Lung cancer is the most incident and the deadliest cancer for both men and women. Therefore, the need for new and more effective therapies with fewer side effects is a concern. Photodynamic Therapy (PDT) relies on the administration of a photosensitizer that is subsequently activated by irradiation with light of appropriate wavelength. As a result, reactive oxygen species (ROS) are produced leading to cell death. PDT use in the treatment of cancer is still limited, due to the low number of new approved drugs. In light with this, this work aims to develop new photosensitizers for PDT. As an alternative class of the porphyrin-based photosensitizers, two compounds of the family of the boron−dipyrromethenes (BODIPY) have been synthesized and evaluated on the human lung cancer cell line A549. In this study, we used the MTT and SRB assays to assess the cytotoxicity of the compounds with concentrations ranging from 0,1 to 50 μM. Both compounds are photocytotoxics at the higher concentration studied. The most effective compound in the photocytotoxicity studies was the BODIPY 2 when used in a concentration of 10 and 50 µM. These promising results uphold further studies.

Cancer is a major public health problem worldwide with 18.1 million new cases of cancer and 9.6 million deaths worldwide in 2018¹.

The protein p53 is involved in many biological processes that are important to maintain the normal function of the cells (e.g. apoptosis, cell arrest, and DNA repair). It is an attractive target in oncology because it can modulate several additional cellular processes that are relevant for the suppression of tumour development, such as opposing oncogenic metabolic reprogramming, activating autophagy, and restraining invasion and metastasis. In all types of human cancers, the p53 tumour suppressor function is inactivated by mutation or gene deletion or by negative regulators such as MDM2 and MDMX. In the last years, the most popular approach among medicinal chemists to activate the wild-type p53 was the inhibition of p53-MDM2 protein-protein interaction (PPI) using small molecules. However, it is currently known that the full reactivation of p53 is only achieved when the interactions of p53 with both negative regulators are inhibited. Due to the lack of dual p53-MDM2/X PPIs inhibitors in clinical trials, it is urgent to develop small molecules that inhibit p53-MDMs PPIs².

Our research team has been working on the development and optimization of spiropyrazoline oxindoles to obtain dual p53-MDM2/X PPIs inhibitors. Hence, we have already developed derivatives with good antiproliferative activities in HCT-116 p53(+/+) human colorectal carcinoma cell line, which induce apoptosis and cell cycle arrest at G0/G1 phase, upregulate p53 steady-state levels, and lead to a decrease of MDM2 levels³. In this communication, we report the structure-based computational optimization of this chemical family for the development of novel p53-MDM2/X interactions inhibitors. Our studies will shed light on the possible binding mode of spirooxindole derivatives to MDM2 and MDMX and will drive the hit-to-lead optimization strategy. Furthermore, we report our most recent optimization of the synthesis of these new spiropyrazoline oxindoles derivatives and the first preliminary biological results.

Acknowledgements: This work was supported by National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) through UID/DTP/04138/2019 (iMed.ULisboa), project PTDC/QUI-QOR/29664/2017, Principal Investigator grant CEECIND/01772/2017 (M. M. M. Santos) and PhD fellowships SFRH/BD/137544/2018 (E.A. Lopes) and SFRH/BD/117931/2016 (M. Espadinha).

¹Ferlay, J., Colombet, M., Soerjomataram, I., Mathers, C., Parkin, D., Piñeros, M., Znaor, A. and Bray, F., Int. J. Cancer, 2019, 144, 1941-1953.

²Espadinha M., Barcherini V., Lopes E. A., Santos M. M. M., Curr. Top. Med. Chem. 2018, 18, 647-660.

³a) Nunes R., Ribeiro C. J. A., Monteiro Â., Rodrigues C. M. P., Amaral J. D., Santos M. M. M., Eur. J. Med. Chem., 2017, 139, 168-179. b) Amaral J. D., Silva D., Rodrigues C. M. P., Solá S., Santos M. M. M., Front. Chem., 2019, 7, article 7

Breast-milk α_S1-casein was suggested as an agonist of the Toll-like receptor 4 (TLR4).¹ Pathogen recognition receptor TLR4 responds to lipopolysaccharides and a wide range of molecules, from proteins to metal ions. In consequence, three criteria are required to validate agonists which directly activate TLR4 and exclude TLR4-agonisticity through contaminants.² Recently, we demonstrated that α_S1-casein fulfilled two of these criteria. (i) α_S1-Casein required TLR4/MD2 complex as well as cofactor CD14 to induce IL-8 secretion via TLR4 and (ii) α_S1-casein bound TLR4, MD2 and CD14.³ Aim of this study was to (iii) identify a synthetic amino acid sequence derived from human α_S1-casein responsible for TLR4-agonistic effects.

