Hit-to-lead optimization of pharmaceutical interesting tryptophanol-derived scaffolds

The indole nucleus is the core structure of many natural and synthetic molecules. In fact, indole-based compounds have been described with different biological activities, ranging from antitumor to antimalarials. Recent studies made by our research group led to the discovery of one enantiopure tryptophanol-derived oxazoloisoindolinone as novel activator of wild-type p53 and reactivator of mutant-type p53. The first objective of this thesis was centered on the hit-to-lead optimization of this specific derivative. Several alkyl substituents with increasing size and electron withdrawing/donating groups were selected for the protection of the indole nitrogen to assemble a library of new analogues for biological screening. A study of the activity based on chirality revealed one compound to be more active than the hit compound 34. Particularly, compound 34d restores the wt-like growth inhibitory effect to mut p53R280K in a percentage of 86.8%. Finally, the stability-profile of this chemical family was evaluated using compounds 34 and 34e as models. The second main goal of this thesis was the investigation of new leads as promising antimalarials, based on the recent report that a library of enantiopure indolizinoindolones exhibits in vitro activity against erythrocytic and liver-stages of malaria parasite. Structural derivatization of the hit-compounds was carried out using an enantioselective two-step route, involving an intramolecular cyclization to assemble the final polycyclic indolizinoindolones derivatives. In vitro screening of these tryptophanol-derived tricyclic compounds permitted the identification of two indolizinoindolone small molecules as the most active compounds

Targeting p53 for melanoma treatment: counteracting tumour proliferation, dissemination and therapeutic resistance

Melanoma is the deadliest form of skin cancer, primarily due to its high metastatic propensity and therapeutic resistance in advanced stages. The frequent inactivation of the p53 tumour suppressor protein in melanomagenesis may predict promising outcomes for p53 activators in melanoma therapy. Herein, we aimed to investigate the antitumor potential of the p53-activating agent SLMP53-2 against melanoma. Two- and three-dimensional cell cultures and xenograft mouse models were used to unveil the antitumor activity and the underlying molecular mechanism of SLMP53-2 in melanoma. SLMP53-2 inhibited the growth of human melanoma cells in a p53-dependent manner through induction of cell cycle arrest and apoptosis. Notably, SLMP53-2 induced p53 stabilization by disrupting the p53–MDM2 interaction, enhancing p53 transcriptional activity. It also promoted the expression of p53-regulated microRNAs (miRNAs), including miR-145 and miR-23a. Moreover, it displayed anti-invasive and antimigratory properties in melanoma cells by inhibiting the epithelial-to-mesenchymal transition (EMT), angiogenesis and extracellular lactate production. Importantly, SLMP53-2 did not induce resistance in melanoma cells. Additionally, it synergized with vemurafenib, dacarbazine and cisplatin, and resensitized vemurafenib-resistant cells. SLMP53-2 also exhibited antitumor activity in human melanoma xenograft mouse models by repressing cell proliferation and EMT while stimulating apoptosis. This work discloses the p53-activating agent SLMP53-2 which has promising therapeutic potential in advanced melanoma, either as a single agent or in combination therapy. By targeting p53, SLMP53-2 may counteract major features of melanoma aggressiveness.This work received financial support from PT national funds (FCT/MCTES, Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) through LAQV/REQUIMTE (UID/QUI/50006/2020), iMed.ULisboa (UIDB/04138/2020), and PTDC/QUIQOR/29664/2017, PTDC/MEC-ONC/32018/2017. We thank FCT for the fellowships SFRH/BD/ 128673/2017 (J. Loureiro), 2020.04613.BD (J. Calheiros), PD/BD/143126/2019 (V. Barcherini)

SLMP53-2 Restores Wild-Type-Like Function to Mutant p53 through Hsp70: Promising Activity in Hepatocellular Carcinoma.

