Emmanuele Crespan
Istituto di Genetica Molecolare “Luigi Luca Cavalli-Sforza”
Via Abbiategrasso, 207 – 27100 PAVIA
tel: +39 0382 546354
fax: +39 0382 546370
E-mail: emmanuele.crespan@igm.cnr.it
Current Research Activity
RNase H2
With more than 107 ribonucleotides misincorporated in the human genome during replication, embedded ribonucleotides are the most abundant DNA lesions. The presence of the 2’-hydroxyl group makes misincorporated ribonucleotides prone to spontaneous hydrolysis, potentially resulting in the accumulation of strand breaks. Moreover, their presence may decrease the rate of DNA replication causing replicative fork stalling and, eventually, collapse. Ribonucleotide removal is initiated by Ribonuclease H2 (RNase H2), the key player in Ribonucleotide Excision Repair (RER). Its absence leads to embryonic lethality in mice, while mutations decreasing its activity cause the severe phenotype of the Aicardi–Goutières syndrome.
We are interested in the identification of factors that participate in RER using biochemical and cell biology approaches. We are also focusing our attention on the ability of RNase H2 to process ribonucleotides embedded in non-canonical DNA form and in the development of tools to map ribonucleotide embedded in the genome, with the aim to identify cold and hot spots for their misincorporation.
Alternative Lengthening of Telomeres
About 10% of cancers do not rely on telomerases activity as maintenance mechanism of telomeres (TMM), but on an alternative lengthening of telomeres (ALT) pathway. ALT-proficient cells show common features such as heterogenic and fluctuating telomere lengths, high levels of telomere recombination events, and abundant extrachromosomal telomeric repeat DNA. Despite a comprehensive view of the factors and the molecular mechanisms involved in ALT is still missing, mounting evidence led to a consensus on repair of double strand breaks (DSBs) originated from replication forks collapse as the leading cause of ALT. A homology directed process, involving the invasion and elongation of G-rich strand using the complementary strand as a template is also a consolidate characteristic of ALT mechanism. Different pathways has been proposed to explain TMM in ALT, all of which require enzymes able to catalyse DNA strand transfer and annealing reactions followed by DNA synthesis. So far, the identity of the DNA polymerases (Pols) involved in the elongation step of ALT has remained elusive.
By using multidisciplinary approaches (including biochemistry, cell biology, proteomics, imaging, CRISPR libraries, and animal models) we investigate of the roles of specialised DNA polymerases in ALT mechanism and, in general, in the reconstruction of the molecular steps occurring in ALT initiation. The goal of this research is the identification of enzyme(s) essential in ALT process that to be considered as targets for new therapeutic approaches.
CANVAS
Co-occurrence of cerebellar Ataxia with Neuropathy and Vestibular Areflexia Syndrome (CANVAS) is a rare, adult-onset, slowly progressive neurological disorder characterized by imbalance, sensory neuropathy (neuronopathy), bilateral vestibulopathy, chronic cough, and occasionally autonomic dysfunction. The genetic cause of the disease was very recently discovered and consists of biallelic AAGGG repeat expansions in the second intron of replication factor C subunit 1 (RFC1). The mechanism leading to symptoms onset is not yet understood.
To understand the mechanism driving CANVAS onset, we aim to characterize RFC1 activity on CANVAS derived patient cell lines through biochemistry and cellular based assays.
Repair DNA polymerases.
DNA repair is crucial to maintain genomic stability, with eepair DNA polymerases, as well as translesion polymerases playing a pivotal role. We have focus our attention on the study of the enzymatic properties and cellular roles of DNA polymerases in response to DNA damage in an attempt to identify specific activities and molecular processes that could be targets for innovative therapies. In particular, we identified and characterized selective inhibitors of specific polymerases, including compounds that can be incorporated specifically against certain types of DNA damages, such as abasic sites, to be used as markers for these lesions. The same research has led to the elucidation of the role of repair polymerases and other accessory factors for the faithful bypass of various lesions. In particular, we found that DNA polymerase Lambda is specifically recruited for the faithful bypass of the mutagenic, and most common oxidised lesion, 8-oxoG.We also explored the contribution of repair polymerases in the expansion of tri-nucleotides repeats that are characteristic of different neurodegenerative diseases, suggesting a possible role of polymerase beta in Poly-Q disorders.
Drugs development.
Part of our research is focused on the characterization of new inhibitors of molecular targets involved in viral, neurodegenerative, and neoplastic diseases. In particular, we are interested in the evaluation of the inhibition potency, toxicity, and mechanism of action of new drug candidates through in vitro assays using recombinant proteins as well as cellular models. We have been involved in the characterization of molecules targeting essential factors in the replication of different viruses including HIV-1, HCV, Zika Virus, enteroviruses, and Dengue virus. We are also collaborating with several groups on the evaluation of small molecules as inhibitors of kinases responsible of the onset and progression of specific cancers or targets of viral or bacterial infections (host targets), or involved in neurodegenerative diseases progression.
