Discovery and Validation of Lmj_04_BRCT Domain,a Novel Therapeutic Target: Identification of Candidate Drugs for Leishmaniasis

Since many of the currently available antileishmanial treatments exhibit toxicity, low effectiveness, and resistance, search and validation of new therapeutic targets allowing the development of innovative drugs have become a worldwide priority. This work presents a structure-based drug discovery strategy to validate the Lmj_04_BRCT domain as a novel therapeutic target in Leishmania spp. The structure of this domain was explored using homology modeling, virtual screening, and molecular dynamics studies. Candidate compounds were validated in vitro using promastigotes of Leishmania major, L. amazonensis, and L. infantum, as well as primary mouse macrophages infected with L. major. The novel inhibitor CPE2 emerged as the most active of a group of compounds against Leishmania, being able to significantly reduce the viability of promastigotes. CPE2 was also active against the intracellular forms of the parasites and significantly reduced parasite burden in murine macrophages without exhibiting toxicity in host cells. Furthermore, L. major promastigotes treated with CPE2 showed significant lower expression levels of several genes (α-tubulin, Cyclin CYCA, and Yip1) related to proliferation and treatment resistance. Our in silico and in vitro studies suggest that the Lmj_04_BRCT domain and its here disclosed inhibitors are new potential therapeutic options against leishmaniasis.     

Diallyl sulfides: Selective inhibitors of family X DNA polymerases from garlic (Allium sativum L.)

Diallyl sulfides, organosulfur compounds isolated from garlic (Allium sativum L.), selectively inhibit the activities of mammalian family X DNA polymerases (pols), such as pol β, pol λ and terminal deoxynucleotidyl transferase (TdT), in vitro. The purified fraction (i.e., Sample-A) consisted of diallyl trisulfide, diallyl tetrasulfide and diallyl pentasulfide (molecular ratio: 5.3:3:1). Commercially purchased diallyl sulfides also inhibited the activities of family X pols, and the order of their effect was as follows: Sample-A > diallyl trisulfide > diallyl disulfide > diallyl monosulfide, suggesting that the number of sulfur atoms in the compounds might play an important structural role in enzyme inhibition. The suppression of human cancer cell (promyelocytic leukaemia cell line, HL-60) growth had the same tendency as the inhibition of pol X family among the compounds. Diallyl sulfides were suggested to bind to the pol β-like region of family X pols.     

Structure-Guided Synthesis and Evaluation of Small-Molecule Inhibitors Targeting Protein–Protein Interactions of BRCA1 tBRCT Domain

The tandem BRCT domains (tBRCT) of BRCA1 engage phosphoserine-containing motifs in target proteins to propagate intracellular signals initiated by DNA damage, thereby controlling cell cycle arrest and DNA repair. Recently, we identified Bractoppin, the first small-molecule inhibitor of the BRCA1 tBRCT domain, which selectively interrupts BRCA1-mediated cellular responses evoked by DNA damage. Here, we combine structure-guided chemical elaboration, protein mutagenesis and cellular assays to define the structural features responsible for Bractoppin's activity. Bractoppin fails to bind mutant forms of BRCA1 tBRCT bearing K1702A, a key residue mediating phosphopeptide recognition, or F1662R or L1701K that adjoin the pSer-recognition site. However, the M1775R mutation, which engages the Phe residue in the consensus phosphopeptide motif pSer-X-X-Phe, does not affect Bractoppin binding, confirming a binding mode distinct from the substrate phosphopeptide binding. We explored these structural features through structure-guided chemical elaboration and characterized structure–activity relationships (SARs) in biochemical assays. Two analogues, CCBT2088 and CCBT2103 were effective in abrogating BRCA1 foci formation and inhibiting G2 arrest induced by irradiation of cells. Collectively, our findings reveal structural features underlying the activity of a novel inhibitor of phosphopeptide recognition by the BRCA1 tBRCT domain, providing fresh insights to guide the development of inhibitors that target protein–protein interactions.     

Role of the N-terminal region of Rap1p in the transcriptional activation of glycolytic genes in Saccharomyces cerevisiae

In the yeast two-hybrid system, the N-terminal region of Rap1p was shown to interact with Gcr1p and Gcr2p. Disruption of gcr1 and/or gcr2 in the two-hybrid reporter strain demonstrated that the interaction with Gcr1p does not require Gcr2p, whereas the interaction with Gcr2p is mediated through Gcr1p. Deletion of the N-terminal region of Rap1p alone did not show a growth phenotype, but a growth defect was observed when this mutation was combined with a gcr2 deletion. The poor growth of the gcr1 null mutant was not affected further by the N-terminal deletion of Rap1p, but the growth of gcr1 strains with mutations in the DNA binding region of Gcr1p was affected by the removal of the N-terminal region of Rap1p. These results suggest that one function of the N-terminal region of Rap1p, presumably the BRCT domain, is to facilitate the binding of Gcr1p to the promoter by a protein-protein interaction.