Hedgehog Pathway Inhibition by Novel Small Molecules Impairs Melanoma Cell Migration and Invasion under Hypoxia.

Melanoma is the principal cause of death in skin cancer due to its ability to invade and cause metastasis. Hypoxia, which characterises the tumour microenvironment (TME), plays an important role in melanoma development, as cancer cells can adapt and acquire a more aggressive phenotype. Carbonic anhydrases (CA) activity, involved in pH regulation, is related to melanoma cell migration and invasion. Furthermore, the Hedgehog (Hh) pathway, already known for its role in physiological processes, is a pivotal character in cancer cell growth and can represent a promising pharmacological target. In this study, we targeted Hh pathway components with cyclopamine, glabrescione B and C22 in order to observe their effect on carbonic anhydrase XII (CAXII) expression especially under hypoxia. We then performed a migration and invasion assay on two melanoma cell lines (SK-MEL-28 and A375) where Smoothened, the upstream protein involved in Hh regulation, and GLI1, the main transcription factor that determines Hh pathway activation, were chemically inhibited. Data suggest the existence of a relationship between CAXII, hypoxia and the Hedgehog pathway demonstrating that the chemical inhibition of the Hh pathway and CAXII reduction resulted in melanoma migration and invasion impairment especially under hypoxia. As in recent years drug resistance to small molecules has arisen, the development of new chemical compounds is crucial. The multitarget Hh inhibitor C22 proved to be effective without signs of cytotoxicity and, for this reason, it can represent a promising compound for future studies, with the aim to reach a better melanoma disease management.

Selection of Natural Compounds with HMGA-Interfering Activities and Cancer Cell Cytotoxicity.

HMGA proteins are intrinsically disordered (ID) chromatin architectural factors characterized by three DNA binding domains (AT-hooks) that allow them to bind into the DNA minor groove of AT-rich stretches. HMGA are functionally involved in regulating transcription, RNA processing, DNA repair, and chromatin remodeling and dynamics. These proteins are highly expressed and play essential functions during embryonic development. They are almost undetectable in adult tissues but are re-expressed at high levels in all cancers where they are involved in neoplastic transformation and cancer progression. We focused on identifying new small molecules capable of binding into the minor groove of AT-rich DNA sequences that could compete with HMGA for DNA binding and, thus, potentially interfere with their activities. Here, a docking-based virtual screening of a unique high diversity in-house library composed of around 1000 individual natural products identified 16 natural compounds as potential minor groove binders that could inhibit the interaction between HMGA and DNA. To verify the ability of these selected compounds to compete with HMGA proteins, we screened them using electrophoretic mobility shift assays. We identified Sorocein C, a Diels-Alder (D-A)-type adducts, isolated from Sorocea ilicifolia and Sorocea bonplandii with an HMGA/DNA-displacing activity and compared its activity with that of two structurally related compounds, Sorocein A and Sorocein B. All these compounds showed a cytotoxicity effect on cancer cells, suggesting that the Sorocein-structural family may provide new and yet unexplored chemotypes for the development of minor groove binders to be evaluated as anticancer agents.

Exploring the Potential of Anthraquinone-Based Hybrids for Identifying a Novel Generation of Antagonists for the Smoothened Receptor in HH-Dependent Tumour.

Natural products (NPs) are highly profitable pharmacological tools due to their chemical diversity and ability to modulate biological systems. Accessing new chemical entities while retaining the biological relevance of natural chemotypes is a fundamental goal in the design of novel bioactive compounds. Notably, NPs have played a crucial role in understanding Hedgehog (HH) signalling and its pharmacological modulation in anticancer therapy. However, HH antagonists developed so far have shown several limitations, thus growing interest in the design of second-generation HH inhibitors. Through smart manipulation of the NPs core-scaffold, unprecedented and intriguing architectures have been achieved following different design strategies. This study reports the rational design and synthesis of a first and second generation of anthraquinone-based hybrids by combining the rhein scaffold with variously substituted piperazine nuclei that are structurally similar to the active portion of known SMO antagonists, the main transducer of the HH pathway. A thorough functional and biological investigation identified RH2_2 and RH2_6 rhein-based hybrids as valuable candidates for HH inhibition through SMO antagonism, with the consequent suppression of HH-dependent tumour growth. These findings also corroborated the successful application of the NPs-based hybrid design strategy in the development of novel NP-based SMO antagonists.