Orcid id: 0000-0003-0597-6929
Pubblicazioni Recenti
2022 |
Princiotto S; Musso L; Manetti F; Marcellini V; Maga G; Crespan E; Perini C; Zaffaroni N; Beretta GL; Dallavalle S Synthesis and biological activity evaluation of 3-(hetero) arylideneindolin-2-ones as potential c-Src inhibitors Journal Article In: Journal of enzyme inhibition and medicinal chemistry, vol. 37, iss. 1, pp. 2382-2394, 2022. @article{%a1.%Yb_41,Inhibition of c-Src is considered one of the most studied approaches to cancer treatment, with several heterocyclic compounds approved during the last 15 years as chemotherapeutic agents. Starting from the biological evaluation of an in-house collection of small molecules, indolinone was selected as the most promising scaffold. In this work, several functionalised indolinones were synthesised and their inhibitory potency and cytotoxic activity were assayed. The pharmacological profile of the most active compounds, supported by molecular modelling studies, revealed that the presence of an amino group increased the affinity towards the ATP-binding site of c-Src. At the same time, bulkier derivatizations seemed to improve the interactions within the enzymatic pocket. Overall, these data represent an early stage towards the optimisation of new, easy-to-be functionalised indolinones as potential c-Src inhibitors. |
2021 |
Mentegari E; Bertoletti F; Kissova M; Zucca E; Galli S; Tagliavini G; Garbelli A; Maffia A; Bione S; Ferrari E; d'Adda di Fagagna F; Francia S; Sabbioneda S; Chen LY; Lingner J; Bergoglio V; Hoffmann JS; Hubscher U; Crespan E; Maga G A Role for Human DNA Polymerase lambda in Alternative Lengthening of Telomeres Journal Article In: International journal of molecular sciences, vol. 22, no. 5, pp. 2365, 2021. @article{%a1:%Y_131,Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol lambda) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the absence of Pol lambda, strongly affects the survival of ALT cells. In vitro, Pol lambda can promote annealing of even a single G-rich telomeric repeat to its complementary strand and use it to prime DNA synthesis. The noncoding telomeric repeat containing RNA TERRA and replication protein A negatively regulate this activity, while the Protection of Telomeres protein 1 (POT1)/TPP1 heterodimer stimulates Pol lambda. Pol lambda associates with telomeres and colocalizes with TPP1 in cells. In summary, our data suggest a role of Pol lambda in the maintenance of telomeres by the ALT mechanism. |
Grazia Martina M; Vicenti I; Bauer L; Crespan E; Rango E; Boccuto A; Olivieri N; Incerti M; Zwaagstra M; Allodi M; Bertoni S; Dreassi E; Zazzi M; van Kuppeveld FJM; Maga G; Radi M In: ChemMedChem, vol. 16, iss. 23, no. 3548, pp. 3552, 2021. @article{%a1:%Ybvwb,Over half a century since the description of the first antiviral drug, "old" re-emerging viruses and "new" emerging viruses still represent a serious threat to global health. Their high mutation rate and rapid selection of resistance toward common antiviral drugs, together with the increasing number of co-infections, make the war against viruses quite challenging. Herein we report a host-targeted approach, based on the inhibition of the lipid kinase PI4KIIIβ, as a promising strategy for inhibiting the replication of multiple viruses hijacking this protein. We show that bithiazole inhibitors of PI4KIIIβ block the replication of human rhinoviruses (hRV), Zika virus (ZIKV) and SARS-CoV-2 at low micromolar and sub-micromolar concentrations. However, while the anti-hRV/ZIKV activity can be directly linked to PI4KIIIβ inhibition, the role of PI4KIIIβ in SARS-CoV-2 entry/replication is debated. |
Dede M; Napolitano S; Melati A; Pirota V; Maga G; Crespan E High Flexibility of RNaseH2 Catalytic Activity with Respect to Non-Canonical DNA Structures Journal Article In: International journal of molecular sciences, vol. 22, no. 10, pp. 5201, 2021. @article{%a1:%Yb,Ribonucleotides misincorporated in the human genome are the most abundant DNA lesions. The 2'-hydroxyl group makes them prone to spontaneous hydrolysis, potentially resulting in strand breaks. Moreover, their presence may decrease the rate of DNA replication causing replicative fork stalling and collapse. Ribonucleotide removal is initiated by Ribonuclease H2 (RNase H2), the key player in Ribonucleotide Excision Repair (RER). Its absence leads to embryonic lethality in mice, while mutations decreasing its activity cause Aicardi-Goutières syndrome. DNA geometry can be altered by DNA lesions or by peculiar sequences forming secondary structures, like G-quadruplex (G4) and trinucleotide repeats (TNR) hairpins, which significantly differ from canonical B-form. Ribonucleotides pairing to lesioned nucleotides, or incorporated within non-B DNA structures could avoid RNase H2 recognition, potentially contributing to genome instability. In this work, we investigate the ability of RNase H2 to process misincorporated ribonucleotides in a panel of DNA substrates showing different geometrical features. RNase H2 proved to be a flexible enzyme, recognizing as a substrate the majority of the constructs we generated. However, some geometrical features and non-canonical DNA structures severely impaired its activity, suggesting a relevant role of misincorporated ribonucleotides in the physiological instability of specific DNA sequences. |
Pirota V; Lunghi E; Benassi A; Crespan E; Freccero M; Doria F Selective Binding and Redox-Activity on Parallel G-Quadruplexes by Pegylated Naphthalene Diimide-Copper Complexes Journal Article In: Molecules, vol. 26, no. 16, pp. 5025, 2021. @article{%a1:%Ybvwe,G-quadruplexes (G4s) are higher-order supramolecular structures, biologically important in the regulation of many key processes. Among all, the recent discoveries relating to RNA-G4s, including their potential involvement as antiviral targets against COVID-19, have triggered the ever-increasing need to develop selective molecules able to interact with parallel G4s. Naphthalene diimides (NDIs) are widely exploited as G4 ligands, being able to induce and strongly stabilize these structures. Sometimes, a reversible NDI-G4 interaction is also associated with an irreversible one, due to the cleavage and/or modification of G4s by functional-NDIs. This is the case of NDI-Cu-DETA, a copper(II) complex able to cleave G4s in the closest proximity to the target binding site. Herein, we