Novel Sortase A Inhibitors to Counteract Gram-Positive Bacterial Biofilms

Sortase A (SrtA) is a membrane enzyme responsible for the covalent anchoring of surface proteins on the cell wall of Gram-positive bacteria. Nowadays it is considered an interesting target for the development of new anti-infective drugs which aim to interfere with important Gram-positive virulence mechanisms. Along the years, we studied the anti-staphylococcal and anti-biofilm activity of some natural and synthetic polyhalogenated pyrrolic compounds, called pyrrolomycins. Some of them were active on Gram-positive pathogens at a μg/mL range of concentration (1.5-0.045 μg/mL) and showed a biofilm inhibition in the range of 50-80%. [1-3] In light of these encouraging results, herein we present our efforts in the design and synthesis of novel pyrrolomycins. To dispose of sufficient amount for the in-depth in vitro investigation, we developed an efficient and easy-to-use microwave synthetic methodology. All compounds showed a good inhibitory activity toward SrtA, in accordance with the molecular modelling studies, having IC50 values ranging from 130 to 300 µM comparable to berberine hydrochloride, our reference compound. Particularly, the pentabromo-derivative exhibited the highest capability to interfere with biofilm formation of S. aureus with an IC50 of 3.4 nM. This compound was also effective in altering S. aureus murein hydrolase activity, responsible for degradation, turnover, and maturation of bacterial peptidoglycan and involved in the initial stages of S. aureus biofilm formation. [4

Landomycins as Glutathione-Depleting Agents and Natural Fluorescent Probes for Cellular Michael Adduct-Dependent Quinone Metabolism

Landomycins are angucyclines with promising antineoplastic activity produced by Streptomyces bacteria. The aglycone landomycinone is the distinctive core, while the oligosaccharide chain differs within derivatives. Herein, we report that landomycins spontaneously form Michael adducts with biothiols, including reduced cysteine and glutathione, both cell-free or intracellularly involving the benz[a]anthraquinone moiety of landomycinone. While landomycins generally do not display emissive properties, the respective Michael adducts exerted intense blue fluorescence in a glycosidic chain-dependent manner. This allowed label-free tracking of the short-lived nature of the mono-SH-adduct followed by oxygen-dependent evolution with addition of another SH-group. Accordingly, hypoxia distinctly stabilized the fluorescent mono-adduct. While extracellular adduct formation completely blocked the cytotoxic activity of landomycins, intracellularly it led to massively decreased reduced glutathione levels. Accordingly, landomycin E strongly synergized with glutathione-depleting agents like menadione but exerted reduced activity under hypoxia. Summarizing, landomycins represent natural glutathione-depleting agents and fluorescence probes for intracellular anthraquinone-based angucycline metabolism

Progettazione e sintesi di nuovi derivati 4-chinazolinonici potenziali inibitori della diidrofolato reduttasi

I chinazolinoni sono composti eterociclici azotati che, insieme alle chinazoline, rappresentano degli importanti farmacofori in possesso di un ampio spettro di proprietà biologiche tra cui quella antitumorale. Recentemente sono stati riportati in letteratura dei derivati 4-chinazolinonici in grado di inibire in vitro l’enzima diidrofolato reduttasi (DHFR) con IC50 comprese tra 0.4 e 1.0 µM [1]. Allo scopo di progettare la sintesi di nuovi potenziali inibitori della DHFR, è stato condotto uno studio di modellistica molecolare considerando tale enzima come biotarget. Tale studio ha portato alla selezione di 42 nuovi derivati 4-chinazolinonici (Figura 1). Attualmente, sono stati sintetizzati 20 dei 42 nuovi derivati 4-chinazolinonici, che sono stati saggiati preliminarmente sulla linea cellulare K562. Il derivato più attivo ha mostrato una IC50 di 18 µM. Sono in corso saggi enzimatici per valutare in vitro l’inibizione dell’enzima DHFR. Bibliografia 1 Al-Omary F.A.M.; et al, Bioorganic & Medicinal Chemistry, 2010, 18, 2849

Sintesi di un isostero del 3,5-dimetil-6-fenil-8-(trifluorometil)-5,6-diidropirazolo[3,4-f][1,2,3,5]tetrazepin-4(3H)-one (CF3-TZP) con potenziale attività biologica

