Synthesis and biological evaluation of 1-(2,4,5-trisubstituted phenyl)-3-(5-cyanopyrazin-2-yl)ureas as potent Chk1 kinase inhibitors

Based on the X-ray crystallography of our lead compound 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-cyanopyrazin-2-yl)urea in the checkpoint kinase 1 (Chk1) enzyme, we modified R4, and to a lesser extent, R2, and R5 of the phenyl ring, and made a variety of N-aryl-N'-pyrazinylurea Chk1 inhibitors. Enzymatic activity less than 20 nM was observed in 15 of 41 compounds. Compound 8i provided the best overall results in the cellular assays as it abrogated doxorubicin-induced cell cycle arrest (IC50=1.7 microM) and enhanced doxorubicin cytotoxicity (IC50=0.44 microM) while displaying no single agent activity.     

4-(Aminoalkylamino)-3-benzimidazole-quinolinones as potent CHK-1 inhibitors

CHK-1 is one of the key enzymes regulating checkpoints in cellular growth cycles. Novel 4-(amino-alkylamino)-3-benzimidazole-quinolinones were prepared and assayed for their ability to inhibit CHK-1. These compounds are potent cell permeable CHK-1 inhibitors and showed synergistic effect with a DNA-damaging agent, camptothecin.     

Cyanopyridyl containing 1,4-dihydroindeno[1,2-c]pyrazoles as potent checkpoint kinase 1 inhibitors: improving oral biovailability

A series of 1,4-dihydroindeno[1,2-c]pyrazole compounds with a cyanopyridine moiety at the 3-position of the tricyclic pyrazole core was explored as potent CHK-1 inhibitors. The impact of substitutions at the 6 and/or 7-position of the core on pharmacokinetic properties was studied in detail. Compounds carrying a side chain with an ether linker at the 7-position and a terminal morpholino group, such as 29 and 30, exhibited much-improved oral biovailability in mice as compared to earlier generation inhibitors. These compounds also possessed desirable cellular activity in potentiating doxorubicin and will serve as valuable tool compounds for in vivo evaluation of CHK-1 inhibitors to sensitize DNA-damaging agents.     

SMK-1/PPH-4.1-mediated silencing of the CHK-1 response to DNA damage in early C. elegans embryos

During early embryogenesis in Caenorhabditis elegans, the ATL-1-CHK-1 (ataxia telangiectasia mutated and Rad3 related-Chk1) checkpoint controls the timing of cell division in the future germ line, or P lineage, of the animal. Activation of the CHK-1 pathway by its canonical stimulus DNA damage is actively suppressed in early embryos so that P lineage cell divisions may occur on schedule. We recently found that the rad-2 mutation alleviates this checkpoint silent DNA damage response and, by doing so, causes damage-dependent delays in early embryonic cell cycle progression and subsequent lethality. In this study, we report that mutations in the smk-1 gene cause the rad-2 phenotype. SMK-1 is a regulatory subunit of the PPH-4.1 (protein phosphatase 4) protein phosphatase, and we show that SMK-1 recruits PPH-4.1 to replicating chromatin, where it silences the CHK-1 response to DNA damage. These results identify the SMK-1-PPH-4.1 complex as a critical regulator of the CHK-1 pathway in a developmentally relevant context.     

Pharmacological activation of a novel p53-dependent S-phase checkpoint involving CHK-1

We have recently shown that induction of the p53 tumour suppressor protein by the small-molecule RITA (reactivation of p53 and induction of tumour cell apoptosis; 2,5-bis(5-hydroxymethyl-2-thienyl)furan) inhibits hypoxia-inducible factor-1α and vascular endothelial growth factor expression in vivo and induces p53-dependent tumour cell apoptosis in normoxia and hypoxia. Here, we demonstrate that RITA activates the canonical ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related DNA damage response pathway. Interestingly, phosphorylation of checkpoint kinase (CHK)-1 induced in response to RITA was influenced by p53 status. We found that induction of p53, phosphorylated CHK-1 and γH2AX proteins was significantly increased in S-phase. Furthermore, we found that RITA stalled replication fork elongation, prolonged S-phase progression and induced DNA damage in p53 positive cells. Although CHK-1 knockdown did not significantly affect p53-dependent DNA damage or apoptosis induced by RITA, it did block the ability for DNA integrity to be maintained during the immediate response to RITA. These data reveal the existence of a novel p53-dependent S-phase DNA maintenance checkpoint involving CHK-1.     

2-Trifluoroacetylthiophenes, a novel series of potent and selective class II histone deacetylase inhibitors

The identification of class II HDAC inhibitors has been hampered by lack of efficient enzyme assays, in the preceding paper two assays have been developed to improve the efficiency of these enzymes: mutating an active site histidine to tyrosine, or by the use of a trifluoroacetamide lysine substrate, allowing screening to identify class II HDAC inhibitors. Herein, 2-trifluoroacetylthiophenes have been demonstrated to inhibit class II HDACs, resulting in the development of a series of 5-(trifluoroacetyl)thiophene-2-carboxamides as novel, potent and selective class II HDAC inhibitors. X-ray crystal structures of the HDAC 4 catalytic domain with a bound inhibitor demonstrate these compounds are active site inhibitors and bind in their hydrated form.     

Structure-based design, synthesis, and SAR evaluation of a new series of 8-hydroxyquinolines as HIF-1alpha prolyl hydroxylase inhibitors

A new series of potent 8-hydroxyquinolines was designed based on the newly resolved X-ray crystal structure of EGLN-1. Both alkyl and aryl 8-hydroxyquinoline-7-carboxyamides were good HIF-1alpha prolyl hydroxylase (EGLN) inhibitors. In subsequent VEGF induction assays, these exhibited potent VEGF activity. In addition, this class of compounds did show the ability to stabilize HIF-1alpha.     