present two original Cu(II)-NDI complexes, inspired by NDI-Cu-DETA, differently functionalized with 2-(2-aminoethoxy)ethanol side-chains, to selectively drive redox-catalyzed activity towards parallel G4s. The selective interaction toward parallel G4 topology, controlled by the presence of 2-(2-aminoethoxy)ethanol side chains, was already firmly demonstrated by us using core-extended NDIs. In the present study, the presence of protonable moieties and the copper(II) cavity, increases the binding affinity and specificity of these two NDIs for a telomeric RNA-G4. Once defined the copper coordination relationship and binding constants by competition titrations, ability in G4 stabilization, and ROS-induced cleavage were analyzed. The propensity in the stabilization of parallel topology was highlighted for both of the new compounds HP2Cu and PE2Cu. The results obtained are particularly promising, paving the way for the development of new selective functional ligands for binding and destructuring parallel G4s. |
Vicenti I; Martina MG; Boccuto A; De Angelis M; Giavarini G; Dragoni F; Marchi S; Trombetta CM; Crespan E; Maga G; Eydoux C; Decroly E; Montomoli E; Nencioni L; Zazzi M; Radi M In: European journal of medicinal chemistry, vol. 224, pp. 113683, 2021. @article{%a1:%Ybv,he worldwide circulation of different viruses coupled with the increased frequency and diversity of new outbreaks, strongly highlight the need for new antiviral drugs to quickly react against potential pandemic pathogens. Broad-spectrum antiviral agents (BSAAs) represent the ideal option for a prompt response against multiple viruses, new and re-emerging. Starting from previously identified anti-flavivirus hits, we report herein the identification of promising BSAAs by submitting the multi-target 2,6-diaminopurine chemotype to a system-oriented optimization based on phenotypic screening on cell cultures infected with different viruses. Among the synthesized compounds, 6i showed low micromolar potency against Dengue, Zika, West Nile and Influenza A viruses (IC50 = 0.5-5.3 μM) with high selectivity index. Interestingly, 6i also inhibited SARS-CoV-2 replication in different cell lines, with higher potency on Calu-3 cells that better mimic the SARS-CoV-2 infection in vivo (IC50 = 0.5 μM, SI = 240). The multi-target effect of 6i on flavivirus replication was also analyzed in whole cell studies (in vitro selection and immunofluorescence) and against isolated host/viral targets. |
2020 |
Nalli M; Armijos Rivera JI; Masci D; Coluccia A; Badia R; Riveira-Munoz E; Brambilla A; Cinquina E; Turriziani O; Falasca F; Catalano M; Limatola C; Este JA; Maga G; Silvestri R; Crespan E; La Regina G New indolylarylsulfone non-nucleoside reverse transcriptase inhibitors show low nanomolar inhibition of single and double HIV-1 mutant strains Journal Article In: European journal of medicinal chemistry, vol. 208, pp. 112696, 2020. @article{%a1:%Y_460,We designed and synthesized 21 new indolylarylsulfones (IASs) as new HIV-1 NNRTIs. Among these, IAS 12 exhibited a remarkable antiviral activity against single and double mutants (K103N EC50 = <0.7 nM; Y181C EC50 = <0.7 nM; Y188L EC50 = 21.3 nM; K103N-Y181C EC50 = 6.2 nM), resulting equally or more active than previuosly reported IAS 6 and some approved anti-HIV-1 drugs. Docking and molecular dynamics simulations of compound 12 in complex with WT, Y181C, Y188L, K103N and K103N-Y181C RTs clarified a general binding mode that was consistent with biological results. Kinetic experiments disclosed that derivative 12 preferentially binds WT and K103N-Y181C RTs to binary and ternary complexes, respectively. |
2019 |
Nawrozkij M; Forgione M; Yablokov AS; Lucidi A; Tomaselli D; Patsilinakos A; Panella C; Hailu GS; Kirillov IA; Badia R; Riveira Muñoz E; Crespan E; Armijos-Rivera JI; Cirilli R; Ragno R; Este JA; Maga G; Mai A; Rotili D Effect of alpha-Methoxy Substitution on the anti-HIV Activity of Dihydropyrimidin-4(3H)-ones. Journal Article In: Journal of medicinal chemistry, vol. 62, no. 2, pp. 604-621, 2019. @article{%a1:%Y_54,Conformational restriction applied to dihydrobenzylpyrimidin-4-(3H)-ones (DABOs) by the intoduction of a methyl group at the alpha-benzylic position is known to massively improve the anti-HIV-1 activity of these compounds. Here, we report the effects of methoxy substitution at the alpha-benzylic position in S-, NH-, and N,N-DABOs carrying 2,6-difluoro, 2-chloro-6-fluoro, or 2,6-dichloro substituted benzyl moieties. The various alpha-methoxy DABO series (12-14) present different SAR at the dihalo benzyl substitution, with the most potent compounds (12d,e and 13c) showing similar (picomolar/nanomolar) anti-HIV-1 potency as the corresponding alpha-methyl analogs against wt HIV-1, and 10- to 100-fold increased potency (up to low nanomolar) against clinically relevant K103N, Y181C, Y188L, IRLL98 and K103N+Y181C HIV-1 mutant strains, highlithing the importance of the alpha-methoxy substitution to provide highly efficient DABOs as "second generation" NNRTIs. HPLC enantioseparation of three of the most potent derivatives (12d, 13c and 14c) provided single enantiomers with significant enantioselectivity in HIV-1 inhibition. Computational studies allowed to correlate the best antiviral activity with the (R) absolute configuration at the alpha-methoxy stereogenic center. |
Fallacara AL; Passannanti R; Mori M; Iovenitti G; Musumeci F; Greco C; Crespan E; Kissova M; Maga G; Tarantelli C; Spriano F; Gaudio E; Bertoni F; Botta M; Schenone S In: European journal of medicinal chemistry, vol. 181, pp. 111545, 2019. @article{%a1:%Y%_34,Abnormal activation of B-cell receptor (BCR) signaling plays a key role in the development of lymphoid malignancies, and could be reverted by the simultaneous inhibition of Lyn, Fyn and Blk, three members of the Src family kinase (SFK). Fyn and Blk are also promising targets for the treatment of some forms of T-cell non-Hodgkin lymphoma which point to the druggability of SFKs for the treatment of these cancers. We recently identified Si308 as a potent Fyn inhibitor, while preliminary data showed that it might also inhibit Lyn and Blk. Here, molecular modelling studies were coupled with enzymatic assays to further investigate the effect of Si308 on Lyn and Blk. A small library of pyrazolo[3,4-d]pyrimidines structurally related to Si308 was synthesized and tested on human lymphoma cell lines. Compound 2h emerged as a new multitarget inhibitor of Lyn, Fyn and Blk endowed with remarkable antiproliferative effects on human B and T lymphoma cell lines. Its favorable ADME properties make the compound suitable for further developments. |
