Synthetic lethality
is an innovative framework for discovering
novel anticancer drug candidates. One example is the use of PARP inhibitors
(PARPi) in oncology patients with
BRCA
mutations.
Here, we exploit a new paradigm based on the possibility of triggering
synthetic lethality using only small organic molecules (dubbed “fully
small-molecule-induced synthetic lethality”). We exploited
this paradigm to target pancreatic cancer, one of the major unmet
needs in oncology. We discovered a dihydroquinolone pyrazoline-based
molecule (
35d
) that disrupts the RAD51-BRCA2 protein–protein
interaction, thus mimicking the effect of
BRCA2
mutation.
35d
inhibits the homologous recombination in a human pancreatic
adenocarcinoma cell line. In addition, it synergizes with olaparib
(a PARPi) to trigger synthetic lethality. This strategy aims to widen
the use of PARPi in
BRCA
-competent and olaparib-resistant
cancers, making fully small-molecule-induced synthetic lethality an
innovative approach toward unmet oncological needs.
The cytotoxic action of anticancer drugs can be potentiated by inhibiting DNA repair mechanisms. RAD51 is a crucial protein for genomic stability due to its critical role in the homologous recombination (HR) pathway. BRCA2 assists RAD51 fibrillation and defibrillation in the cytoplasm and nucleus and assists its nuclear transport. BRC4 is a peptide derived from the fourth BRC repeat of BRCA2, and it lacks the nuclear localization sequence. Here, we used BRC4 to (i) reverse RAD51 fibrillation; (ii) avoid the nuclear transport of RAD51; and (iii) inhibit HR and enhance the efficacy of chemotherapeutic treatments. Specifically, using static and dynamic light scattering, transmission electron microscopy, and microscale thermophoresis, we show that BRC4 eroded RAD51 fibrils from their termini through a “domino” mechanism and yielded monomeric RAD51 with a cumulative nanomolar affinity. Using cellular assays (BxPC-3, pancreatic cancer), we show that a myristoylated BRC4 (designed for a more efficient cell entry) abolished the formation of nuclear RAD51 foci. The present study provides a molecular description of RAD51 defibrillation, an essential step in BRCA2-mediated homologous recombination and DNA repair.
RAD51 is an ATP-dependent recombinase, recruited by BRCA2
to mediate
DNA double-strand breaks repair through homologous recombination and
represents an attractive cancer drug target. Herein, we applied for
the first-time protein-templated dynamic combinatorial chemistry on
RAD51 as a hit identification strategy. Upon design of
N
-acylhydrazone-based dynamic combinatorial libraries, RAD51 showed
a clear templating effect, amplifying 19
N
-acylhydrazones.
Screening against the RAD51–BRCA2 protein–protein interaction
via ELISA assay afforded 10 inhibitors in the micromolar range. Further
19
F NMR experiments revealed that
7
could bind
RAD51 and be displaced by BRC4, suggesting an interaction in the same
binding pocket of BRCA2. These results proved not only that ptDCC
could be successfully applied on full-length oligomeric RAD51, but
also that it could address the need of alternative strategies toward
the identification of small-molecule PPI inhibitors.
RAD51, a key player in the homologous recombination (HR) mechanism, is a critical protein to preserve genomic stability. BRCA2, upon DNA damage, promotes RAD51 fibrils disassembly and its nuclear recruitment. Here, we use BRC4, a peptide derived from the fourth BRC repeat of BRCA2; BRC4 induces RAD51 defibrillation through a 'domino' effect, eroding fibrils from their termini, and yielding monomeric RAD51. The congruence among several techniques (static and dynamic light scattering, negative staining transmission electron microscopy (TEM), and microscale thermophoresis) allows an accurate estimation of the kinetic and thermodynamic parameters of this process. BRC4 lacks, however, a nuclear localization sequence; therefore, it cannot transport RAD51 into the nucleus, thus behaving as a RAD51 inhibitor. Cellular assays (BxPC-3, pancreatic cancer cells) indeed show that BRC4 efficiently inhibits HR and enhances the cytotoxic effect of cisplatin, a DNA-damaging drug. The present study sheds further light on the complexity of the HR pathway, paving the way for designing peptide and small organic molecule inhibitors of RAD51 as innovative anticancer and chemo/radiosensitizer compounds.
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