Inhibitors of UHRF1 base flipping activity showing cytotoxicity against cancer cells.

Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) is a nuclear multi-domain protein overexpressed in numerous human cancer types. We previously disclosed the anthraquinone derivative UM63 that inhibits UHRF1-SRA domain base-flipping activity, although having DNA intercalating properties. Herein, based on the UM63 structure, new UHRF1-SRA inhibitors were identified through a multidisciplinary approach, combining molecular modelling, biophysical assays, molecular and cell biology experiments. We identified AMSA2 and MPB7, that inhibit UHRF1-SRA mediated base flipping at low micromolar concentrations, but do not intercalate into DNA, which is a key advantage over UM63. These molecules prevent UHRF1/DNMT1 interaction at replication forks and decrease the overall DNA methylation in cells. Moreover, both compounds specifically induce cell death in numerous cancer cell lines, displaying marginal effect on non-cancer cells, as they preferentially affect cells with high level of UHRF1. Overall, these two compounds are promising leads for the development of anti-cancer drugs targeting UHRF1.

Selective Targeting of Cancer-Related G-Quadruplex Structures by the Natural Compound Dicentrine.

Aiming to identify highly effective and selective G-quadruplex ligands as anticancer candidates, five natural compounds were investigated here, i.e., the alkaloids Canadine, D-Glaucine and Dicentrine, as well as the flavonoids Deguelin and Millettone, selected as analogs of compounds previously identified as promising G-quadruplex-targeting ligands. A preliminary screening with the G-quadruplex on the Controlled Pore Glass assay proved that, among the investigated compounds, Dicentrine is the most effective ligand of telomeric and oncogenic G-quadruplexes, also showing good G-quadruplex vs. duplex selectivity. In-depth studies in solution demonstrated the ability of Dicentrine to thermally stabilize telomeric and oncogenic G-quadruplexes without affecting the control duplex. Interestingly, it showed higher affinity for the investigated G-quadruplex structures over the control duplex (Kb~106 vs. 105 M-1), with some preference for the telomeric over the oncogenic G-quadruplex model. Molecular dynamics simulations indicated that Dicentrine preferentially binds the G-quadruplex groove or the outer G-tetrad for the telomeric and oncogenic G-quadruplexes, respectively. Finally, biological assays proved that Dicentrine is highly effective in promoting potent and selective anticancer activity by inducing cell cycle arrest through apoptosis, preferentially targeting G-quadruplex structures localized at telomeres. Taken together, these data validate Dicentrine as a putative anticancer candidate drug selectively targeting cancer-related G-quadruplex structures.

Inhibition of adenovirus transport from the endosome to the cell nucleus by rotenone.

Regardless of the clinical impact of human adenovirus (HAdV) infections in the healthy population and its high morbidity in immunosuppressed patients, a specific treatment is still not yet available. In this study, we screened the CM1407 COST Action's chemical library, comprising 1,233 natural products to identify compounds that restrict HAdV infection. Among them, we identified rotenolone, a compound that significantly inhibited HAdV infection. Next, we selected four isoflavonoid-type compounds (e.g., rotenone, deguelin, millettone, and tephrosin), namely rotenoids, structurally related to rotenolone in order to evaluate and characterized in vitro their antiviral activities against HAdV and human cytomegalovirus (HCMV). Their IC50 values for HAdV ranged from 0.0039 µM for rotenone to 0.07 µM for tephrosin, with selective indices ranging from 164.1 for rotenone to 2,429.3 for deguelin. In addition, the inhibition of HCMV replication ranged from 50% to 92.1% at twice the IC50 concentrations obtained in the plaque assay for each compound against HAdV. Our results indicated that the mechanisms of action of rotenolone, deguelin, and tephrosin involve the late stages of the HAdV replication cycle. However, the antiviral mechanism of action of rotenone appears to involve the alteration of the microtubular polymerization, which prevents HAdV particles from reaching the nuclear membrane of the cell. These isoflavonoid-type compounds exert high antiviral activity against HAdV at nanomolar concentrations, and can be considered strong hit candidates for the development of a new class of broad-spectrum antiviral drugs.