Geronikaki A; Petrou A; Kartsev V; Eleftheriou P; Boga R; Bartolo B; Crespan E; Franco G; Maga G Molecular docking, design, synthesis and biological evaluation of novel 2,3-aryl-thiazolidin-4-ones as potent NNRTIs. Journal Article In: SAR and QSAR in environmental research, vol. 30, no. 10, pp. 697-714, 2019. @article{%a1:%Y%_36,Nonnucleoside reverse transcriptase inhibitors (NNRTIs) remain the most promising anti-AIDS agents that target the HIV-1 reverse transcriptase enzyme (RT). However, the efficiency of approved NNRTI drugs has decreased by the appearance of drug-resistant viruses and side effects upon long-term usage. Thus, there is an urgent need for developing new, potent NNRTIs with broad spectrum against HIV-1 virus and with improved properties. In this study, a series of thiazolidinone derivatives was designed based on a butterfly mimicking scaffold consisting of a substituted benzothiazolyl moiety connected with a substituted phenyl ring via a thiazolidinone moiety. The most promising derivatives were selected using molecular docking analysis and PASS prediction program, synthesized and evaluated for HIV-1 RT inhibition. Five out of fifteen tested compounds exhibited good inhibitory action. It was observed that the presence of Cl or CN substituents at the position 6 of the benzothiazole ring in combination with two fluoro atoms at the ortho-positions or a hydrogen acceptor substituent at the 4-position of the phenyl ring are favourable for the HIV RT inhibitory activity. |
Tassini S; Langron E; Delang L; Mirabelli C; Lanko K; Crespan E; Kissova M; Tagliavini G; Fontò G; Bertoni S; Palese S; Giorgio C; Ravanetti F; Ragionieri L; Zamperini C; Mancini A; Dreassi E; Maga G; Vergani P; Neyts J; Radi M Multitarget CFTR Modulators Endowed with Multiple Beneficial Side Effects for Cystic Fibrosis Patients: Toward a Simplified Therapeutic Approach Journal Article In: Journal of medicinal chemistry, vol. 62, no. 23, pp. 10833-10847, 2019. @article{%a1:%Y_66,Cystic fibrosis (CF) is a multiorgan disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). In addition to respiratory impairment due to mucus accumulation, viruses and bacteria trigger acute pulmonary exacerbations, accelerating disease progression and mortality rate. Treatment complexity increases with patients’ age, and simplifying the therapeutic regimen represents one of the key priorities in CF. We have recently reported the discovery of multitarget compounds able to “kill two birds with one stone” by targeting F508del-CFTR and PI4KIIIβ and thus acting simultaneously as CFTR correctors and broad-spectrum enterovirus (EV) inhibitors. Starting from these preliminary results, we report herein a hit-to-lead optimization and multidimensional structure–activity relationship (SAR) study that led to compound 23a. This compound showed good antiviral and F508del-CFTR correction potency, additivity/synergy with lumacaftor, and a promising in vitro absorption, distribution, metabolism, and excretion (ADME) profile. It was well tolerated in vivo with no sign of acute toxicity and histological alterations in key biodistribution organs. |
2018 |
Molinari A; Fallacara AL; Di Maria S; Zamperini C; Poggialini F; Musumeci F; Schenone S; Angelucci A; Colapietro A; Crespan E; Kissova M; Maga G; Botta M Efficient optimization of pyrazolo[3,4-d]pyrimidines derivatives as c-Src kinase inhibitors in neuroblastoma treatment. Journal Article In: Bioorganic & medicinal chemistry letters, vol. 28, no. 21, pp. 3454-3457, 2018. @article{%a1:%Y_162,The proto-oncogene c-Src is a non-receptor tyrosine kinase which is involved in the regulation of many cellular processes, such as differentiation, adhesion and survival. c-Src hyperactivation has been detected in many tumors, including neuroblastoma (NB), one of the major causes of death from neoplasia in infancy. We already reported a large family of pyrazolo[3,4-d]pyrimidines active as c-Src inhibitors. Interestingly, some of these derivatives resulted also active on SH-SY5Y NB cell line. Herein, starting from our previous Free Energy Perturbation/Monte Carlo calculations, we report an optimization study which led to the identification of a new series of derivatives endowed with nanomolar Ki values against c-Src, interesting antiproliferative activity on SH-SY5Y cells and a suitable ADME profile. |
Kaptein SJF; Vincetti P; Crespan E; Rivera JIA; Costantino G; Maga G; Neyts J; Radi M Identification of Broad-Spectrum Dengue/Zika Virus Replication Inhibitors by Functionalization of Quinoline and 2,6-Diaminopurine Scaffolds. Journal Article In: ChemMedChem, vol. 13, no. 14, pp. 1371-1376, 2018. @article{%a1:%Y_149,Social and demographic changes across the world over the past 50 years have resulted in significant outbreaks of arboviruses such as dengue virus (DENV) and Zika virus (ZIKV). Despite the increased threat of infection, no approved drugs or fully protective vaccines are available to counteract the spread of DENV and ZIKV. The development of "broad-spectrum" antivirals (BSAs) that target common components of multiple viruses can be a more effective strategy to limit the rapid emergence of viral pathogens than the classic "one-bug/one-drug" approach. Starting from previously identified multitarget DENV inhibitors, herein we report the identification of novel 2,6-diaminopurine derivatives that are able to block the replication of both Zika virus and all serotypes of dengue virus (DENV 1-4) in infected cells. |
2017 |
