Maria Laura Greco

Hit Identification of a Novel Dual Binder for h-telo/c-myc G-Quadruplex by a Combination of Pharmacophore Structure-Based Virtual Screening and Docking Refinement

It is well known that G‐quadruplexes are targets of great interest for their roles in crucial biological processes, such as aging and cancer. Hence, a promising strategy for anticancer drug therapy is the stabilization of these structures by small molecules. We report a high‐throughput in silico screening of commercial libraries from several different vendors by means of a combined structure‐based pharmacophore model approach followed by docking simulations. The compounds selected by the virtual screening procedure were then tested for their ability to interact with human telomeric G‐quadruplex folding by circular dichroism, fluorescence spectroscopy, and fluorescence intercalator displacement. Our approach resulted in the identification of a 13‐[(dimethylamino)methyl]‐12‐hydroxy‐8H‐benzo[c]indolo[3,2,1‐ij][1,5]naphthyridin‐8‐one derivative as a novel promising stabilizer of G‐quadruplex structures within the human telomeric and the c‐myc promoter sequences.

Further SAR studies on bicyclic basic merbarone analogues as potent antiproliferative agents.

Pyrimidopyrimidine derivatives 1 were prepared as rigid thioanalogues of merbarone (a catalytic topoisomerase II inhibitor) and screened as antiproliferative agents against different tumor cell lines. A number of the synthesized compounds emerged as cytotoxic in cell-based assays (MT-4, HeLa and MCF-7 cells) at low micromolar concentrations. In a National Cancer Institute screening, selected member of the series showed a broad spectrum of antiproliferative activity against various tumours (melanoma, renal, CNS, colon and breast cancers). The acid-base and steric properties of the substituent at position 7 of the pyrimidopyrimidine scaffold deeply affected potency. Enzymatic assays evidenced that a subset of tested derivatives efficiently inhibit topoisomerase IIα accordingly to merbarone mechanism of action. However this property does not fully rationalize the cytotoxicity data of the full ligand panel, suggesting that different target(s) should be additionally involved.

Pharmacophore Hybridization To Discover Novel Topoisomerase II Poisons with Promising Antiproliferative Activity

We used a pharmacophore hybridization strategy to combine key structural elements of merbarone and etoposide and generated new type II topoisomerase (topoII) poisons. This first set of hybrid topoII poisons shows promising antiproliferative activity on human cancer cells, endorsing their further exploration for anticancer drug discovery.

Double stranded promoter region of BRAF undergoes to structural rearrangement in nearly physiological conditions

The folding of oncogene promoters into non‐canonical DNA secondary structures is considered a strategy to control gene expression. Herein, we focused on a 30 bases sequence located upstream of the transcription start site of BRAF (Braf‐176) that contains 80% of guanines. We analyzed the structural behavior of the G‐ and C‐rich strands. By the use of spectroscopic and electrophoretic techniques we confirmed that they actually fold into a predominant antiparallel G‐quadruplex and into an i‐motif, respectively, and that they can coexist at nearly physiological conditions. Finally, the influence of several factors (KCl, pH, PEG200) on the conversion of the double stranded form of the oncogene promoter into the two above mentioned non‐canonical structures has been explored.

Coexistence of two main folded G-quadruplexes within a single G-rich domain in the EGFR promoter

Abstract EGFR is an oncogene which codifies for a tyrosine kinase receptor that represents an important target for anticancer therapy. Indeed, several human cancers showed an upregulation of the activity of this protein. The promoter of this gene contains some G-rich domains, thus representing a yet unexplored point of intervention to potentially silence this gene. Here, we explore the conformational equilibria of a 30-nt long sequence located at position −272 (EGFR-272). By merging spectroscopic and electrophoretic analysis performed on the wild-type sequence as well as on a wide panel of related mutants, we were able to prove that in potassium ion containing solution this sequence folds into two main G-quadruplex structures, one parallel and one hybrid. They show comparable thermal stabilities and affinities for the metal ion and, indeed, they are always co-present in solution. The folding process is driven by a hairpin occurring in the domain corresponding to the terminal loop which works as an important stabilizing element for both the identified G-quadruplex arrangements.