In un precedente lavoro abbiamo mostrato i risultati relativi alla sintesi ed all’attività biologica del CF3-TZP 1[1] (Figura 1). Le attività antiproliferativa e apoptotica del composto 1 sono state testate su differenti linee cellulari, HL60 sensibili, HL60-R (MDR), K562 e K562-R (resistenti al Gleevec®), mostrando un profilo di attività biologica similare sulle cellule sensibili e resistenti nel range di 21-40 µM per l’IC50 e 36-62 µM per l’AC50. L’analisi citofluorimetrica sulle K562 sensibili ha indicato che il composto 1 determina un arresto dose-dipendente del ciclo cellulare in fase G0-G1 nelle prime 24 h di trattamento, mentre nelle successive 24 h si è notato una riduzione del picco G0-G1 ed un incremento del picco apoptotico subG0-G1. Gli incoraggianti risultati biologici ci hanno spinto a continuare gli studi su questa tipologia di molecole sintetizzando l’isostero 2 (Figura 1) attraverso una lunga via di sintesi (15 steps). Attualmente, sono in corso i saggi biologici per valutare le attività antiproliferativa e apoptotica. Bibliografia 1 Maggio, B.; et al, Eur. J. Med. Chem., 2008, 43, 120

Pirrolomicine che inibiscono la Sortasi A nelle infezioni sostenute da batteri Gram-positivi

La Sortasi A è un enzima di membrana responsabile dell’ancoraggio delle proteine di superficie sulla parete cellulare dei batteri Gram-positivi. Essa è considerata un interessante obiettivo per lo sviluppo di nuovi farmaci anti-infettivi che mirino ad interferire con importanti meccanismi di virulenza Gram-positivi. In un precedente lavoro abbiamo indagato sull’attività antistafilococcica e antibiofilm di alcune Pirrolomicine naturali e sintetiche, composti pirrolici polialogenati attivi su patogeni Gram-positivi, alle concentrazioni di 1.5 e 0.045 µg/mL. I risultati biologici hanno mostrato percentuali di inibizione di biofilm comprese tra 50-80% [1]. Allo scopo di indagare sul loro meccanismo d’azione sono stati condotti studi di modellistica molecolare e saggi di inibizione in vitro sull’enzima Sortasi A (Figura 1). I risultati ottenuti indicano che la Sortasi A potrebbe essere il bersaglio sul quale le Pirrolomicine agiscono, con IC50 comprese tra 130-250 µM, nell’inibizione della formazione di biofilm. Bibliografia 1 Schillaci, D.; et al, Biofouling, 2010, 26, 433

Design, synthesis and biological evaluation of new anticancer drugs: FGFR inhibitors