Half of human cancers harbor TP53 mutations that render p53 inactive as a tumor suppressor. In these cancers, reactivation of mutant p53 (mutp53) through restoration of wild-type-like function constitutes a valuable anticancer therapeutic strategy. In order to search for mutp53 reactivators, a small library of tryptophanol-derived oxazoloisoindolinones was synthesized and the potential of these compounds as mutp53 reactivators and anticancer agents was investigated in human tumor cells and xenograft mouse models. By analysis of their anti-proliferative effect on a panel of p53-null NCI-H1299 tumor cells ectopically expressing highly prevalent mutp53, the compound SLMP53-2 was selected based on its potential reactivation of multiple structural mutp53. In mutp53-Y220C-expressing hepatocellular carcinoma (HCC) cells, SLMP53-2-induced growth inhibition was mediated by cell cycle arrest, apoptosis, and endoplasmic reticulum stress response. In these cells, SLMP53-2 restored wild-type-like conformation and DNA-binding ability of mutp53-Y220C by enhancing its interaction with the heat shock protein 70 (Hsp70), leading to the reestablishment of p53 transcriptional activity. Additionally, SLMP53-2 displayed synergistic effect with sorafenib, the only approved therapy for advanced HCC. Notably, it exhibited potent antitumor activity in human HCC xenograft mouse models with a favorable toxicological profile. Collectively, SLMP53-2 is a new mutp53-targeting agent with promising antitumor activity, particularly against HCC

Development of protein p53 activators to tackle colon cancer

Colorectal cancer (CRC) figures currently as the third most diagnosed cancer and ranks second as leading cause of cancer death globally. Due to unmet screening programs, limited therapeutic strategies, and increasing incidence rates, CRC accounts for 10% of global cancer incidence and 9.4% of cancer deaths, just lower than lung cancer. Despite the latest progresses made in understanding CRC pathophysiology, poor therapeutic options are currently available. Targeted therapy represents a fundamental therapeutic option for the cure of CRC. In this respect, the tumor suppressor p53 figures as key therapeutic target. p53 is a multifunctional protein that regulates cell cycle, DNA repair, apoptosis and metabolic pathways. In CRC, mutations of the TP53 gene occur in 60% of patients and are associated with a more aggressive tumor phenotype and multi drug resistance. Unfortunately, there are still very few examples of mutant p53 reactivators, that restore wild-type p53 function, with low adverse effects on normal cells. Following our research on the design and synthesis of novel wild-type p53 activators, tryptophanol-derived isoindolinones SLMP53-1 (43a), DIMP53-1 (43d) and SLMP53-2 (44a) were identified, with promising p53-dependent in vitro and in vivo biological activity in wild-type and mutant p53 expressing human cancer cells. In this PhD project, the optimization of the tryptophanol-derived isoindolinone family was addressed. Towards this goal, an initial screening of the pharmacokinetic profile of hit compounds SLMP53-1 (43a) and DIMP53-1 (43d) was conducted. Investigations through in vitro procedures on the stability profile of these new chemical entities were performed in physiological conditions, in human plasma and in human liver microsomes. Determination of the Phase I and Phase II metabolites and identification of the possible reactive metabolites allowed to identify the metabolic liabilities of the tryptophanol-derived isoindolinone scaffold. For the first time, non-heme containing iron(II) complexes were employed to prepare the major Phase I metabolites of tryptophanol derivatives, and their biological potential was subsequently evaluated. This allowed to understand that the parent compounds SLMP53-1 (43a) and DIMP53-1 (43d) are responsible for the observed antiproliferative activity. Compounds SLMP53-1 (43a) and SLMP53-2 (44a) were selected for hit-to-lead optimization to improve the efficacy, selectivity and metabolic stability of the scaffold. The first series of compounds was prepared through stereoselective cyclocondensation of enantiopure forms of amino alcohol tryptophanol and selected oxoacids, with yields of 41-86%. Chemical derivatization of para and meta positions of the C-9b phenyl ring and the impact of the stereochemistry were considered. From series 1, compound 66f’ resulted to exhibit 6-fold increase of the antiproliferative activity and 3.3-fold increase selectivity for the p53 pathway in human colorectal carcinoma HCT116 cell line, when compared to hit compound SLMP53-1 (43a). Importantly, the compound showed low toxicity in normal colon cells. Subsequently, a small series of compound 66f’ analogues were prepared exploring further derivatization of the para and meta positions of the C-9b phenyl ring and derivatizing the N-indole moiety, with yields of 66-95%. Based on the structure-metabolism relationships acquired for hit compound SLMP53-1 (43a), a series of halogen-enriched tryptophanol-derived isoindolinones was prepared by pyridinium bromide perbromide-promoted bromination with yields 75-92%. Two compounds, 73k and 73d, showed 1.9- and 3.9-fold higher antiproliferative activity in HCT116 cell line, once compared to hit compound SLMP53-1 (43a), and exhibited 3.8- and 1.9-fold selectivity towards p53 pathway, respectively. Through differential scanning fluorimetry experiments most active tryptophanol-derived isoindolinone compounds were screened against wild-type p53 core domain. Compound 66f’ enhances the thermostability of wt p53 core domain by melting temperature (Tm) increment of 1.64°C and compound 73d increases wt p53 core domain by Tm value of 10.39°C. This result may indicate that the compounds promote p53 stability. Additionally, screening of the metabolic stability of compounds 66f’, 73k and 73d revealed that the optimized compounds display a more adequate metabolic profile once compared with hit SLMP53-1 (43a). Derivatization of the indole nitrogen and introduction of a bromine atom promotes higher metabolic stability of the tryptophanol-derived isoindolinone scaffold and induces a decrement of the oxidative metabolism in positions 2 and 3 of the indole core. A metabolic switch is promoted under these conditions and metabolization of the 6-membered ring of the indole is observed. In general, the results collected in this PhD, give relevant contributions in the development of p53 modulators with adequate metabolic profile and optimized efficacy and selectivity