Targeting the tubulin C-terminal tail by charged small molecules.

The disordered tubulin C-terminal tail (CTT), which possesses a higher degree of heterogeneity, is the target for the interaction of many proteins and cellular components. Compared to the seven well-described binding sites of microtubule-targeting agents (MTAs) that localize on the globular tubulin core, tubulin CTT is far less explored. Therefore, tubulin CTT can be regarded as a novel site for the development of MTAs with distinct biochemical and cell biological properties. Here, we designed and synthesized linear and cyclic peptides containing multiple arginines (RRR), which are complementary to multiple acidic residues in tubulin CTT. Some of them showed moderate induction and promotion of tubulin polymerization. The most potent macrocyclic compound 1f was found to bind to tubulin CTT and thus exert its bioactivity. Such RRR containing compounds represent a starting point for the discovery of tubulin CTT-targeting agents with therapeutic potential.

Identification of a Novel Curcumin Derivative Influencing Notch Pathway and DNA Damage as a Potential Therapeutic Agent in T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy considered curable by modern clinical management. Nevertheless, the prognosis for T-ALL high-risk cases or patients with relapsed and refractory disease is still dismal. Therefore, there is a keen interest in developing more efficient and less toxic therapeutic approaches. T-ALL pathogenesis is associated with Notch signaling alterations, making this pathway a highly promising target in the fight against T-ALL. Here, by exploring the anti-leukemic capacity of the natural polyphenol curcumin and its derivatives, we found that curcumin exposure impacts T-ALL cell line viability and decreases Notch signaling in a dose- and time-dependent fashion. However, our findings indicated that curcumin-mediated cell outcomes did not depend exclusively on Notch signaling inhibition, but might be mainly related to compound-induced DNA-damage-associated cell death. Furthermore, we identified a novel curcumin-based compound named CD2066, endowed with potentiated anti-proliferative activity in T-ALL compared to the parent molecule curcumin. At nanomolar concentrations, CD2066 antagonized Notch signaling, favored DNA damage, and acted synergistically with the CDK1 inhibitor Ro3306 in T-ALL cells, thus representing a promising novel candidate for developing therapeutic agents against Notch-dependent T-ALL.

Novel pyrazolo[3,4-d]pyrimidines as dual Src/Bcr-Abl kinase inhibitors: Synthesis and biological evaluation for chronic myeloid leukemia treatment.

The Bcr-Abl tyrosine kinase (TK) is the molecular hallmark of chronic myeloid leukemia (CML). Src is another TK kinase whose involvement in CML was widely demonstrated. Small molecules active as dual Src/Bcr-Abl inhibitors emerged as effective targeted therapies for CML and a few compounds are currently in clinical use. In this study, we applied a target-oriented approach to identify a family of pyrazolo[3,4-d]pyrimidines as dual Src/Bcr-Abl inhibitors as anti-leukemia agents. Considering the high homology between Src and Bcr-Abl, in-house Src inhibitors 8a-l and new analogue compounds 9a-n were screened as dual Src/Bcr-Abl inhibitors. The antiproliferative activity on K562 CML cells and the ADME profile were determined for the most promising compounds. Molecular modeling studies elucidated the binding mode of the inhibitors into the Bcr-Abl (wt) catalytic pocket. Compounds 8j and 8k showed nanomolar activities in enzymatic and cellular assays, together with favorable ADME properties, emerging as promising candidates for CML therapy. Finally, derivatives 9j and 9k, emerging as valuable inhibitors of the most aggressive Bcr-Abl mutation, T315I, constitute a good starting point in the search for compounds able to treat drug-resistant forms of CML. Overall, this study allowed us to identify more potent compounds than those previously reported by the group, marking a step forward in searching for new antileukemic agents.

Discovery of MDM2-p53 and MDM4-p53 protein-protein interactions small molecule dual inhibitors.