Famiglini V; La Regina G; Coluccia A; Masci D; Brancale A; Badia R; Riveira-Munoz E; Esté JA; Crespan E; Brambilla A; Maga G; Catalano M; Limatola C; Formica FR; Cirilli R; Novellino E; Silvestri R Chiral Indolylarylsulfone Non-Nucleoside Reverse Transcriptase Inhibitors as New Potent and Broad Spectrum Anti-HIV-1 Agents. Journal Article In: Journal of medicinal chemistry, vol. 60, no. 15, pp. 6528-6547, 2017. @article{%a1:%Y_198,We designed and synthesized a series of chiral indolyarylsulfones (IASs) as new HIV-1 NNRTIs. The new IASs 8-37 showed potent inhibition of the HIV-1 WT NL4-3 strain and of the mutant K103N, Y181C, Y188L, and K103N-Y181C HIV-1 strains. Six racemic mixtures, 8, 23-25, 31, and 33, were separated at semipreparative level into their pure enantiomers. The (R)-8 enantiomer bearing the chiral (α-methylbenzyl) was superior to the (S)-counterpart. IAS derivatives bearing the (S) alanine unit, (S)-23, (S,R)-25, (S)-31, and (S)-33, were remarkably more potent than the corresponding (R)-enantiomers. Compound 23 protected hippocampal neuronal cells from the excitotoxic insult, while efavirenz (EFV) did not contrast the neurotoxic effect of glutamate. The present results highlight the chiral IASs as new NNRTIs with improved resistance profile against the mutant HIV-1 strains and reduced neurotoxic effects. |
Tassini S; Sun L; Lanko K; Crespan E; Langron E; Falchi F; Kissova M; Armijos-Rivera JI; Delang L; Mirabelli C; Neyts J; Pieroni M; Cavalli A; Costantino G; Maga G; Vergani P; Leyssen P; Radi M In: Journal of medicinal chemistry, vol. 60, no. 4, pp. 1400-1416, 2017. @article{%a1:%Y_199,Enteroviruses (EVs) are among the most frequent infectious agents in humans worldwide and represent the leading cause of upper respiratory tract infections. No drugs for the treatment of EV infections are currently available. Recent studies have also linked EV infection with pulmonary exacerbations, especially in cystic fibrosis (CF) patients, and the importance of this link is probably underestimated. The aim of this work was to develop a new class of multitarget agents active both as broad-spectrum antivirals and as correctors of the F508del-cystic fibrosis transmembrane conductance regulator (CFTR) folding defect responsible for >90% of CF cases. We report herein the discovery of the first small molecules able to simultaneously act as correctors of the F508del-CFTR folding defect and as broad-spectrum antivirals against a panel of EVs representative of all major species. |
Garbelli A; Riva V; Crespan E; Maga G How to win the HIV-1 drug resistance hurdle race: running faster or jumping higher? Journal Article In: Biochemical journal, vol. 474, no. 10, pp. 1559-1577, 2017. @article{%a1:%Y_219,Infections by the human immunodeficiency virus type 1 (HIV-1), the causative agent of the acquired immunodeficiency syndrome (AIDS), are still totaling an appalling 36.7 millions worldwide, with 1.1 million AIDS deaths/year and a similar number of yearly new infections. All this, in spite of the discovery of HIV-1 as the AIDS etiological agent more than 30 years ago and the introduction of an effective combinatorial antiretroviral therapy (cART), able to control disease progression, more than 20 years ago. Although very effective, current cART is plagued by the emergence of drug-resistant viral variants and most of the efforts in the development of novel direct-acting antiviral agents (DAAs) against HIV-1 have been devoted toward the fighting of resistance. In this review, rather than providing a detailed listing of all the drugs and the corresponding resistance mutations, we aim, through relevant examples, at presenting to the general reader the conceptual shift in the approaches that are being taken to overcome the viral resistance hurdle. From the classic 'running faster' strategy, based on the development of novel DAAs active against the mutant viruses selected by the previous drugs and/or presenting to the virus a high genetic barrier toward the development of resilience, to a 'jumping higher' approach, which looks at the cell, rather than the virus, as a source of valuable drug targets, in order to make the cellular environment non-permissive toward the replication of both wild-type and mutated viruses. 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society. |
Musumeci F; Fallacara AL; Brullo C; Grossi G; Botta L; Calandro P; Chiariello M; Kissova M; Crespan E; Maga G; Schenone S Identification of new pyrrolo[2,3-d]pyrimidines as Src tyrosine kinase inhibitors in vitro active against Glioblastoma. Journal Article In: European journal of medicinal chemistry, vol. 127, pp. 369-378, 2017. @article{%a1:%Y_209,In the last few years, several pyrrolo-pyrimidine derivatives have been either approved by the US FDA and in other countries for the treatment of different diseases or are currently in phase I/II clinical trials. Herein we present the synthesis and the characterization of a novel series of pyrrolo[2,3-d]pyrimidines, compounds 8a-j, and their activity against Glioblastoma multiforme (GBM). Docking studies and MM-GBSA analysis revealed the ability of such compounds to efficiently interact with the ATP binding site of Src. Enzymatic assays against a mini-panel of kinases (Src, Fyn, EGFR, Kit, Flt3, Abl, AblT315I) have been performed, showing an unexpected selectivity of our pyrrolo[2,3-d]pyrimidines for Src. Finally, the derivatives were tested for their antiproliferative potency on U87 GBM cell line. Compound 8h showed a considerable cytotoxicity effect against U87 cell line with an IC50 value of 7.1 microM. |
Mentegari E; Crespan E; Bavagnoli L; Kissova M; Bertoletti F; Sabbioneda S; Imhof R; Sturla SJ; Nilforoushan A; Hubscher U; van Loon B; Maga G Ribonucleotide incorporation by human DNA polymerase eta impacts translesion synthesis and RNase H2 activity. Journal Article In: Nucleic Acids Research, vol. 45, no. 5, pp. 2600-2614, 2017. @article{%a1:%Y_190,Ribonucleotides (rNs) incorporated in the genome by DNA polymerases (Pols) are removed by RNase H2. Cytidine and guanosine preferentially accumulate over the other rNs. Here we show that human Pol η can incorporate cytidine monophosphate (rCMP) opposite guanine, 8-oxo-7,8-dihydroguanine, 8-methyl-2'-deoxyguanosine and a cisplatin intrastrand guanine crosslink (cis-PtGG), while it cannot bypass a 3-methylcytidine or an abasic site with rNs as substrates. Pol eta is also capable of synthesizing polyribonucleotide chains, and its activity is enhanced by its auxiliary factor DNA Pol delta interacting protein 2 (PolDIP2). Human RNase H2 removes cytidine and guanosine less efficiently than the other rNs and incorporation of rCMP opposite DNA lesions further reduces the efficiency of RNase H2. Experiments with XP-V cell extracts indicate Pol eta as the major basis of rCMP incorporation opposite cis-PtGG. These results suggest that translesion synthesis by Pol eta can contribute to the accumulation of rCMP in the genome, particularly opposite modified guanines. |
