monotherapy or in combination with other effective drugs against SARS-CoV-2 infection.

High-content screening of Thai medicinal plants reveals Boesenbergia rotunda extract and its component Panduratin A as anti-SARS-CoV-2 agents.

Since December 2019, the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused severe pneumonia, a disease named COVID-19, that became pandemic and created an acute threat to public health. The effective therapeutics are in urgent need. Here, we developed a high-content screening for the antiviral candidates using fluorescence-based SARS-CoV-2 nucleoprotein detection in Vero E6 cells coupled with plaque reduction assay. Among 122 Thai natural products, we found that Boesenbergia rotunda extract and its phytochemical compound, panduratin A, exhibited the potent anti-SARS-CoV-2 activity. Treatment with B. rotunda extract and panduratin A after viral infection drastically suppressed SARS-CoV-2 infectivity in Vero E6 cells with IC50 of 3.62 µg/mL (CC50 = 28.06 µg/mL) and 0.81 µΜ (CC50 = 14.71 µM), respectively. Also, the treatment of panduratin A at the pre-entry phase inhibited SARS-CoV-2 infection with IC50 of 5.30 µM (CC50 = 43.47 µM). Our study demonstrated, for the first time, that panduratin A exerts the inhibitory effect against SARS-CoV-2 infection at both pre-entry and post-infection phases. Apart from Vero E6 cells, treatment with this compound was able to suppress viral infectivity in human airway epithelial cells. This result confirmed the potential of panduratin A as the anti-SARS-CoV-2 agent in the major target cells in human. Since B. rotunda is a culinary herb generally grown in China and Southeast Asia, its extract and the purified panduratin A may serve as the promising candidates for therapeutic purposes with economic advantage during COVID-19 situation.

Structure-guided fragment-based drug discovery at the synchrotron: screening binding sites and correlations with hotspot mapping.

Structure-guided drug discovery emerged in the 1970s and 1980s, stimulated by the three-dimensional structures of protein targets that became available, mainly through X-ray crystal structure analysis, assisted by the development of synchrotron radiation sources. Structures of known drugs or inhibitors were used to guide the development of leads. The growth of high-throughput screening during the late 1980s and the early 1990s in the pharmaceutical industry of chemical libraries of hundreds of thousands of compounds of molecular weight of approximately 500 Da was impressive but still explored only a tiny fraction of the chemical space of the predicted 1040 drug-like compounds. The use of fragments with molecular weights less than 300 Da in drug discovery not only decreased the chemical space needing exploration but also increased promiscuity in binding targets. Here we discuss advances in X-ray fragment screening and the challenge of identifying sites where fragments not only bind but can be chemically elaborated while retaining their positions and binding modes. We first describe the analysis of fragment binding using conventional X-ray difference Fourier techniques, with Mycobacterium abscessus SAICAR synthetase (PurC) as an example. We observe that all fragments occupy positions predicted by computational hotspot mapping. We compare this with fragment screening at Diamond Synchrotron Light Source XChem facility using PanDDA software, which identifies many more fragment hits, only some of which bind to the predicted hotspots. Many low occupancy sites identified may not support elaboration to give adequate ligand affinity, although they will likely be useful in drug discovery as 'warm spots' for guiding elaboration of fragments bound at hotspots. We discuss implications of these observations for fragment screening at the synchrotron sources. This article is part of the theme issue 'Fifty years of synchrotron science: achievements and opportunities'.

Industrial scale high-throughput screening delivers multiple fast acting macrofilaricides.

Nematodes causing lymphatic filariasis and onchocerciasis rely on their bacterial endosymbiont, Wolbachia, for survival and fecundity, making Wolbachia a promising therapeutic target. Here we perform a high-throughput screen of AstraZeneca's 1.3 million in-house compound library and identify 5 novel chemotypes with faster in vitro kill rates (<2 days) than existing anti-Wolbachia drugs that cure onchocerciasis and lymphatic filariasis. This industrial scale anthelmintic neglected tropical disease (NTD) screening campaign is the result of a partnership between the Anti-Wolbachia consortium (A∙WOL) and AstraZeneca. The campaign was informed throughout by rational prioritisation and triage of compounds using cheminformatics to balance chemical diversity and drug like properties reducing the chance of attrition from the outset. Ongoing development of these multiple chemotypes, all with superior time-kill kinetics than registered antibiotics with anti-Wolbachia activity, has the potential to improve upon the current therapeutic options and deliver improved, safer and more selective macrofilaricidal drugs.

