Design and synthesis of 4-anilinoquinazolines as Raf kinase inhibitors. Part 1. Selective B-Raf/B-RafV600E and potent EGFR/VEGFR2 inhibitory 4-(3-hydroxyanilino)-6-(1H-1,2,3-triazol-4-yl)quinazolines.

This paper presents the design and synthesis of 4-(3-hydroxyanilino)-6-(1H-1,2,3-triazol-4-yl)quinazolines of scaffold 9 as selective B-Raf/B-RafV600E and potent EGFR/VEGFR2 kinase inhibitors. Total 14 compounds of scaffold 9 having different side chains at the triazolyl group with/without fluoro substituents at the anilino group were synthesized and investigated. Among them, 9m with a 2-carbamoylethyl side chain and C-4'/C-6' difluoro substituents was the most potent, which selectively inhibited B-Raf (IC50: 57 nM) and B-RafV600E (IC50: 51 nM) over C-Raf (IC50: 1.0 µM). Compound 9m also actively inhibited EGFR (IC50: 73 nM) and VEGFR2 (IC50: 7.0 nM) but not EGFRT790M and PDGFR-ß (IC50: >10 µM). Despite having good potency for B-Raf and B-RafV600E in the enzymatic assays, 9m was less active to inhibit melanoma A375 cells which proliferate due to constitutively activated B-Raf600E. The inferior activity of 9m for A375 was similar to that of sorafenib (6), suggesting that 9m might bind to the inactive conformations of B-Raf and B-RafV600E. Docking simulations could thus be performed to reveal the binding poses of 9m in B-Raf, B-RafV600E, and VEGFR2 kinases.

Highly reliable GIGA-sized synthetic human therapeutic antibody library construction.

Background: Monoclonal antibodies (mAbs) and their derivatives are the fastest expanding category of pharmaceuticals. Efficient screening and generation of appropriate therapeutic human antibodies are important and urgent issues in the field of medicine. The successful in vitro biopanning method for antibody screening largely depends on the highly diverse, reliable and humanized CDR library. To rapidly obtain potent human antibodies, we designed and constructed a highly diverse synthetic human single-chain variable fragment (scFv) antibody library greater than a giga in size by phage display. Herein, the novel TIM-3-neutralizing antibodies with immunomodulatory functions derived from this library serve as an example to demonstrate the library's potential for biomedical applications. Methods: The library was designed with high stability scaffolds and six complementarity determining regions (CDRs) tailored to mimic human composition. The engineered antibody sequences were optimized for codon usage and subjected to synthesis. The six CDRs with variable length CDR-H3s were individually subjected to ß-lactamase selection and then recombined for library construction. Five therapeutic target antigens were used for human antibody generation via phage library biopanning. TIM-3 antibody activity was verified by immunoactivity assays. Results: We have designed and constructed a highly diverse synthetic human scFv library named DSyn-1 (DCB Synthetic-1) containing 2.5 × 1010 phage clones. Three selected TIM-3-recognizing antibodies DCBT3-4, DCBT3-19, and DCBT3-22 showed significant inhibition activity by TIM-3 reporter assays at nanomolar ranges and binding affinities in sub-nanomolar ranges. Furthermore, clone DCBT3-22 was exceptionally superior with good physicochemical property and a purity of more than 98% without aggregation. Conclusion: The promising results illustrate not only the potential of the DSyn-1 library for biomedical research applications, but also the therapeutic potential of the three novel fully human TIM-3-neutralizing antibodies.

Synthesis of selenophene derivatives as novel CHK1 inhibitors.

A series of selenophene derivatives 3 were synthesized as potential CHK1 inhibitors. The effects of substitution on the 4'- or 5'-position of selenophene moiety and shifting the hydroxyl group position on C6- phenolic ring of oxindole were explored. This study led to the discovery of the most potent CHK1 inhibitors 29-33 and 39-43, which had IC(50) values in the subnanomolar range.

Direct involvement of the tumor suppressor p53 in nucleotide excision repair.