MDM2 and MDM4 are key negative regulators of p53, an important protein involved in several cell processes (e.g. cell cycle and apoptosis). Not surprisingly, the p53 tumor suppressor function is inactivated in tumors overexpressing these two proteins. Therefore, both MDM2 and MDM4 are considered important therapeutic targets for an effective reactivation of the p53 function. Herein, we present our studies on the development of spiropyrazoline oxindole small molecules able to inhibit MDM2/4-p53 protein-protein interactions (PPIs). Twenty-seven potential spiropyrazoline oxindole dual inhibitors were prepared based on in silico structural optimization studies of a hit compound with MDM2 and MDM4 proteins. The antiproliferative activity of the target compounds was evaluated in cancer cell lines harboring wild-type p53 and overexpressing MDM2 and/or MDM4. The most active compounds in SJSA-1 cells, 2q and 3b, induce cell death via apoptosis and control cell growth by targeting the G0/G1 cell cycle checkpoint in a concentration-dependent manner. The ability of the five most active spiropyrazoline oxindoles in dissociating p53 from MDM2 and MDM4 was analyzed by an immunoenzymatic assay. Three compounds inhibited MDM2/4-p53 PPIs with IC50 values in the nM range, while one compound inhibited more selectively the MDM2-p53 PPI over the MDM4-p53 PPI. Collectively, these results show: i) 3b may serve as a valuable lead for obtaining selective MDM2-p53 PPI inhibitors and more efficient anti-osteosarcoma agents; ii) 2a, 2q and 3f may serve as valuable leads for obtaining dual MDM2/4 inhibitors and more effective p53 activators.

Novel Insights on Human Carbonic Anhydrase Inhibitors Based on Coumalic Acid: Design, Synthesis, Molecular Modeling Investigation, and Biological Studies.

Human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms IX and XII are overexpressed in solid hypoxic tumors, and they are considered as prognostic tools and therapeutic targets for cancer. Based on a molecular simplification of the well-known coumarin scaffold, we developed a new series of derivatives of the pyran-2-one core. The new compounds are endowed with potent and selective inhibitory activity against the tumor-related hCA isoforms IX and XII, in the low nanomolar range, whereas they are inactive against the two cytosolic off-targets hCA I and II. The compounds exhibiting the best hCA inhibition were further investigated against the breast adenocarcinoma cell line (MCF7) in hypoxic conditions, evaluating their ability to eventually synergize with doxorubicin. The compounds' biocompatibility on healthy cells was also tested and confirmed on Human Gingival Fibroblasts (HGFs). Furthermore, the possible binding mode of all compounds to the active site of the tumor-associated human CA IX was investigated by computational techniques which predicted the binding conformations and the persistency of binding poses within the active site of the enzyme, furnishing relevant data for the design of tight binding inhibitors.

Hidden in Plants-A Review of the Anticancer Potential of the Solanaceae Family in In Vitro and In Vivo Studies.

Many of the anticancer agents that are currently in use demonstrate severe side effects and encounter increasing resistance from the target cancer cells. Thus, despite significant advances in cancer therapy in recent decades, there is still a need to discover and develop new, alternative anticancer agents. The plant kingdom contains a range of phytochemicals that play important roles in the prevention and treatment of many diseases. The Solanaceae family is widely used in the treatment of various diseases, including cancer, due to its bioactive ingredient content. The purpose of this literature review is to highlight the antitumour activity of Solanaceae extracts-single isolated compounds and nanoparticles with extracts-and their synergistic effect with chemotherapeutic agents in various in vitro and in vivo cancer models. In addition, the biological properties of many plants of the Solanaceae family have not yet been investigated, which represents a challenge and an opportunity for future anticancer therapy.

SARS-CoV-2 Nsp13 encodes for an HLA-E-stabilizing peptide that abrogates inhibition of NKG2A-expressing NK cells.

Natural killer (NK) cells are innate immune cells that contribute to host defense against virus infections. NK cells respond to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and are activated in patients with acute coronavirus disease 2019 (COVID-19). However, by which mechanisms NK cells detect SARS-CoV-2-infected cells remains largely unknown. Here, we show that the Non-structural protein 13 of SARS-CoV-2 encodes for a peptide that is presented by human leukocyte antigen E (HLA-E). In contrast with self-peptides, the viral peptide prevents binding of HLA-E to the inhibitory receptor NKG2A, thereby rendering target cells susceptible to NK cell attack. In line with these observations, NKG2A-expressing NK cells are particularly activated in patients with COVID-19 and proficiently limit SARS-CoV-2 replication in infected lung epithelial cells in vitro. Thus, these data suggest that a viral peptide presented by HLA-E abrogates inhibition of NKG2A+ NK cells, resulting in missing self-recognition.