A Click Chemistry-Based Proteomic Approach Reveals that 1,2,4-Trioxolane and Artemisinin Antimalarials Share a Common Protein Alkylation Profile.

In spite of the recent increase in endoperoxide antimalarials under development, it remains unclear if all these chemotypes share a common mechanism of action. This is important since it will influence cross-resistance risks between the different classes. Here we investigate this proposition using novel clickable 1,2,4-trioxolane activity based protein-profiling probes (ABPPs). ABPPs with potent antimalarial activity were able to alkylate protein target(s) within the asexual erythrocytic stage of Plasmodium falciparum (3D7). Importantly, comparison of the alkylation fingerprint with that generated from an artemisinin ABPP equivalent confirms a highly conserved alkylation profile, with both endoperoxide classes targeting proteins in the glycolytic, hemoglobin degradation, antioxidant defence, protein synthesis and protein stress pathways, essential biological processes for plasmodial survival. The alkylation signatures of the two chemotypes show significant overlap (ca. 90 %) both qualitatively and semi-quantitatively, suggesting a common mechanism of action that raises concerns about potential cross-resistance liabilities.

A Click Chemistry-Based Proteomic Approach Reveals that 1,2,4-Trioxolane and Artemisinin Antimalarials Share a Common Protein Alkylation Profile.

In spite of the recent increase in endoperoxide antimalarials under development, it remains unclear if all these chemotypes share a common mechanism of action. This is important since it will influence cross-resistance risks between the different classes. Here we investigate this proposition using novel clickable 1,2,4-trioxolane activity based protein-profiling probes (ABPPs). ABPPs with potent antimalarial activity were able to alkylate protein target(s) within the asexual erythrocytic stage of Plasmodium falciparum (3D7). Importantly, comparison of the alkylation fingerprint with that generated from an artemisinin ABPP equivalent confirms a highly conserved alkylation profile, with both endoperoxide classes targeting proteins in the glycolytic, hemoglobin degradation, antioxidant defence, protein synthesis and protein stress pathways, essential biological processes for plasmodial survival. The alkylation signatures of the two chemotypes show significant overlap (ca. 90 %) both qualitatively and semi-quantitatively, suggesting a common mechanism of action that raises concerns about potential cross-resistance liabilities.

Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7.

The artemisinin (ART)-based antimalarials have contributed significantly to reducing global malaria deaths over the past decade, but we still do not know how they kill parasites. To gain greater insight into the potential mechanisms of ART drug action, we developed a suite of ART activity-based protein profiling probes to identify parasite protein drug targets in situ. Probes were designed to retain biological activity and alkylate the molecular target(s) of Plasmodium falciparum 3D7 parasites in situ. Proteins tagged with the ART probe can then be isolated using click chemistry before identification by liquid chromatography-MS/MS. Using these probes, we define an ART proteome that shows alkylated targets in the glycolytic, hemoglobin degradation, antioxidant defense, and protein synthesis pathways, processes essential for parasite survival. This work reveals the pleiotropic nature of the biological functions targeted by this important class of antimalarial drugs.

Generation of quinolone antimalarials targeting the Plasmodium falciparum mitochondrial respiratory chain for the treatment and prophylaxis of malaria.