For this, we analyzed the amino acid sequence (AAS) of α_S1-casein in silico. α_S1‑Casein showed to be α-helical and was likely to be intrinsically disordered in the region corresponding to R¹⁶-K⁹⁹ of α_S1-casein. Six truncated variants of α_S1-casein coding for parts of the AAS were purified from Escherichia coli. These variants were tested for binding to HEK293 cells transfected with TLR4 (TLR4⁺) by flow cytometry and their induction of IL-8 secretion via TLR4. Variants of α_S1-casein truncated at the N-terminus (E³⁵-W¹⁸⁵, R⁵⁷-W¹⁸⁵, V⁷⁷-W¹⁸⁵) bound TLR4⁺ induced lower IL-8 secretion with less AAS (7.5 ng/ml, 4.8 ng/ml, 3.6 ng/ml). Variant corresponding to E⁹³-W¹⁸⁵ of α_S1-casein was neither binding TLR4⁺ nor inducing IL-8 secretion. Therefore, we postulated V⁷⁷-E⁹² derived from α_S1-casein as TLR4-agonist. This was confirmed by a synthetic peptide V⁷⁷-E⁹² derived from α_S1-casein, which induced an IL-8 secretion of 0.95 ng/ml. Hence, the third criteria of TLR4-agonists fulfilled and activation of TLR4 through contamination was excluded.

In conclusion, α_S1-casein was proofed as an agonist directly activating TLR4. This supported our postulate that α_S1-casein has at least two functions, a nutritional and an immune active one.

1. Vordenbaumen, S. et al. Human casein alpha s1 induces proinflammatory cytokine expression in monocytic cells by TLR4 signaling. Mol Nutr Food Res 60, 1079-89 (2016).
2. Mancek-Keber, M. & Jerala, R. Postulates for validating TLR4 agonists. Eur J Immunol 45, 356-70 (2015).
3. Saenger, T. et al. Human αS1-casein induces IL-8 secretion by binding to the ecto-domain of the TLR4/MD2 receptor complex. Biochim Biophys Acta Gen Subj 1863, 632-643 (2019).

TAp73 is a key tumour suppressor protein, regulating the transcription of unique and shared p53 target genes with crucial roles in tumorigenesis and therapeutic response. As such, in tumours with impaired p53 signalling, like neuroblastoma (NBL), TAp73 activation represents an encouraging strategy, alternative to p53 activation, to suppress tumour growth and chemoresistance [1]. Actually, NBL represents one of the most common childhood solid cancers but despite the available treatments, the high-risk patients are still characterized by low survival rates, making the search of new therapeutic options an urgent need [2].

In this work, we report the synthesis and biological evaluation of a new TAp73-activating agent, the 1-carbaldehyde-3,4-dimethoxyxanthone (LEM2), as a potent antitumour agent either alone or in combination with conventional drugs [3]. LEM2 anticancer activity was evaluated in the wild-type p53 expressing human colon adenocarcinoma HCT116 cells (HCT116 p53^+/+) and in the respective p53-null isogenic derivative cells (HCT116 p53^-/-), by sulforhodamine B assay. Results showed that LEM2 had potent p53-independent tumour growth inhibitory effect, with similar IC₅₀values in p53^+/+(0.98 ± 0.12 μM) and p53^−/−(0.68 ± 0.08 μM) HCT116 cells. The antiproliferative effect of LEM2 on the growth of human tumour cells expressing distinct mutant p53 forms was also investigated, with IC₅₀values around 1–3 μM. LEM2 antitumor effect was associated with enhanced TAp73 transcriptional activity, cell cycle arrest, and apoptosis in p53-null and mutant p53-expressing tumour cells. Importantly, the LEM2 antiproliferative effect was not associated with genotoxicity. Mechanistically, LEM2 was able to disrupt the TAp73 interaction with MDM2 and mutant p53, both in yeast and in human tumour cells. Additionally, by cellular thermal shift assay (CETSA), it was verified that LEM2 induced thermal stabilization of TAp73α but not of MDM2 or mutant p53, suggesting the potential interaction of LEM2 with TAp73α. LEM2 also displayed potent antitumour activity against immortalized and patient-derived NBL cells [4,5], consistent with an activation of the TAp73 pathway. LEM2 alcohol and carboxylic acid derivativeswere also tested to support that the LEM2 biological activity was due to the molecule itself and not to its potential derivatives. Indeed, both the alcohol 1-(hydroxymethyl)-3,4-dimethoxy-9H-xanthen-9-one (LEMred) and the carboxylic acid 3,4-dimethoxy-9-oxo-9H-xanthene-1-carboxylic acid (LEMox) presented a much lower activity when compared to LEM2 and were unable to inhibit the TAp73-MDM2 interaction.

