Natural allosteric modulators and their biological targets: molecular signatures and mechanisms.

Covering: 2008 to 2018Over the last decade more than two hundred single natural products were confirmed as natural allosteric modulators (alloNPs) of proteins. The compounds are presented and discussed with the support of a chemical space, constructed using a principal component analysis (PCA) of molecular descriptors from chemical compounds of distinct databases. This analysis showed that alloNPs are dispersed throughout the majority of the chemical space defined by natural products in general. Moreover, a cluster of alloNPs was shown to occupy a region almost devoid of allosteric modulators retrieved from a dataset composed mainly of synthetic compounds, further highlighting the importance to explore the entire natural chemical space for probing allosteric mechanisms. The protein targets which alloNPs bind to comprised 81 different proteins, which were classified into 5 major groups, with enzymes, in particular hydrolases, being the main representative group. The review also brings a critical interpretation on the mechanisms by which alloNPs display their molecular action on proteins. In the latter analysis, alloNPs were classified according to their final effect on the target protein, resulting in 3 major categories: (i) local alteration of the orthosteric site; (ii) global alteration in protein dynamics that change function; and (iii) oligomer stabilisation or protein complex destabilisation via protein-protein interaction in sites distant from the orthosteric site. G-protein coupled receptors (GPCRs), which use a combination of the three types of allosteric regulation found, were also probed by natural products. In summary, the natural allosteric modulators reviewed herein emphasise their importance for exploring alternative chemotherapeutic strategies, potentially pushing the boundaries of the druggable space of pharmacologically relevant drug targets.

Design, synthesis and in vitro evaluation against human cancer cells of 5-methyl-5-styryl-2,5-dihydrofuran-2-ones, a new series of goniothalamin analogues.

The present work describes the preparation of a novel series of compounds based on the structure of goniothalamin (1), a natural styryl lactone with known cytotoxic and antiproliferative activities against a variety of cancer cell lines. A focused library of 17 goniothalamin analogues displaying the 5-methyl-2,5-dihydrofuran-2-one motif were prepared, and their cytotoxicity evaluated. While the analogues bearing methoxy and/or hydroxy groups on the aromatic moiety usually were at least three times less potent than the lead compound (1), ortho and para-trifluoromethyl analogues 10 and 11 exhibited levels of cytotoxicity similar to goniothalamin (1) against most cancer cell lines evaluated. One could suggest that the electronic effect of the trifluoromethyl group activates the inhibitor's electrophilic site via reduction of the electron density of the α,ß-unsaturated ester oxygen atom. These results provide new information on the structure activity relationship of these α,ß-unsaturated styryl lactones, thereby further focusing the design of novel candidates.

Competing interests during the key N-glycosylation of 6-chloro-7-deaza-7-iodopurine for the synthesis of 7-deaza-2'-methyladenosine using Vorbrüggen conditions.

A short 3-step synthesis of the antiviral agent 7DMA is described herein. The nature of a major by-product formed during the key N-glycosylation of 6-chloro-7-deaza-7-iodopurine with perbenzoylated 2-methyl-ribose under Vorbrüggen conditions was also investigated. Spectroscopic analyses support that the solvent itself is converted into a nucleophilic species competing with the nucleobase and further reacting with the activated riboside in an unanticipated fashion. These findings call for a revision of reaction conditions when working with weakly reactive nucleobases in the presence of Lewis acids. 7DMA thus obtained was evaluated for its efficacy against an emerging flavivirus in vitro.

Discovery and structural characterization of chicoric acid as a SARS-CoV-2 nucleocapsid protein ligand and RNA binding disruptor.

The nucleocapsid (N) protein plays critical roles in coronavirus genome transcription and packaging, representing a key target for the development of novel antivirals, and for which structural information on ligand binding is scarce. We used a novel fluorescence polarization assay to identify small molecules that disrupt the binding of the N protein to a target RNA derived from the SARS-CoV-2 genome packaging signal. Several phenolic compounds, including L-chicoric acid (CA), were identified as high-affinity N-protein ligands. The binding of CA to the N protein was confirmed by isothermal titration calorimetry, 1H-STD and 15N-HSQC NMR, and by the crystal structure of CA bound to the N protein C-terminal domain (CTD), further revealing a new modulatory site in the SARS-CoV-2 N protein. Moreover, CA reduced SARS-CoV-2 replication in cell cultures. These data thus open venues for the development of new antivirals targeting the N protein, an essential and yet underexplored coronavirus target.

Identification and characterization of the anti-SARS-CoV-2 activity of cationic amphiphilic steroidal compounds.