The tumor suppressor p53 enhances repair of UVC-induced DNA damage. The comet-NE assay, a conventional alkaline comet assay which includes a nuclear digestion step, was used to examine the effects of p53 on the excision activity of nuclear extracts (NEs). In contrast with untreated NEs, NEs immunodepleted of p53 or NEs of p53-null cells were unable to excise UVC-induced DNA adducts. Introduction of p53 by transfection restored the excision activity to NEs of p53-null cells. Deletion of the N-terminal 99 amino acids and/or the C-terminal 85 amino acids of p53 barely affected the excision activity, whereas further deletion of the C-terminus of p53 by another 10 amino acids completely abolished the excision activity of NEs. Immunostaining following localized UV irradiation was used to examine the effects of p53 on the recruitment of repair proteins for nucleotide excision repair (NER). Although recruitment of XPC occurred regardless of the presence of p53, the recruitment of XPB was p53-dependent. However, p53 with the 95 amino acid deletion at its C-terminus was unable to support this recruitment of XPB. Consistently, intact p53 (but not the C-terminal 95 residue truncated version) was detected in co-immunoprecipitation assays with an anti-XPB antibody. These results support the hypothesis that p53 facilitates NER through direct involvement by protein-protein interactions.

Expression of tumor suppressor p53 facilitates DNA repair but not UV-induced G2/M arrest or apoptosis in Chinese hamster ovary CHO-K1 cells.

Tumor suppressor p53 is an essential regulator in mammalian cellular responses to DNA damage including cell cycle arrest and apoptosis. Our study with Chinese hamster ovary CHO-K1 cells indicates that when p53 expression and its transactivation capacity was inhibited by siRNA, UVC-induced G2/M arrest or apoptosis were unaffected as revealed by flow cyotmetric analyses and other measurements. However, inhibition of p53 rendered the cells slower to repair UV-induced damages upon a plasmid as shown in host cell reactivation assay. Furthermore, the nuclear extract (NE) of p53 siRNA-treated cells was inactive to excise the UV-induced DNA adducts as analyzed by comet assay. Consistently, the immunodepletion of p53 also deprived the excision activity of the NE in the similar experiment. Thus, tumor suppressor p53 of CHO-K1 cells may facilitate removal of UV-induced DNA damages partly via its involvement in the repair mechanism.

Colcemid inhibits the rejoining of the nucleotide excision repair of UVC-induced DNA damages in Chinese hamster ovary cells.

In our previous study, we found that colcemid, an inhibitor of mitotic spindle, promotes UVC-induced apoptosis in Chinese hamster ovary cells (CHO.K1). In this study, a brief treatment of colcemid on cells after but not before UV irradiation could synergistically reduce the cell viability. Although colcemid did not affect the excision of UV-induced DNA damages such as [6-4] photoproducts or cyclobutane pyrimidine dimers, colcemid accumulated the DNA breaks when it was added to cells following UV-irradiation. This colcemid effect required nucleotide excision repair (NER) since the same accumulation of DNA breaks was barely or not detected in two NER defective strains of CHO cells, UV5 or UV24. Furthermore, the colcemid effect was not due to semi-conservative DNA replication or mitosis since the colcemid-caused accumulation of DNA breaks was also seen in non-replicating cells. Moreover, colcemid inhibited rejoining of DNA breaks accumulated by hydroxyurea/cytosine arabinoside following UV irradiation. Nevertheless, colcemid did not affect the unscheduled DNA synthesis as assayed by the incorporation of bromodeoxyuridine. Taken together, our results suggest that colcemid might inhibit the step of ligation of NER pathways.

The roles of p53, DNA repair, and oxidative stress in ultraviolet C induction of proliferating cell nuclear antigen expression.

The expression of proliferating cell nuclear antigen (PCNA) promoter was moderately induced in UV-irradiated, quiescent human and rodent cells. The induction was independent of tumor suppressor gene p53, because the PCNA expression was UV-inducible in the subclones of human fibroblasts in which the activity of p53 was abrogated by human papilloma virus E6. Furthermore, the induction did not depend on DNA repair, since PCNA was UV inducible in UVL-10 and xrs-6 cells, in which nucleotide excision repair and double-stranded repair, respectively, are largely compromised. However, the induction was inhibited by antioxidant N-acetylcysteine. The role of oxidative stress observed here is consistent with the previous finding that the proximal AP-1 site is critical to the UV inducibility of PCNA promoter.