Fibroblast growth factor receptors (FGFRs) constitute a family of tyrosine kinases receptors (RTKs) that exert pivotal physiological functions in human embryonic and adult tissues. Hyperactivated FGFR signaling drives tumorigenesis in multiple cancer types, including lung and brain cancers. Great effort has been laid on the development of new compounds that specifically target the FGFR axis. However, cancer cell- based and microenvironmental resistance mechanisms against FGFR inhibitors often arise and are currently poorly understood. Furthermore, FGFR-targeted therapy often presents different side effects, e due to the broad biological spectrum of the FGFR signaling axis as well as to its involvement in the homeostasis of many tissue types. It is well known that metal complexes, e.g. of copper(II), zinc(II), nickel(II) and platinum(II) ions, may exhibit in vitro anti-proliferative activity against different human cancer cell lines. Usually, their cytotoxic activity has been related to their binding capabilities toward biological macromolecules. In this thesis, the recently reported in vitro anticancer properties of copper(II) compounds, coupled to the known Cu2+ ! Cu+ redox activity, have been exploited to design and synthesize, new compounds as specific inhibitors of FGFR targets. The ligands were designed on the basis of the crystal structures of FGFR1 and FGFR4 co-crystallized with their known inhibitor Ponatinib. It is worth mentioning that the onset of drug resistance to of Ponatinib has been associated to its low water solubility, which partially hinders cell membrane permeability and release to the cytosol and also increases its accumulations in lipid compartments like adiposomes. For this reason, in the following thesis work, an attempt has been taken to design, synthesize and test new ligands with structural similarity to Ponatinib, as well as their positively charged derivatives, as a consequence of their coordination to cationic metal ions such as Cu2+. The anti-proliferative activity of the most promising molecules was evaluated in vitro and in vivo, in order to understand their possible mechanism of action. In details, 22 ligands and 3 copper(II) complexes have been synthesized as potential drugs against FGFR targets. In particular, the copper complexes were identified to act as pro-drugs that are spontaneously activated by hypoxia. After a screening of several tumor cell lines to test the activity of the newly synthesized drug candidates, lung cancer and brain tumor turned out to be the most sensitive cancer types. This study shows that the most active drugs are able to inhibit the FGF/FGFR axis efficiently.Fibroblast growth factor receptors (FGFRs) constitute a family of tyrosine kinases receptors (RTKs) that exert pivotal physiological functions in human embryonic and adult tissues. Hyperactivated FGFR signaling drives tumorigenesis in multiple cancer types, including lung and brain cancers. Great effort has been laid on the development of new compounds that specifically target the FGFR axis. However, cancer cell- based and microenvironmental resistance mechanisms against FGFR inhibitors often arise and are currently poorly understood. Furthermore, FGFR-targeted therapy often presents different side effects, e due to the broad biological spectrum of the FGFR signaling axis as well as to its involvement in the homeostasis of many tissue types. It is well known that metal complexes, e.g. of copper(II), zinc(II), nickel(II) and platinum(II) ions, may exhibit in vitro anti-proliferative activity against different human cancer cell lines. Usually, their cytotoxic activity has been related to their binding capabilities toward biological macromolecules. In this thesis, the recently reported in vitro anticancer properties of copper(II) compounds, coupled to the known Cu2+ ! Cu+ redox activity, have been exploited to design and synthesize, new compounds as specific inhibitors of FGFR targets. The ligands were designed on the basis of the crystal structures of FGFR1 and FGFR4 co-crystallized with their known inhibitor Ponatinib. It is worth mentioning that the onset of drug resistance to of Ponatinib has been associated to its low water solubility, which partially hinders cell membrane permeability and release to the cytosol and also increases its accumulations in lipid compartments like adiposomes. For this reason, in the following thesis work, an attempt has been taken to design, synthesize and test new ligands with structural similarity to Ponatinib, as well as their positively charged derivatives, as a consequence of their coordination to cationic metal ions such as Cu2+. The anti-proliferative activity of the most promising molecules was evaluated in vitro and in vivo, in order to understand their possible mechanism of action. In details, 22 ligands and 3 copper(II) complexes have been synthesized as potential drugs against FGFR targets. In particular, the copper complexes were identified to act as pro-drugs that are spontaneously activated by hypoxia. After a screening of several tumor cell lines to test the activity of the newly synthesized drug candidates, lung cancer and brain tumor turned out to be the most sensitive cancer types. This study shows that the most active drugs are able to inhibit the FGF/FGFR axis efficiently

Design, synthesis and biological evaluation of new anticancer drugs: FGFR inhibitors

Fibroblast growth factor receptors (FGFRs) constitute a family of tyrosine kinases receptors (RTKs) that exert pivotal physiological functions in human embryonic and adult tissues. Hyperactivated FGFR signaling drives tumorigenesis in multiple cancer types, including lung and brain cancers. Great effort has been laid on the development of new compounds that specifically target the FGFR axis. However, cancer cell- based and microenvironmental resistance mechanisms against FGFR inhibitors often arise and are currently poorly understood. Furthermore, FGFR-targeted therapy often presents different side effects, e due to the broad biological spectrum of the FGFR signaling axis as well as to its involvement in the homeostasis of many tissue types. It is well known that metal complexes, e.g. of copper(II), zinc(II), nickel(II) and platinum(II) ions, may exhibit in vitro anti-proliferative activity against different human cancer cell lines. Usually, their cytotoxic activity has been related to their binding capabilities toward biological macromolecules. In this thesis, the recently reported in vitro anticancer properties of copper(II) compounds, coupled to the known Cu2+ ! Cu+ redox activity, have been exploited to design and synthesize, new compounds as specific inhibitors of FGFR targets. The ligands were designed on the basis of the crystal structures of FGFR1 and FGFR4 co-crystallized with their known inhibitor Ponatinib. It is worth mentioning that the onset of drug resistance to of Ponatinib has been associated to its low water solubility, which partially hinders cell membrane permeability and release to the cytosol and also increases its accumulations in lipid compartments like adiposomes. For this reason, in the following thesis work, an attempt has been taken to design, synthesize and test new ligands with structural similarity to Ponatinib, as well as their positively charged derivatives, as a consequence of their coordination to cationic metal ions such as Cu2+. The anti-proliferative activity of the most promising molecules was evaluated in vitro and in vivo, in order to understand their possible mechanism of action. In details, 22 ligands and 3 copper(II) complexes have been synthesized as potential drugs against FGFR targets. In particular, the copper complexes were identified to act as pro-drugs that are spontaneously activated by hypoxia. After a screening of several tumor cell lines to test the activity of the newly synthesized drug candidates, lung cancer and brain tumor turned out to be the most sensitive cancer types. This study shows that the most active drugs are able to inhibit the FGF/FGFR axis efficiently