Tryptophanol-Derived Oxazolopyrrolidone Lactams as Potential Anticancer Agents against Gastric Adenocarcinoma

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).Gastric cancer is one of the deadliest cancers in modern societies, so there is a high level of interest in discovering new drugs for this malignancy. Previously, we demonstrated the ability of tryptophanol-derived polycyclic compounds to activate the tumor suppressor protein p53, a relevant therapeutic target in cancer. In this work, we developed a novel series of enantiomerically pure tryptophanol-derived small molecules to target human gastric adenocarcinoma (AGS) cells. From an initial screening of fourteen compounds in AGS cell line, a hit compound was selected for optimization, leading to two derivatives selective for AGS gastric cells over other types of cancer cells (MDA-MB-231, A-549, DU-145, and MG-63). More importantly, the compounds were non-toxic in normal cells (HEK 293T). Additionally, we show that the growth inhibition of AGS cells induced by these compounds is mediated by apoptosis. Stability studies in human plasma and human liver microsomes indicate that the compounds are stable, and that the major metabolic transformations of these molecules are mono- and di-hydroxylation of the indole ring.This research was funded by FCT (Fundação para a Ciência e a Tecnologia) through iMed.ULisboa (UIDB/04138/2020), CQE (UID/QUI/00100/2020), project PTDC/QUI-QOR/29664/2017, principal investigator grants CEECIND/02001/2017 (A. M. M. Antunes), and CEECIND/03143/2017 (L. M. Gonçalves), and PhD fellowships SFRH/BD/117931/2016 (M. Espadinha) and PD/BD/143126/2019 (V. Barcherini), and funded by Severo Ochoa FunFuture CEX2019-917-S.info:eu-repo/semantics/publishedVersio

Tryptophanol-Derived Oxazolopyrrolidone Lactams as Potential Anticancer Agents against Gastric Adenocarcinoma

Gastric cancer is one of the deadliest cancers in modern societies, so there is a high level of interest in discovering new drugs for this malignancy. Previously, we demonstrated the ability of tryptophanol-derived polycyclic compounds to activate the tumor suppressor protein p53, a relevant therapeutic target in cancer. In this work, we developed a novel series of enantiomerically pure tryptophanol-derived small molecules to target human gastric adenocarcinoma (AGS) cells. From an initial screening of fourteen compounds in AGS cell line, a hit compound was selected for optimization, leading to two derivatives selective for AGS gastric cells over other types of cancer cells (MDA-MB-231, A-549, DU-145, and MG-63). More importantly, the compounds were non-toxic in normal cells (HEK 293T). Additionally, we show that the growth inhibition of AGS cells induced by these compounds is mediated by apoptosis. Stability studies in human plasma and human liver microsomes indicate that the compounds are stable, and that the major metabolic transformations of these molecules are mono- and di-hydroxylation of the indole ring.This research was funded by FCT (Fundação para a Ciência e a Tecnologia) through iMed.ULisboa (UIDB/04138/2020), CQE (UID/QUI/00100/2020), project PTDC/QUI-QOR/29664/ 2017, principal investigator grants CEECIND/02001/2017 (A. M. M. Antunes), and CEECIND/03143/ 2017 (L. M. Gonçalves), and PhD fellowships SFRH/BD/117931/2016 (M. Espadinha) and PD/BD/ 143126/2019 (V. Barcherini), and funded by Severo Ochoa FunFuture CEX2019-917-S.Peer reviewe