The antiproliferative effect of LEM2, alone and in combination with doxorubicin and cisplatin, was analysed by MTT assay and the synergistic effect was evaluated through determination of combination and dose reduction index values. The results showed a potent TAp73-dependent cytotoxic activity of LEM2, superior to that of nutlin-3a (a known TAp73 activator), associated with induction of cell cycle arrest and apoptosis and upregulation of TAp73 target genes, in NBL cells. Also, we observed a pronounced synergistic effect between doxorubicin/cisplatin and LEM2, which may result from enhancing TAp73 activation through alternative pathways.

In conclusion, besides its relevant contribution to the advance of TAp73 pharmacology, LEM2 may pave the way to improved therapeutic alternatives against NBL, both alone and in combination with conventional chemotherapeutics.

1 – Wolter J et al. Future Oncol., 2010; 6: 429-444; 2- Nakagawara A et al. Jpn. J. Clin. Oncol., 2018; 48:214-24; 3 – Gomes S et al. Cancer letters., 2019; 0304-3835; 4 - Veselska R et al.Cancer, 2006 6: 32; 5 - Veselska R et al. BMC Cancer, 2008; 8: 300

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 grant UID/QUI/50006/2019. This work received financial support from the European Union (FEDER funds through the Operational Competitiveness Program (COMPETE) POCI-01-0145-FEDER-006684/POCI-01-0145-FEDER-007440 and (3599-PPCDT) PTDC/DTP-FTO/1981/2014 – POCI-01-0145-FEDER-016581) and the FCT grants PTDC/QUIQOR/29664/2017, POCI-01-0145-FEDER-028736 (PTDC/SAU-PUB/28736/2017). We thank FCT and ESF (European Social Fund) through POCH (Programa Operacional Capital Humano) for: L. Raimundo PhD grant ref. SFRH/BD/117949/2016; J. Loureiro PhD grant ref SFRH/BD/128673/2017; H. Ramos PhD grant ref SFRH/BD/119144/2016. J. Calheiro thanks ICETA for her grant ref. ICETA2019-71. M. Monteiro thanks ICETA for her grant ref. ICETA2019-70 We thank (POCH), specifically the BiotechHealth Programme (Doctoral Programme on Cellular and Molecular Biotechnology Applied to Health Sciences; PD/00016/2012).

Polyketide synthases type I (PKS-I) gene cluster is responsible for the synthesis of highly assorted group of secondary metabolites such as antimicrobial and anticancer agents. In our study, screening was carried out using degenerate primers to determine the presence of PKS-I gene cluster in endophytic actinomycetes isolated from two medicinal plants Ocmium teniflorum (Tulsi) and Azadirachta indica (Neem). A total of 28 endophytes that were isolated and identified from our previous study were further confirmed through 16S rRNA gene sequencing to exhibit a 99% similarity with Streptomyces sp. The molecular screening using PCR revealed the presence of PKS- I gene with a product size of 750bps in the isolates, FHK-1, FHK-2, FHK-3, FHK-4, FHK-5, FHK-6, FHK-7, FHK-8, FHK-9, FHK-11, FHK-13, FHK-16, FHK-18, FHK-20, FHK-21, FHK-23, FHK,25 and FHK-28. These isolates were further checked for their antimicrobial potential using their crude extracts. They displayed prominent bioactivity against ATCC pathogens, Escherichia coli, Proteus vulgaris, Rhodococcus equi, Staphylococcus epidermidis, Enterococcus faecalis, and Acinetobacter baumanii. Our study revealed that the endophytes from O. tenuiflorum and A. indica are bioactive and versatile harboring the PKS-I gene cluster.