Design of copper(II) complexes as potential anticancer prodrugs

The fibroblast growth factor receptors (FGFR) are tyrosine kinases that are present in many types of endothelial and tumor cells and play an important role in cell growth, survival, and migration as well as in maintaining tumor angiogenesis (1). FGFR genetic alterations are frequently observed in cancer, suggesting that FGFR inhibition may be a promising therapy in patients harboring these lesions (2). In particular, molecules with structural properties similar to Ponatinib, a known inhibitor of FGFR, that shows a selective interaction for the ATP binding site of the isoform 4 of these receptors (FGFR4), are being considered. Molecular modeling studies have been conducted to design novel potential inhibitors of the FGFR4, starting from the crystallographic structure of Ponatinib complexed with this specific isoform (2), Fig. 1 left. The considered ligands must contain chelating coordination sites for copper(II) ions (Fig. 2 right). In fact, copper (II) complex are considered as promising anticancer agents (3,4). It is expected that, under intracellular hypoxic conditions, the spontaneous reduction to a lower oxidation state of the metal ion should occur, with consequent release of the ligand molecule and the subsequent interaction of the latter with the target biomolecule. (1) Genshi Zhao, Wei-ying Li, Daohong Chen, et al.Mol Cancer Ther 2011; 10:2200-2210. (2) Jayaprakash D. Karkera, Gabriela Martinez Cardona, Katherine Bell, et al. Mol Cancer Ther 2017;16:1717-1726. (3) C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato, C. Marzano, Chem. Rev. 2014, 114, 815-86 (4) N.H. Campbell, N.H.A. Karim, G.N. Parkinson, M. Gunaratnam, V. Petrucci, A.K. Todd, R. Vilar, S. Neidle, J. Med. Chem. 2011, 55, 209-22

DESIGN AND SYNTHESIS OF NEW QUINAZOLIN-4(3H)-ONE HYBRIDS AS DUAL INHIBITORS OF TUBULIN AND DIHYDROFOLATE REDUCTASE

New strategies are needed for fighting cancer with the goal to improve efficacy of anti-cancer therapy and to limit the onset of drug resistance. Indeed, cancer cells are able to set cellular mechanisms for survival and multiple pathways support their survival. The inhibition of one pathway may then result in the activation of an alternative pathway. One strategy useful for combatting this phenomenon is represented by multi-target drugs. Herein we will present our work aim at identifying new anticancer compounds, which combine dihydrofolate reductase (DHFR) properties with tubulin inhibition. DHFR is a key enzyme involved in the synthesis of raw material for cell proliferation and the inhibition of tubulin protein family is able to suppress mitotic spindle dynamics and causes mitotic arrest and cell death. 1,2 To identify compounds able to simultaneously recognize the above-mentioned targets and using a molecular modelling approach, we designed a small library of potential DHFR/tubulin inhibitors. The quinazolin-4(3H)-one moiety is a common scaffold of DHFR and tubulin inhibitors and therefore it was select as starting pharmacophore. 2,3 Specifically, we designed compounds 1a-n, and performed docking studies on both tubulin and DHFR, in comparison with colchicine (a tubulin inhibitor reference compound) and LIH, a lipophilic antifolate. The docking results showed that seven compounds (1h-n), characterized by a 2-styryl group in the quinazolinone moiety, exhibited a good interaction with both tubulin and DHFR active sites with score values in the range of -10.7 \u2012 -10.2 and -11.0 \u2012 -9.9 kcal/mol, respectively. Among all, compound 1k emerged as the most interesting one, showing a potential good affinity for both enzymes. Building on these in silico results, we synthetized the whole compound series. Future preclinical studies are ongoing to confirm the DHFR and tubulin inhibition properties and therefor their efficacy on cancer cells cytotoxic activity