There is an urgent need for new antimalarial drugs with novel mechanisms of action to deliver effective control and eradication programs. Parasite resistance to all existing antimalarial classes, including the artemisinins, has been reported during their clinical use. A failure to generate new antimalarials with novel mechanisms of action that circumvent the current resistance challenges will contribute to a resurgence in the disease which would represent a global health emergency. Here we present a unique generation of quinolone lead antimalarials with a dual mechanism of action against two respiratory enzymes, NADH:ubiquinone oxidoreductase (Plasmodium falciparum NDH2) and cytochrome bc(1). Inhibitor specificity for the two enzymes can be controlled subtly by manipulation of the privileged quinolone core at the 2 or 3 position. Inhibitors display potent (nanomolar) activity against both parasite enzymes and against multidrug-resistant P. falciparum parasites as evidenced by rapid and selective depolarization of the parasite mitochondrial membrane potential, leading to a disruption of pyrimidine metabolism and parasite death. Several analogs also display activity against liver-stage parasites (Plasmodium cynomolgi) as well as transmission-blocking properties. Lead optimized molecules also display potent oral antimalarial activity in the Plasmodium berghei mouse malaria model associated with favorable pharmacokinetic features that are aligned with a single-dose treatment. The ease and low cost of synthesis of these inhibitors fulfill the target product profile for the generation of a potent, safe, and inexpensive drug with the potential for eventual clinical deployment in the control and eradication of falciparum malaria.

Identification, design and biological evaluation of bisaryl quinolones targeting Plasmodium falciparum type II NADH:quinone oxidoreductase (PfNDH2).

A program was undertaken to identify hit compounds against NADH:ubiquinone oxidoreductase (PfNDH2), a dehydrogenase of the mitochondrial electron transport chain of the malaria parasite Plasmodium falciparum. PfNDH2 has only one known inhibitor, hydroxy-2-dodecyl-4-(1H)-quinolone (HDQ), and this was used along with a range of chemoinformatics methods in the rational selection of 17 000 compounds for high-throughput screening. Twelve distinct chemotypes were identified and briefly examined leading to the selection of the quinolone core as the key target for structure-activity relationship (SAR) development. Extensive structural exploration led to the selection of 2-bisaryl 3-methyl quinolones as a series for further biological evaluation. The lead compound within this series 7-chloro-3-methyl-2-(4-(4-(trifluoromethoxy)benzyl)phenyl)quinolin-4(1H)-one (CK-2-68) has antimalarial activity against the 3D7 strain of P. falciparum of 36 nM, is selective for PfNDH2 over other respiratory enzymes (inhibitory IC(50) against PfNDH2 of 16 nM), and demonstrates low cytotoxicity and high metabolic stability in the presence of human liver microsomes. This lead compound and its phosphate pro-drug have potent in vivo antimalarial activity after oral administration, consistent with the target product profile of a drug for the treatment of uncomplicated malaria. Other quinolones presented (e.g., 6d, 6f, 14e) have the capacity to inhibit both PfNDH2 and P. falciparum cytochrome bc(1), and studies to determine the potential advantage of this dual-targeting effect are in progress.

Identification, design and biological evaluation of heterocyclic quinolones targeting Plasmodium falciparum type II NADH:quinone oxidoreductase (PfNDH2).

Following a program undertaken to identify hit compounds against NADH:ubiquinone oxidoreductase (PfNDH2), a novel enzyme target within the malaria parasite Plasmodium falciparum, hit to lead optimization led to identification of CK-2-68, a molecule suitable for further development. In order to reduce ClogP and improve solubility of CK-2-68 incorporation of a variety of heterocycles, within the side chain of the quinolone core, was carried out, and this approach led to a lead compound SL-2-25 (8b). 8b has IC(50)s in the nanomolar range versus both the enzyme and whole cell P. falciparum (IC(50) = 15 nM PfNDH2; IC(50) = 54 nM (3D7 strain of P. falciparum) with notable oral activity of ED(50)/ED(90) of 1.87/4.72 mg/kg versus Plasmodium berghei (NS Strain) in a murine model of malaria when formulated as a phosphate salt. Analogues in this series also demonstrate nanomolar activity against the bc(1) complex of P. falciparum providing the potential added benefit of a dual mechanism of action. The potent oral activity of 2-pyridyl quinolones underlines the potential of this template for further lead optimization studies.