Meroni A; Mentegari E; Crespan E; Muzi-Falconi M; Lazzaro F; Podestà A The Incorporation of Ribonucleotides Induces Structural and Conformational Changes in DNA. Journal Article In: Biophysical journal, vol. 113, no. 7, pp. 1373-1382, 2017. @article{%a1:%Y_222,Ribonucleotide incorporation is the most common error occurring during DNA replication. Cells have hence developed mechanisms to remove ribonucleotides from the genome and restore its integrity. Indeed, the persistence of ribonucleotides into DNA leads to severe consequences, such as genome instability and replication stress. Thus, it becomes important to understand the effects of ribonucleotides incorporation, starting from their impact on DNA structure and conformation. Here we present a systematic study of the effects of ribonucleotide incorporation into DNA molecules. We have developed, to our knowledge, a new method to efficiently synthesize long DNA molecules (hundreds of basepairs) containing ribonucleotides, which is based on a modified protocol for the polymerase chain reaction. By means of atomic force microscopy, we could therefore investigate the changes, upon ribonucleotide incorporation, of the structural and conformational properties of numerous DNA populations at the single-molecule level. Specifically, we characterized the scaling of the contour length with the number of basepairs and the scaling of the end-to-end distance with the curvilinear distance, the bending angle distribution, and the persistence length. Our results revealed that ribonucleotides affect DNA structure and conformation on scales that go well beyond the typical dimension of the single ribonucleotide. In particular, the presence of ribonucleotides induces a systematic shortening of the molecules, together with a decrease of the persistence length. Such structural changes are also likely to occur in vivo, where they could directly affect the downstream DNA transactions, as well as interfere with protein binding and recognition. |
2016 |
Radi M; Schneider R; Fallacara AL; Botta L; Crespan E; Tintori C; Maga G; Kissova M; Calgani A; Richters A; Musumeci F; Rauh D; Schenone S A cascade screening approach for the identification of Bcr-Abl myristate pocket binders active against wild type and T315I mutant. Journal Article In: Bioorganic & Medicinal Chemistry Letters, vol. 26, no. 15, pp. 3436-3440, 2016. @article{%a1:%Y_304,The major clinical challenge in drug-resistant chronic myelogenous leukemia (CML) is currently represented by the Bcr-Abl T315I mutant, which is unresponsive to treatment with common first and second generation ATP-competitive tyrosine kinase inhibitors (TKIs). Allosteric inhibition of Bcr-Abl represent a new frontier in the fight against resistant leukemia and few candidates have been identified in the last few years. Among these, myristate pocket (MP) binders discovered by Novartis (e.g. GNF2/5) showed promising results, although they proved to be active against the T315I mutant only in combination with first and second generation ATP-competitive inhibitors. Here we used a cascade screening approach based on sequential fluorescence polarization (FP) screening, in silico docking/dynamics studies and kinetic-enzymatic studies to identify novel MP binders. A pyrazolo[3,4-d]pyrimidine derivative (6) has been identified as a promising allosteric inhibitor active on 32D leukemia cell lines (expressing Bcr-Abl WT and T315I) with no need of combination with any ATP-competitive inhibitor. |
Tintori C; Brai A; Dasso Lang MC; Deodato D; Greco AM; Bizzarri BM; Cascone L; Casian A; Zamperini C; Dreassi E; Crespan E; Maga G; Vanham G; Ceresola E; Canducci F; Arien KK; Botta M In: Journal of medicinal chemistry, vol. 59, no. 6, pp. 2747-2759, 2016. @article{%a1:%Y_314,Preventing HIV transmission by the use of a vaginal microbicide is a topic of considerable interest in the fight against AIDS. Both a potent anti-HIV agent and an efficient formulation are required to develop a successful microbicide. In this regard, molecules able to inhibit the HIV replication before the integration of the viral DNA into the genetic material of the host cells, such as entry inhibitors or reverse transcriptase inhibitors (RTIs), are ideal candidates for prevention purpose. Among RTIs, S- and N-dihydroalkyloxybenzyloxopyrimidines (S-DABOs and N-DABOs) are interesting compounds active at nanomolar concentration against wild type of RT and with a very interesting activity against RT mutations. Herein, novel N-DABOs were synthesized and tested as anti-HIV agents. Furthermore, their mode of binding was studied by molecular modeling. At the same time, a vaginal microbicide gel formulation was developed and tested for one of the most promising candidates. |
Goodson WH 3rd; Lowe L; Carpenter DO; Gilbertson M; Manaf Ali A; Lopez de Cerain Salsamendi A; Lasfar A; Carnero A; Azqueta A; Amedei A; Charles AK; Collins AR; Ward A; Salzberg AC; Colacci A; Olsen AK; Berg A; Barclay BJ; Zhou BP; Blanco-Aparicio C; Baglole CJ; Dong C; Mondello C; Hsu CW; Naus CC; Yedjou C; Curran CS; Laird DW; Koch DC; Carlin DJ; Felsher DW; Roy D; Brown DG; Ratovitski E; Ryan EP; Corsini E; Rojas E; Moon EY; Laconi E; Marongiu F; Al-Mulla F; Chiaradonna F; Darroudi F; Martin FL; Van Schooten FJ; Goldberg GS; Wagemaker G; Nangami G; Calaf GM; Williams G; Wolf GT; Koppen G; Brunborg G; Kim Lyerly H; Krishnan H; Ab Hamid H; Yasaei H; Sone H; Kondoh H; Salem HK; Hsu HY; Park HH; Koturbash I; Miousse IR; Scovassi AI; Klaunig JE; Vondráček J; Raju J; Roman J; Wise JP Sr; Whitfield JR; Woodrick J; Christopher JA; Ochieng J; Martinez-Leal JF; Weisz J; Kravchenko J; Sun J; Prudhomme KR; Narayanan KB; Cohen-Solal KA; Moorwood K; Gonzalez L; Soucek L; Jian L; D'Abronzo LS; Lin LT; Li L; Gulliver L; McCawley LJ; Memeo L; Vermeulen L; Leyns L; Zhang L; Valverde M; Khatami M; Romano MF; Chapellier M; Williams MA; Wade M; Manjili MH; Lleonart M; Xia M; Gonzalez MJ; Karamouzis MV; Kirsch-Volders M; Vaccari M; Kuemmerle NB; Singh N; Cruickshanks N; Kleinstreuer N; van Larebeke N; Ahmed N; Ogunkua O; Krishnakumar PK; Vadgama P; Marignani PA; Ghosh PM; Ostrosky-Wegman P; Thompson P; Dent P; Heneberg P; Darbre P; Sing Leung P; Nangia-Makker P; Cheng QS; Robey RB; Al-Temaimi R; Roy R; Andrade-Vieira R; Sinha RK; Mehta R; Vento R; Di Fiore R; Ponce-Cusi R; Dornetshuber-Fleiss R; Nahta R; Castellino RC; Palorini R; Abd Hamid R; Langie