Esc peptides as novel potentiators of defective cystic fibrosis transmembrane conductance regulator: an unprecedented property of antimicrobial peptides.

Mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein lead to persistent lung bacterial infections, mainly due to Pseudomonas aeruginosa, causing loss of respiratory function and finally death of people affected by CF. Unfortunately, even in the era of CFTR modulation therapies, management of pulmonary infections in CF remains highly challenging especially for patients with advanced stages of lung disease. Recently, we identified antimicrobial peptides (AMPs), namely Esc peptides, with potent antipseudomonal activity. In this study, by means of electrophysiological techniques and computational studies we discovered their ability to increase the CFTR-controlled ion currents, by direct interaction with the F508del-CFTR mutant. Remarkably, this property was not explored previously with any AMPs or peptides in general. More interestingly, in contrast with clinically used CFTR modulators, Esc peptides would give particular benefit to CF patients by combining their capability to eradicate lung infections and to act as promoters of airway wound repair with their ability to ameliorate the activity of the channel with conductance defects. Overall, our findings not only highlighted Esc peptides as the first characterized AMPs with a novel property, that is the potentiator activity of CFTR, but also paved the avenue to investigate the functions of AMPs and/or other peptide molecules, for a new up-and-coming pharmacological approach to address CF lung disease.

Identification of Effective Anticancer G-Quadruplex-Targeting Chemotypes through the Exploration of a High Diversity Library of Natural Compounds.

In the quest for selective G-quadruplex (G4)-targeting chemotypes, natural compounds have been thus far poorly explored, though representing appealing candidates due to the high structural diversity of their scaffolds. In this regard, a unique high diversity in-house library composed of ca. one thousand individual natural products was investigated. The combination of molecular docking-based virtual screening and the G4-CPG experimental screening assay proved to be useful to quickly and effectively identify-out of many natural compounds-five hit binders of telomeric and oncogenic G4s, i.e., Bulbocapnine, Chelidonine, Ibogaine, Rotenone and Vomicine. Biophysical studies unambiguously demonstrated the selective interaction of these compounds with G4s compared to duplex DNA. The rationale behind the G4 selective recognition was suggested by molecular dynamics simulations. Indeed, the selected ligands proved to specifically interact with G4 structures due to peculiar interaction patterns, while they were unable to firmly bind to a DNA duplex. From biological assays, Chelidonine and Rotenone emerged as the most active compounds of the series against cancer cells, also showing good selectivity over normal cells. Notably, the anticancer activity correlated well with the ability of the two compounds to target telomeric G4s.

Hepatic miR-144 Drives Fumarase Activity Preventing NRF2 Activation During Obesity.

BACKGROUND AND AIMS: Oxidative stress plays a key role in the development of metabolic complications associated with obesity, including insulin resistance and the most common chronic liver disease worldwide, nonalcoholic fatty liver disease. We have recently discovered that the microRNA miR-144 regulates protein levels of the master mediator of the antioxidant response, nuclear factor erythroid 2-related factor 2 (NRF2). On miR-144 silencing, the expression of NRF2 target genes was significantly upregulated, suggesting that miR-144 controls NRF2 at the level of both protein expression and activity. Here we explored a mechanism whereby hepatic miR-144 inhibited NRF2 activity upon obesity via the regulation of the tricarboxylic acid (TCA) metabolite, fumarate, a potent activator of NRF2. METHODS: We performed transcriptomic analysis in liver macrophages (LMs) of obese mice and identified the immuno-responsive gene 1 (Irg1) as a target of miR-144. IRG1 catalyzes the production of a TCA derivative, itaconate, an inhibitor of succinate dehydrogenase (SDH). TCA enzyme activities and kinetics were analyzed after miR-144 silencing in obese mice and human liver organoids using single-cell activity assays in situ and molecular dynamic simulations. RESULTS: Increased levels of miR-144 in obesity were associated with reduced expression of Irg1, which was restored on miR-144 silencing in vitro and in vivo. Furthermore, miR-144 overexpression reduces Irg1 expression and the production of itaconate in vitro. In alignment with the reduction in IRG1 levels and itaconate production, we observed an upregulation of SDH activity during obesity. Surprisingly, however, fumarate hydratase (FH) activity was also upregulated in obese livers, leading to the depletion of its substrate fumarate. miR-144 silencing selectively reduced the activities of both SDH and FH resulting in the accumulation of their related substrates succinate and fumarate. Moreover, molecular dynamics analyses revealed the potential role of itaconate as a competitive inhibitor of not only SDH but also FH. Combined, these results demonstrate that silencing of miR-144 inhibits the activity of NRF2 through decreased fumarate production in obesity. CONCLUSIONS: Herein we unravel a novel mechanism whereby miR-144 inhibits NRF2 activity through the consumption of fumarate by activation of FH. Our study demonstrates that hepatic miR-144 triggers a hyperactive FH in the TCA cycle leading to an impaired antioxidant response in obesity.