SLMP53-1 Inhibits Tumor Cell Growth through Regulation of Glucose Metabolism and Angiogenesis in a P53-Dependent Manner

The Warburg effect is an emerging hallmark of cancer, which has the tumor suppressor p53 as its major regulator. Herein, we unveiled that p53 activation by (S)-tryptophanol-derived oxazoloisoindolinone (SLMP53-1) mediated the reprograming of glucose metabolism in cancer cells and xenograft human tumor tissue, interfering with angiogenesis and migration. Particularly, we showed that SLMP53-1 regulated glycolysis by downregulating glucose transporter 1 (GLUT1), hexokinase-2 (HK2), and phosphofructokinase-2 isoform 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3 (PFKFB3) (key glycolytic enzymes), while upregulating the mitochondrial markers synthesis of cytochrome c oxidase 2 (SCO2), cytochrome c oxidase subunit 4 (COX4), and OXPHOS mitochondrial complexes. SLMP53-1 also downregulated the monocarboxylate transporter 4 (MCT4), causing the subsequent reduction of lactate export by cancer cells. Besides the acidification of the extracellular environment, SLMP53-1 further increased E-cadherin and reduced metalloproteinase-9 (MMP-9) expression levels in both cancer cells and xenograft human tumor tissue, which suggested the interference of SLMP53-1 in extracellular matrix remodeling and epithelial-to-mesenchymal transition. Consistently, SLMP53-1 depleted angiogenesis, decreasing endothelial cell tube formation and vascular endothelial growth factor (VEGF) expression levels. SLMP53-1 also exhibited synergistic growth inhibitory activity in combination with the metabolic modulator dichloroacetic acid. These data reinforce the promising application of the p53-activating agent SLMP53-1 in cancer therapy, by targeting p53-mediated pathways of growth and dissemination

Metabolism-Guided Optimization of Tryptophanol-Derived Isoindolinone p53 Activators

For the first time, the pharmacokinetic (PK) profile of tryptophanol-derived isoindolinones, previously reported as p53 activators, was investigated. From the metabolites’ identification, performed by liquid chromatography coupled to high resolution tandem mass spectrometry (LC-HRMS/MS), followed by their preparation and structural elucidation, it was possible to identify that the indole C2 and C3 are the main target of the cytochrome P450 (CYP)-promoted oxidative metabolism in the tryptophanol-derived isoindolinone scaffold. Based on these findings, to search for novel p53 activators a series of 16 enantiopure tryptophanol-derived isoindolinones substituted with a bromine in indole C2 was prepared, in yields of 62–89%, and their antiproliferative activity evaluated in human colon adenocarcinoma HCT116 cell lines with and without p53. Structural optimization led to the identification of two (S)-tryptophanol-derived isoindolinones 3.9-fold and 1.9-fold more active than hit SLMP53-1, respectively. Compounds’ metabolic stability evaluation revealed that this substitution led to a metabolic switch, with the impact of Phase I oxidative metabolism being minimized. Through differential scanning fluorimetry (DSF) experiments, the most active compound of the series in cell assays led to an increase in the protein melting temperature (Tm) of 10.39 °C, suggesting an effective binding to wild-type p53 core domain

Potency and Selectivity Optimization of Tryptophanol‐Derived Oxazoloisoindolinones: Novel p53 Activators in Human Colorectal Cancer