SA; Eltom S; Brooks SA; Ryeom S; Wise SS; Bay SN; Harris SA; Papagerakis S; Romano S; Pavanello S; Eriksson S; Forte S; Casey SC; Luanpitpong S; Lee TJ; Otsuki T; Chen T; Massfelder T; Sanderson T; Guarnieri T; Hultman T; Dormoy V; Odero-Marah V; Sabbisetti V; Maguer-Satta V; Rathmell WK; Engström W; Decker WK; Bisson WH; Rojanasakul Y; Luqmani Y; Chen Z; Hu Z Llona-Minguez S; Hoglund A; Jacques SA; Johansson L; Calderon-Montano JM; Claesson M; Loseva O; Valerie NC; Lundbäck T; Piedrafita J; Maga G; Crespan E; Meijer L; Burgos Morón E; Baranczewski P; Hagbjork AL; Svensson R; Wiita E; Almlof I; Visnes T; Jeppsson F; Sigmundsson K; Jensen AJ; Artursson P; Jemth AS; Stenmark P; Warpman Berglund U; Scobie M; Helleday T Discovery of the First Potent and Selective Inhibitors of Human dCTP Pyrophosphatase 1. Journal Article In: Journal of medicinal chemistry, vol. 59, no. 3, pp. 1140-1148, 2016. @article{%a1:%Y_292,The dCTPase pyrophosphatase 1 (dCTPase) regulates the intracellular nucleotide pool through hydrolytic degradation of canonical and noncanonical nucleotide triphosphates (dNTPs). dCTPase is highly expressed in multiple carcinomas and is associated with cancer cell stemness. Here we report on the development of the first potent and selective dCTPase inhibitors that enhance the cytotoxic effect of cytidine analogues in leukemia cells. Boronate 30 displays a promising in vitro ADME profile, including plasma and mouse microsomal half-lives, aqueous solubility, cell permeability and CYP inhibition, deeming it a suitable compound for in vivo studies. |
Mentegari E; Kissova M; Bavagnoli L; Maga G; Crespan E DNA Polymerases lambda and beta: The Double-Edged Swords of DNA Repair. Journal Article In: Genes (Basel), vol. 7, no. 9, pp. e57, 2016. @article{%a1:%Y_299,DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell's genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases beta and lambda are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase lambda also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy. |
Crespan E; Furrer A; Rösinger M; Bertoletti F; Mentegari E; Chiapparini G; Imhof R; Ziegler N; Sturla SJ; Hubscher U; van Loon B; Maga G Impact of ribonucleotide incorporation by DNA polymerases beta and lambda on oxidative base excision repair. Journal Article In: Nature Communications, vol. 7, pp. 10805, 2016. @article{%a1:%Y_263,Oxidative stress is a very frequent source of DNA damage. Many cellular DNA polymerases (Pols) can incorporate ribonucleotides (rNMPs) during DNA synthesis. However, whether oxidative stress-triggered DNA repair synthesis contributes to genomic rNMPs incorporation is so far not fully understood. Human specialized Pols beta and lamdda are the important enzymes involved in the oxidative stress tolerance, acting both in base excision repair and in translesion synthesis past the very frequent oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxo-G). We found that Pol beta, to a greater extent than Pol lambda can incorporate rNMPs opposite normal bases or 8-oxo-G, and with a different fidelity. Further, the incorporation of rNMPs opposite 8-oxo-G delays repair by DNA glycosylases. Studies in Pol beta- and lambda-deficient cell extracts suggest that Pol beta levels can greatly affect rNMP incorporation opposite oxidative DNA lesions. |
El-Moghazy SM; George RF; Osman EE; Elbatrawy AA; Kissova M; Colombo A; Crespan E; Maga G Novel pyrazolo[3,4-d]pyrimidines as dual Src-Abl inhibitors active against mutant form of Abl and the leukemia K-562 cell line. Journal Article In: European Journal of Medicinal Chemistry, vol. 123, pp. 1-13, 2016. @article{%a1:%Y_272,Some novel 6-substituted pyrazolo[3,4-d]pyrimidines 4, 5, 6a-d, 7a-c, 8 and pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]pyrimidines 9a-c, 10a-c, 11, 12a,b, 13a-c and 14 were synthesized and characterized by spectral and elemental analyses. They were screened for their biological activity in vitro against Abl and Src kinases. Compounds 7a and 7b revealed the highest activity against both wild and mutant Abl kinases as well as the Src kinase and the leukemia K-562 cell line. They can be considered as new hits for further structural optimization to obtain better activity. |
Kissova M; Maga G; Crespan E The human tyrosine kinase Kit and its gatekeeper mutant T670I, show different kinetic properties: Implications for drug design. Journal Article In: Bioorganic & Medicinal Chemistry, vol. 24, no. 19, pp. 4555-4532, 2016. @article{%a1:%Y_321,The tyrosine kinase Kit, a receptor for Stem Cell Factor, is involved, among others, in processes associated to cell survival, proliferation and migration. Upon physiological conditions, the activity of Kit is tightly regulated. However, primary mutations that lead to its constitutive activation are the causal oncogenic driver of gastrointestinal stromal tumours (GISTs). GISTs are known to be refractory to conventional therapies but the introduction of Imatinib, a selective inhibitor of tyrosine kinases Abl and Kit, significantly ameliorated the treatment options of GISTs patients. However, the acquisition of secondary mutations renders Kit resistant towards all available drugs. Mutation involving gatekeeper residues (such as V654a and T670I) influence both the structure and the catalytic activity of the enzyme. Therefore, detailed knowledge of the enzymatic properties of the mutant forms, in comparison with the wild type enzyme, is an important pre-requisite for the rational development of specific inhibitors. In this paper we report a thorough kinetic analysis of the reaction catalyzed by the Kit kinase and its gatekeeper mutated form T670I. Our results revealed the different mechanisms of action of these two enzymes and may open a new avenue for the future design of specific Kit inhibitors. |
2015 |