The ongoing COVID-19 pandemic caused a significant loss of human lives and a worldwide decline in quality of life. Treatment of COVID-19 patients is challenging, and specific treatments to reduce COVID-19 aggravation and mortality are still necessary. Here, we describe the discovery of a novel class of epiandrosterone steroidal compounds with cationic amphiphilic properties that present antiviral activity against SARS-CoV-2 in the low micromolar range. Compounds were identified in screening campaigns using a cytopathic effect-based assay in Vero CCL81 cells, followed by hit compound validation and characterization. Compounds LNB167 and LNB169 were selected due to their ability to reduce the levels of infectious viral progeny and viral RNA levels in Vero CCL81, HEK293, and HuH7.5 cell lines. Mechanistic studies in Vero CCL81 cells indicated that LNB167 and LNB169 inhibited the initial phase of viral replication through mechanisms involving modulation of membrane lipids and cholesterol in host cells. Selection of viral variants resistant to steroidal compound treatment revealed single mutations on transmembrane, lipid membrane-interacting Spike and Envelope proteins. Finally, in vivo testing using the hACE2 transgenic mouse model indicated that SARS-CoV-2 infection could not be ameliorated by LNB167 treatment. We conclude that anti-SARS-CoV-2 activities of steroidal compounds LNB167 and LNB169 are likely host-targeted, consistent with the properties of cationic amphiphilic compounds that modulate host cell lipid biology. Although effective in vitro, protective effects were cell-type specific and did not translate to protection in vivo, indicating that subversion of lipid membrane physiology is an important, yet complex mechanism involved in SARS-CoV-2 replication and pathogenesis.

Trypanosoma cruzi Malic Enzyme Is the Target for Sulfonamide Hits from the GSK Chagas Box.

Chagas disease, an infectious condition caused by Trypanosoma cruzi, lacks treatment with drugs with desired efficacy and safety profiles. To address this unmet medical need, a set of trypanocidal compounds were identified through a large multicenter phenotypic-screening initiative and assembled in the GSK Chagas Box. In the present work, we report the screening of the Chagas Box against T. cruzi malic enzymes (MEs) and the identification of three potent inhibitors of its cytosolic isoform (TcMEc). One of these compounds, TCMDC-143108 (1), came out as a nanomolar inhibitor of TcMEc, and 14 new derivatives were synthesized and tested for target inhibition and efficacy against the parasite. Moreover, we determined the crystallographic structures of TcMEc in complex with TCMDC-143108 (1) and six derivatives, revealing the allosteric inhibition site and the determinants of specificity. Our findings connect phenotypic hits from the Chagas Box to a relevant metabolic target in the parasite, providing data to foster new structure-activity guided hit optimization initiatives.

Synthesis of the oxepinochromone natural products ptaeroxylin (desoxykarenin), ptaeroxylinol, and eranthin.

An improved synthesis of the oxepinochromone ptaeroxylin is reported, together with the syntheses of the related natural products ptaeroxylinol and eranthin. Ptaeroxylin and ptaeroxylinol were obtained from the chromenone noreugenin by selective reaction of the 7-hydroxyl group, allylation of the 5-hydroxyl, followed by Claisen rearrangement under microwave conditions with concomitant deprotection of the 7-hydroxyl. Alkylation of the 7-hydroxyl with the appropriate allyl bromide provides a precursor for ring-closing metathesis to deliver the oxepinochromone ring system. Eranthin was obtained by a similar strategy involving Claisen rearrangement to transfer an allyl group from the C-7 hydroxyl of noreugenin to C-8 regioselectively.

Development of Selective Steroid Inhibitors for the Glucose-6-phosphate Dehydrogenase from Trypanosoma cruzi.

Chagas disease is a parasitic infection affecting millions of people across Latin America, imposing a dramatic socioeconomic burden. Despite the availability of drugs, nifurtimox and benznidazole, lack of efficacy and incidence of side-effects prompt the identification of novel, efficient, and affordable drug candidates. To address this issue, one strategy could be probing the susceptibility of Trypanosoma parasites toward NADP-dependent enzyme inhibitors. Recently, steroids of the androstane group have been described as highly potent but nonselective inhibitors of parasitic glucose-6-phosphate dehydrogenase (G6PDH). In order to promote selectivity, we have synthesized and evaluated 26 steroid derivatives of epiandrosterone in enzymatic assays, whereby 17 compounds were shown to display moderate to high selectivity for T. cruzi over the human G6PDH. In addition, three compounds were effective in killing intracellular T. cruzi forms infecting rat cardiomyocytes. Altogether, this study provides new SAR data around G6PDH and further supports this target for treating Chagas disease.

Using Esterase Selectivity to Determine the In Vivo Duration of Systemic Availability and Abolish Systemic Side Effects of Topical ß-Blockers.

