| dc.description.abstract | Lysine acetylation of histone tails is an epigenetic hallmark that plays a crucial role
in the regulation of chromatin structure. Alteration in acetylation levels and/or aberrant
activity in proteins involved in this crucial regulation have been linked to the
development of several human diseases. Bromodomains are protein modules that,
through the recognition of the acetyl-lysine modifications, drive transcriptional
programmes that result in phenotypic changes. In this scenario, BRD9 has been
receiving growing interest in diverse therapeutic areas, particularly oncology. Indeed,
acting as a subunit of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling
complex, BRD9 regulates the expression of oncogenes and anti-apoptotic proteins
leading to abnormal cell proliferation and survival in different tumor types. These
findings stimulated an intensive research activity on BRD9 that emerged as an
appealing target in cancer therapy. In addition, recently it has been assessed that
targeting BRD9 could hold a potential for the treatment of inflammation.
On these bases, the present research work had been focused on the discovery and
development of new agents able to disrupt the activity of BRD9 within the SWI/SNF
complex by applying a multi-disciplinary strategy. Intense efforts and coordinated
research work between the computational and the synthetic units led to the
development of the first 3D structure-based pharmacophore models for BRD9 that
represent valid and effective implements for the entire scientific community interested
in the identification of new potent and selective ligands. Aided by these pioneering in
silico tools, three new classes of compounds were effectively explored, supported by
the application of modern synthetic methodologies that allowed rapid access to large
collections of derivatives.
At the beginning of this BRD9 project, an early and simplified version of the three-
dimensional pharmacophores, namely the 4-point “pharm-fragment” model, was
developed and employed for the design of a class of aryl sulfonamide-based
compounds (49-74). Nevertheless, the poor activity obtained with these small
molecules prompted a careful reevaluation of the computational tool. Therefore, the
combination of iterative rounds of in silico design with effective syntheses of specific
compounds and the final biophysical evaluations of the binding activity on BRD9,
have been the cornerstone to optimize the efficiency of the compounds’ selection
process. Thus, two more precise models consisting of 7 pharmacophoric points were
built. Specifically, the development of the “pharm-druglike2” model was crucial for
the chemical exploration of a class of compounds featuring the 1-ethyl-
[1,2,4]triazolo[4,3-a]quinoxaline scaffold. Deepened SAR studies, led to the
identification of new interesting BRD9 ligands, specifically four compounds (1, 5, 11,
and 42) stood out for their potent binding on BRD9 in the low micromolar range and
high selectivity across a panel of bromodomains. In collaboration with Prof. Irace
(Università degli Studi di Napoli Federico II), compounds 1, 11, and 42 showed
interesting bioactivity in interfering with tumor growth and proliferation, proving
promising outcomes in leukemia cells.
Finally, the employment of the “pharm-druglike2” model, led to the discovery of
the latest scaffold, i.e., the 1-ethyl-1H-pyrazolo[3,4-b]pyridine core. The promising
potential, owned by this new class of small molecules as new BRD9 ligands, was
assessed by preliminary biophysical data of the hit compound 43, which disrupted the
recognition BRD9 – Histone H4 at 10 μM, presenting 55.0 ± 1.5 % residual BRD9
activity.
In parallel to the 3D pharmacophore-based discovery, two alternative approaches
were explored to circumvent the crosstalk and complexes formation occurring between
epigenetic modifiers. These mechanisms often represent a bottleneck for the
achievement of a powerful and long-lasting anticancer activity of inhibitors that target
specific subunits. Therefore, in addition to the conventional protein inhibition
approach, we decided to explore the protein targeted degradation strategy. Herein it is
reported a new class of Proteolysis Targeting Chimeras (PROTACs) taking advantage
of our identified potent and selective ligand 1 for BRD9 engagement. Initial biological
results carried out in collaboration with Prof. Altucci (Università degli Studi della
Campania Luigi Vanvitelli), highlighted the promising degradation activity of the
PROTAC compound 75, which showed an evident depletion of BRD9 at 5 μM in U937
treated cells after 24 h, that was also reflected into a strong cytotoxicity in the same
cell line.
Moreover, herein is reported the accurate design and the efficient synthesis of the
first multi-target probe 81 that could be useful to start a yet unexplored investigation
of the ncBAF SWI/SNF complex. The hybrid compound 81 was conceived with the
aim to perturb, through a single agent, two crucial subunits belonging to the above-
mentioned complex, and both involved in the onset of acute myeloid leukemia: BRD9
and SMARCA4.
As a conclusion of the intense investigations of the biological role exerted in cell
environment by BRD9, thanks to a collaboration with Prof. Maione (Università degli
Studi di Napoli Federico II), during this project it was assessed the striking anti-
inflammatory activity featured by both BRD9 ligands (e.g., compound 1) and the
degrader 75. All the tested compounds proved a remarkable decrease of IL-6 and TNF-
α in J774 macrophage cells, paving a new interesting therapeutic applicability of
BRD9 targeting agents.
Finally, in this Ph.D. project, deepened studies in the emerging protein degradation
field were carried out at the University of Dundee (UK) under the supervision of Prof.
Ciulli. The results obtained, through the syntheses of specific VHL ligand derivatives
followed by both biophysical evaluation and the crystal structure solution of MP-1-39
bound to VHL protein, could provide important steps forwards to understand the
mechanisms of molecular recognition of different stereoisomers and open the rational
design of new VHL-based PROTACs. In parallel, the optimization of the degradation
profile and pharmacokinetic properties of Leucine-rich repeat kinase 2 (LRRK2)
PROTACs for the therapeutic treatment of Parkinson’s disease was carried out.
Among a small set of newly synthesized degraders, compound MP01088 qualified as
an attractive starting point for future drug development, presenting a strong LRRK2
degradation in mouse embryonic fibroblasts already after 4 h of treatment. [edited by Author] | it_IT |