Tintori C; Fallacara AL; Radi M; Zamperini C; Dreassi E; Crespan E; Maga G; Schenone S; Musumeci F; Brullo C; Richters A; Gasparrini F; Angelucci A; Festuccia C; Delle Monache S; Rauh D; Botta M In: Journal of Medicinal Chemistry, vol. 58, no. 1, pp. 347-361, 2015. @article{%a1:%Y_348,c-Src is a tyrosine kinase belonging to the Src-family kinases. It is overexpressed and/or hyperactivated in a variety of cancer cells, thus its inhibition has been predicted to have therapeutic effects in solid tumors. Recently, the pyrazolo[3,4-d]pyrimidine 3 was reported as a dual c-Src/Abl inhibitor. Herein we describe a multidisciplinary drug discovery approach for the optimization of the lead 3 against c-Src. Starting from the X-ray crystal structure of c-Src in complex with 3, Monte Carlo free energy perturbation calculations were applied to guide the design of c-Src inhibitors with improved activities. As a result, the introduction of a meta hydroxyl group on the C4 anilino ring was computed to be particularly favorable. The potency of the synthesized inhibitors was increased with respect to the starting lead 3. The best identified compounds were also found active in the inhibition of neuroblastoma cell proliferation. Furthermore, compound 29 also showed in vivo activity in xenograft model using SH-SY5Y cells. |
Vincetti P; Caporuscio F; Kaptein S; Gioiello A; Mancino V; Suzuki Y; Yamamoto N; Crespan E; Lossani A; Maga G; Rastelli G; Castagnolo D; Neyts J; Leyssen P; Costantino G; Radi M Discovery of Multitarget Antivirals Acting on Both the Dengue Virus NS5-NS3 Interaction and the Host Src/Fyn Kinases. Journal Article In: Journal of Medicinal Chemistry, vol. 58, no. 12, 2015. @article{%a1:%Y_353,This study describes the discovery of novel dengue virus inhibitors targeting both a crucial viral protein-protein interaction and an essential host cell factor as a strategy to reduce the emergence of drug resistance. Starting from known c-Src inhibitors, a virtual screening was performed to identify molecules able to interact with a recently discovered allosteric pocket on the dengue virus NS5 polymerase. The selection of cheap-to-produce scaffolds and the exploration of the biologically relevant chemical space around them suggested promising candidates for chemical synthesis. A series of purines emerged as the most interesting candidates able to inhibit virus replication at low micromolar concentrations with no significant toxicity to the host cell. Among the identified antivirals, compound 16i proved to be 10 times more potent than ribavirin, showed a better selectivity index and represents the first-in-class DENV-NS5 allosteric inhibitor able to target both the virus NS5-NS3 interaction and the host kinases c-Src/Fyn. |
Crespan E; Hübscher U; Maga G Expansion of CAG triplet repeats by human DNA polymerases λ and β in vitro, is regulated by flap endonuclease 1 and DNA ligase 1. Journal Article In: DNA Repair, vol. 29, pp. 101-111, 2015. @article{%a1:%Y_361,Huntington's disease (HD) is a neurological genetic disorder caused by the expansion of the CAG trinucleotide repeats (TNR) in the N-terminal region of coding sequence of the Huntingtin's (HTT) gene. This results in the addition of a poly-glutamine tract within the Huntingtin protein, resulting in its pathological form. The mechanism by which TRN expansion takes place is not yet fully understood. We have recently shown that DNA polymerase (Pol) beta can promote the microhomology-mediated end joining andtriplet expansion of a substrate mimicking a double strand break in the TNR region of the HTT gene. Here we show that TNRexpansion is dependent on the structure of the DNA substrate, as well as on the two essential Pol beta co-factors: flap endonuclease1 (Fen1) and DNA ligase 1 (Lig1). We found that Fen1 significantly stimulated TNR expansion by Pol beta, but not by the related enzyme Pol lambda, and subsequent ligation of the DNA products by Lig1. Interestingly, the deletion of N-terminal domains of Pol lambda, resulted in an enzyme which displayed properties more similar to Pol beta, suggesting a possible evolutionary mechanism. These results may suggest a novel mechanism for somatic TNR expansion in HD. |
Tintori C; La Sala G; Vignaroli G; Botta L; Fallacara AL; Falchi F; Radi M; Zamperini C; Dreassi E; Dello Iacono L; Orioli D; Biamonti G; Garbelli M; Lossani A; Gasparrini F; Tuccinardi T; Laurenzana I; Angelucci A; Maga G; Schenone S; Brullo C; Musumeci F; Desogus A; Crespan E; Botta M In: Journal of Medicinal Chemistry, vol. 58, no. 11, pp. 4590-4609, 2015. @article{%a1:%Y_419,Fyn is a member of the Src-family of nonreceptor protein-tyrosine kinases. Its abnormal activity has been shown to be related to various human cancers as well as to severe pathologies, such as Alzheimer's and Parkinson's diseases. Herein, a structure-based drug design protocol was employed aimed at identifying novel Fyn inhibitors. Two hits from commercial sources (1, 2) were found active against Fyn with K(i) of about 2 μM, while derivative 4a, derived from our internal library, showed a K(i) of 0.9 μM. A hit-to-lead optimization effort was then initiated on derivative 4a to improve its potency. Slightly modifications rapidly determine an increase in the binding affinity, with the best inhibitors 4c and 4d having K(i)s of 70 and 95 nM, respectively. Both compounds were found able to inhibit the phosphorylation of the protein Tau in an Alzheimer's model cell line and showed antiproliferative activities against different cancer cell lines. |
Spallarossa A; Caneva C; Caviglia M; Alfei S; Butini S; Campiani G; Gemma S; Brindisi M; Zisterer DM; Bright SA; Williams CD; Crespan E; Maga G; Sanna G; Delogu I; Collu G; Loddo R Unconventional Knoevenagel-type indoles: Synthesis and cell-based studies for the identification of pro-apoptotic agents. Journal Article In: European Journal of Medicinal Chemistry, vol. 102, pp. 648-660, 2015. @article{%a1:%Y_418,A new series of indole-based analogues were recently identified as potential anticancer agents. The Knoevenagel-type indoles herein presented were prepared via a one-pot condensation of iminium salts with active methylene reagents and were isolated as single geometric isomers. Biological evaluation in different cell-based assays revealed an antiproliferative activity for some analogues already in the nanomolar range against leukaemia, breast and renal cancer cell lines. To explain these effects, the most promising analogues of the series were engaged in further cell-based studies. Compounds 5e, l, p and 6a, b highlighted a pro-apoptotic potential being able to induce apoptosis in HL60, K562 and MCF-7 cell lines in a dose and time-dependent manner. The ability of these compounds to arrest cell cycle at the G2/M phase inspired the immunofluorescence studies which allowed us to identify tubulin as a potential target for compounds 5l and 6b. Copyright 2015 Elsevier Masson SAS. All rights reserved. |