Tuning up or down the UV-induced apoptosis in Chinese hamster ovary cells with cell cycle inhibitors.

Exposure to UVC induces apoptosis in Chinese hamster ovary (CHO.K1) cells. While studying the underlying mechanism, we found that a variety of cell cycle inhibitors, including colcemid, hydroxyurea and mimosine, enhance the UV-induced apoptosis in these cells. Such enhancement was not dependent on the cell cycle progression nor was it related to the difference in UV sensitivity at different phases of the cell cycle. The expression of p21(waf1/cip1), a general cyclin-dependent kinase (CDK) inhibitor, was deficient in CHO.K1 cells. Ectopic overexpression of the human p21 markedly increased the survival rates of the UV-irradiated cells in the presence of colcemid. In addition, roscovitine, a small-molecule inhibitor of CDK, also inhibited the UV-induced apoptosis. These observations suggest that deregulation of CDK activity may be critical in the UV-induced apoptosis in CHO.K1 cells.

Synthesis and structure-activity relationship of 3-O-acylated (-)-epigallocatechins as 5α-reductase inhibitors.

A series of 3-O-acylated (-)-epigallocatechins were synthesized and their inhibition of steroid 5α-reductase was studied. They were prepared from the reaction of EGCG with tert-butyldimethylsilyl chloride followed by reductive cleavage of the ester bond. The resultant (-)-epigallocatechins penta-O-tert-butyldimethylsilyl ether was esterified with different fatty acids then desilylated to provide the corresponding products. The activity of 3-O-acylated (-)-epigallocatechins increased with the increasing carbon numbers of the fatty acid moiety, reaching maximum for 16 carbon atoms (compound 4h) with an IC50 of 0.53 µM, which was ∼12-fold more potent than EGCG (IC50=6.29 µM). Introduction of monounsaturated fatty acid provided the most potent compound 6 (IC50=0.48 µM), which showed moderate anti-tumor activity in vivo.

Discovery of pyrrole-indoline-2-ones as Aurora kinase inhibitors with a different inhibition profile.

A series of pyrrole-indolin-2-ones were synthesized, and their inhibition profile for Aurora kinases was studied. The potent compound 33 with phenylsulfonamido at the C-5 position and a carboxyethyl group at the C-3' position selectively inhibited Aurora A over Aurora B with IC50 values of 12 and 156 nM, respectively. Replacement of the carboxyl group with an amino group led to compound 47, which retained the activity for Aurora B and lost activity for Aurora A (IC50=2.19 microM). Computation modeling was used to address the different inhibition profiles of 33 and 47. Compounds 47 and 36 (the ethyl ester analogue of 33) inhibited the proliferation of HCT-116 and HT-29 cells and suppressed levels of the phosphorylated substrates of Aurora A and Aurora B in the Western blots.

Deregulation of cyclin-dependent kinase 2 activity correlates with UVC-induced apoptosis in Chinese hamster ovary cells.

Progression through the cell cycle relies on the activities of cyclin-dependent kinases (Cdk), which in turn are modulated by inhibitory proteins such as p21(waf1/cip1) that are induced when genomic damage occurs. In this study, we show that exposure of normal mammalian cells, such as NIH3T3 fibroblasts, to UVC (25 J/m2, at 254 nm) induces the expression of p21 without causing significant apoptosis, whereas similar treatment of Chinese hamster ovary (CHO-K1) cells with UVC causes apoptosis without inducing p21. The absence of p21 in UV-irradiated CHO-K1 cells is accompanied by the deregulation of Cdk2 activity. The elevation of Cdk2 activity correlates with the increase of UV-induced apoptosis, which can be suppressed by small-molecule Cdk2 inhibitors such as roscovitine and pyrrolidine dithiocarbamate. The results of this study suggest that the deregulation of Cdk2 activity may be critical to UV-induced apoptosis in CHO-K1 cells.