Design and Synthesis of Piperazine-Based Compounds Conjugated to Humanized Ferritin as Delivery System of siRNA in Cancer Cells.

Gene expression regulation by small interfering RNA (siRNA) holds promise in treating a wide range of diseases through selective gene silencing. However, successful clinical application of nucleic acid-based therapy requires novel delivery options. Herein, to achieve efficient delivery of negatively charged siRNA duplexes, the internal cavity of "humanized" chimeric Archaeal ferritin (HumAfFt) was specifically decorated with novel cationic piperazine-based compounds (PAs). By coupling these rigid-rod-like amines with thiol-reactive reagents, chemoselective conjugation was efficiently afforded on topologically selected cysteine residues properly located inside HumAfFt. The capability of PAs-HumAfFt to host and deliver siRNA molecules through human transferrin receptor (TfR1), overexpressed in many cancer cells, was explored. These systems allowed siRNA delivery into HeLa, HepG2, and MCF-7 cancer cells with improved silencing effect on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression with respect to traditional transfection methodologies and provided a promising TfR1-targeting system for multifunctional siRNA delivery to therapeutic applications.

Sofosbuvir Selects for Drug-Resistant Amino Acid Variants in the Zika Virus RNA-Dependent RNA-Polymerase Complex In Vitro.

The nucleotide analog sofosbuvir, licensed for the treatment of hepatitis C, recently revealed activity against the Zika virus (ZIKV) in vitro and in animal models. However, the ZIKV genetic barrier to sofosbuvir has not yet been characterized. In this study, in vitro selection experiments were performed in infected human hepatoma cell lines. Increasing drug pressure significantly delayed viral breakthrough (p = 0.029). A double mutant in the NS5 gene (V360L/V607I) emerged in 3 independent experiments at 40-80 µM sofosbuvir resulting in a 3.9 ± 0.9-fold half- maximal inhibitory concentration (IC50) shift with respect to the wild type (WT) virus. A triple mutant (C269Y/V360L/V607I), detected in one experiment at 80 µM, conferred a 6.8-fold IC50 shift with respect to the WT. Molecular dynamics simulations confirmed that the double mutant V360L/V607I impacts the binding mode of sofosbuvir, supporting its role in sofosbuvir resistance. Due to the distance from the catalytic site and to the lack of reliable structural data, the contribution of C269Y was not investigated in silico. By a combination of sequence analysis, phenotypic susceptibility testing, and molecular modeling, we characterized a double ZIKV NS5 mutant with decreased sofosbuvir susceptibility. These data add important information to the profile of sofosbuvir as a possible lead for anti-ZIKV drug development.

Design and Synthesis of New Withaferin A Inspired Hedgehog Pathway Inhibitors.

Withanolides constitute a well-known family of plant-based alkaloids characterised by widespread biological properties, including the ability of interfering with Hedgehog (Hh) signalling pathway. Following our interest in natural products and in anticancer compounds, we report here the synthesis of a new class of Hh signalling pathway inhibitors, inspired by withaferin A, the first isolated member of withanolides. The decoration of our scaffolds was rationally supported by in silico studies, while functional evaluation revealed promising candidates, confirming once again the importance of natural products as inspiration source for the discovery of novel bioactive compounds. A stereoselective approach, based on Brown chemistry, allowed the obtainment and the functional evaluation of the enantiopure hit compounds.