For disorders of the skin, eyes, ears, and respiratory tract, topical drugs, delivered directly to the target organ, are a therapeutic option. Compared with systemic oral therapy, the benefits of topical treatments include a faster onset of action, circumventing the liver first pass drug metabolism, and reducing systemic side effects. Nevertheless, some systemic absorption still occurs for many topical agents resulting in systemic side effects. One way to prevent these would be to develop drugs that are instantly degraded upon entry into the bloodstream by serum esterases. Because topical ß-blockers are used in glaucoma and infantile hemeangioma and cause systemic side effects, the ß-adrenoceptor system was used to test this hypothesis. Purified liver esterase reduced the apparent affinity of esmolol, an ester-containing ß-blocker used in clinical emergencies, for the human ß-adrenoceptors in a concentration and time-dependent manner. However, purified serum esterase had no effect on esmolol. Novel ester-containing ß-blockers were synthesized and several were sensitive to both liver and serum esterases. Despite good in vitro affinity, one such compound, methyl 2-(3-chloro-4-(3-((2-(3-(3-chlorophenyl)ureido)ethyl)amino)-2-hydroxypropoxy)phenyl)acetate, had no effect on heart rate when injected intravenously into rats, even at 10 times the equipotent dose of esmolol and betaxolol that caused short and sustained reductions in heart rate, respectively. Thus, ester-based drugs, sensitive to serum esterases, offer a mechanism for developing topical agents that are truly devoid of systemic side effects. Furthermore, differential susceptibility to liver and serum esterases degradation may also allow the duration of systemic availability for other drugs to be fine-tuned.

Synthesis of (+/-)-likonide B (smenochromene D) using a regioselective Claisen rearrangement, separation of the enantiomers and stereochemical assignment.

A synthesis of the unusual ansa-bridged farnesyl hydroquinone derivative likonide B in racemic form is described. The natural product, also known as smenochromene D, was obtained from geranylacetone by a route in which the key steps are a regioselective microwave-mediated Claisen rearrangement of an aryl propargyl ether to deliver the chromene ring, and macrocyclization via an intramolecular Mitsunobu reaction. Subsequent HPLC on a chiral stationary phase gave the pure (+)- and (-)-enantiomers, that were studied by CD spectroscopy, thereby shedding some light on the true stereochemical nature of the two natural products (-)-smenochromene D and (+)-likonide B.

Modulation of nuclear receptor function: Targeting the protein-DNA interface.

Nuclear receptors (NRs) are a superfamily of ligand-dependent transcription factors that modulate several biological processes. Traditionally, modulation of NRs has been focused on the development of ligands that recognize and bind to the ligand binding domain (LBD), resulting in activation or repression of transcription through the recruitment of coregulators. However, for more severe diseases, such as breast and prostate cancer, the conventional treatment addressing LBD modulation is not always successful, due to tumor resistance. To overcome these challenges and aiming to modulate NR activity by inhibiting the NR-DNA interaction, new studies focus on the development of molecules targeting alternative sites and domains on NRs. Here, we discuss two different approaches for this alternative NR modulation: one targeting the NR DNA binding domain (DBD); and the other targeting the DNA sites recognized by NRs. Our aim is to present the challenges and perspectives for developing specific inhibitors for each purpose, alongside with already reported examples.

Descoberta de fármacos a partir de produtos naturais e a abordagem Molecular Power House (MPH) / Natural product-based drug discovery and the Molecular Power House (MPH) approach

A descoberta de fármacos a partir de produtos naturais vivencia uma nova era graças ao uso de tecnologias de ponta e abordagens integradas para superar as dificuldades inerentes à pesquisa com produtos naturais. Estes são substâncias químicas altamente complexas e inovadoras produzidas pela biota, em especial organismos sésseis como plantas e micro-organismos, e representam a principal fonte de inspiração de novos fármacos. Resultante do reavivado interesse na descoberta de medicamentos à base de produtos naturais, novas abordagens para a identificação, caracterização, e reabastecimento de produtos naturais estão sendo desenvolvidas, que podem endereçar alguns dos desafios relacionados ao desenvolvimento de fármacos inspirados em moléculas da biodiversidade. Almeja-se a capacidade de detecção e caracterização de novos produtos naturais bioativos, concomitantemente à redução dos custos e tempo para obtenção destas moléculas. Para isto, é necessário inovar nos processos e nas tecnologias aplicadas à descoberta de fármacos a partir de produtos naturais. A abordagem Molecular Power House (MPH) apresentada neste trabalho faz uso de tecnologias integradas como uma forma de superar gargalos clássicos da pesquisa com produtos naturais de plantas, alinhada ao acesso à maior biodiversidade do planeta, posicionando o Brasil no cenário mundial de descoberta de fármacos inovadores.