To search for novel p53 activators, four series of novel (S)‐ and (R)‐tryptophanol‐derived oxazoloisoindolinones were synthesized in a straightforward manner and their antiproliferative activity was evaluated in the human colorectal cancer HCT116 cell line. Structural optimization of the hit compound SLMP53‐1 led to the identification of a (R)‐tryptophanol‐derived isoindolinone that was found to be six‐fold more active, with increased selectivity for HCT116 cells with p53 and with low toxicity in normal cells. Binding studies with MDM2 showed that the antiproliferative activity of tryptophanol‐derived isoindolinones does not involve inhibition of the main negative regulator of the p53 protein. Molecular docking simulations showed that although these molecules establish hydrophobic interactions with MDM2, they do not possess the required features to bind MDM2.Hunting p53 activators: Hit‐to‐lead optimization of the (S)‐tryptophanol‐derived oxazoloisoindolinone SLMP53‐1 led to the development of a chemical library of 34 enantiomerically pure analogues. The compounds were screened in cancer and normal colon cells. From this screening, emerged a novel (R)‐tryptophanol derivative with increased in vitro potency and selectivity in p53 expressing HCT116 cells compared with SLMP53‐1.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/166244/1/cmdc202000522.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/166244/2/cmdc202000522-sup-0001-misc_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/166244/3/cmdc202000522_am.pd

SLMP53-1 interacts with wild-type and mutant p53 DNA-binding domain and reactivates multiple hotspot mutations

Background Half of human cancers harbour TP53 mutations that render p53 inactive as a tumor suppressor. As such, reactivation of mutant (mut)p53 through restoration of wild-type (wt)-like function represents one of the most promising therapeutic strategies in cancer treatment. Recently, we have reported the (S)-tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a new reactivator of wt and mutp53 R280K with in vitro and in vivo p53-dependent antitumor activity. The present work aimed a mechanistic elucidation of mutp53 reactivation by SLMP53-1. Methods and results By cellular thermal shift assay (CETSA), it is shown that SLMP53-1 induces wt and mutp53 R280K thermal stabilization, which is indicative of intermolecular interactions with these proteins. Accordingly, in silico studies of wt and mutp53 R280K DNA-binding domain with SLMP53-1 unveiled that the compound binds at the interface of the p53 homodimer with the DNA minor groove. Additionally, using yeast and p53-null tumor cells ectopically expressing distinct highly prevalent mutp53, the ability of SLMP53-1 to reactivate multiple mutp53 is evidenced. Conclusions SLMP53-1 is a p53-activating agent with the ability to directly target wt and a set of hotspot mutp53. General Significance This work reinforces the encouraging application of SLMP53-1 in the personalized treatment of cancer patients harboring distinct p53 status.European Union (FEDER funds through Programa Operacional Factores de Competitividade – COMPETE) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) through the projects UID/QUI/50006/2019, COMPETE 2020 (POCI-01-0145-FEDER-006684/POCI-01-0145-FEDER-007440) and the BioTecNorte operation (NORTE-01-0145-FEDER-000004), (3599-PPCDT) PTDC/DTP-FTO/1981/2014 – POCI-01-0145-FEDER-016581 and UID/QUI/0081/2013; the Italian Association for Cancer Research, AIRC (IG#5506 to G.F.), Compagnia S. Paolo, Turin, Italy (Project 2017.0526 to G.F.) and Ministry of Health, (Project 5 × 1000, 2013 and 2015; Current research 2016). We also thank FCT for the financial support through CEECIND/01772/2017 (M.M.M. Santos), PTDC/QUI-QOR/29664/2017, UID/DTP/04138/2013, IF/01272/2015 (A. Carvalho), IF/00780/2015 (F. Marcelo) and fellowships SFRH/BD/119144/2016 (H. Ramos), PD/BD/114046/2015 (A. S. Gomes), SFRH/BD/128673/2017 (J. B. Loureiro), SFRH/BD/96189/2013 (S. Gomes), SFRH/BPD/110640/2015 (C. Oliveira) and PD/BI/135334/2017 (V. Barcherini), and the Programa Operacional Potencial Humano (POCH), specifically the BiotechHealth Programme (PD/00016/2012)info:eu-repo/semantics/publishedVersio