CHK1-regulated S-phase checkpoint response reduces camptothecin cytotoxicity

The cytotoxicity of camptothecin (CPT) is S phase specific and is associated with an inhibition of DNA replication. The relationship between CPT-induced inhibition of DNA replication and CPT cytotoxicity remains unclear. We previously reported that the CPT-induced inhibition reflects an activated S-phase (S) checkpoint response and that this response is mainly regulated by ATR/CHK1 pathway. In this study, by comparing A1-5 and B4, the two transformed rat embryo fibroblasts cell lines, we showed that with higher CHK1 expression, A1-5 cells had a stronger S checkpoint response and were more resistant to CPT-treatment. The data suggested that over-activated CHK1 in CPT-treated A1-5 cells regulated the strong S checkpoint response through the CDC25A/CDK2 pathway. When the CHK-1 regulated strong S checkpoint response was abolished, A1-5 cells became much more sensitive to CPT-induced killing. These data indicated that CHK1 regulated S checkpoint response protected cells from CPT-induced killing.     

CHK1-Regulated S-phase Checkpoint Response Reduces Camptothecin Cytotoxity

The cytotoxicity of camptothecin (CPT) is S phase specific and is associated with an inhibition of DNA replication. The relationship between CPT-induced inhibition of DNA replication and CPT cytotoxicity remains unclear. We previously reported that the CPT-induced inhibition reflects an activated S-phase (S) checkpoint response and that this response is mainly regulated by ATR/CHK1 pathway. In this study, by comparing A1-5 and B4, the two transformed rat embryo fibroblasts cell lines, we showed that with higher CHK1 expression, A1-5 cells had a stronger S checkpoint response and were more resistant to CPT-treatment. The data suggested that over-activated CHK1 in CPT-treated A1-5 cells regulated the strong S checkpoint response through the CDC25A/CDK2 pathway. When the CHK-1 regulated strong S checkpoint response was abolished, A1-5 cells became much more sensitive to CPT-induced killing. These data indicated that CHK1 regulated S checkpoint response protected cells from CPT-induced killing.

Key Words:

CHK1, S-phase checkpoint, Camptothecin, DNA damage     

1,4-Dihydroindeno[1,2-c]pyrazoles as potent checkpoint kinase 1 inhibitors: extended exploration on phenyl ring substitutions and preliminary ADME/PK studies

A study on substitutions at the four open positions on the phenyl ring of the 1,4-dihydroindeno[1,2-c]pyrazoles as potent CHK-1 inhibitors is described. Bis-substitution at both the 6- and 7-positions led to inhibitors with IC(50) values below 0.3nM. The compound with the best overall activities (36) was able to potentiate the anti-proliferative effect of doxorubicin in HeLa cells by at least 47-fold. Physicochemical, metabolic, and pharmacokinetic properties of selected inhibitors are also disclosed.     

X-ray structures of checkpoint kinase 2 in complex with inhibitors that target its gatekeeper-dependent hydrophobic pocket

The serine/threonine checkpoint kinase 2 (Chk2) is an attractive molecular target for the development of small molecule inhibitors to treat cancer. Here, we report the rational design of Chk2 inhibitors that target the gatekeeper-dependent hydrophobic pocket located behind the adenine-binding region of the ATP-binding site. These compounds exhibit IC(50) values in the low nanomolar range and are highly selective for Chk2 over Chk1. X-ray crystallography was used to determine the structures of the inhibitors in complex with the catalytic kinase domain of Chk2 to verify their modes of binding.     

Re-purposing clinical kinase inhibitors to enhance chemosensitivity by overriding checkpoints

Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well-tolerated by patients. We exploited the promiscuous nature of kinase inhibitors to screen 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells to a sub-lethal concentration of gemcitabine. Bosutinib, dovitinib, and BEZ-235 were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA-approved Src/Abl inhibitor approved for chronic myelogenous leukemia. Unbeknownst to us, we used an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as “authentic” bosutinib. In vitro and cell-based assays showed that both the authentic bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1, with Bos-I showing greater potency. Imaging data showed that Bos-I forced cells to override gemcitabine-induced DNA damage checkpoint arrest and destabilized stalled replication forks. These inhibitors enhanced sensitivity to the DNA damaging agents’ gemcitabine, cisplatin, and doxorubicin in pancreatic cancer cell lines. The in vivo efficacy of Bos-I was validated using cells derived directly from a pancreatic cancer patient’s tumor. Notably, the xenograft studies showed that the combination of gemcitabine and Bos-I was significantly more effective in suppressing tumor growth than either agent alone. Finally, we show that the gatekeeper residue in Wee1 dictates its sensitivity to the 2 compounds. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers.     

PLK-1 asymmetry contributes to asynchronous cell division of C. elegans embryos

Acquisition of lineage-specific cell cycle duration is an important feature of metazoan development. In Caenorhabditis elegans, differences in cell cycle duration are already apparent in two-cell stage embryos, when the larger anterior blastomere AB divides before the smaller posterior blastomere P1. This time difference is under the control of anterior-posterior (A-P) polarity cues set by the PAR proteins. The mechanisms by which these cues regulate the cell cycle machinery differentially in AB and P1 are incompletely understood. Previous work established that retardation of P1 cell division is due in part to preferential activation of an ATL-1/CHK-1 dependent checkpoint in P1, but how the remaining time difference is controlled is not known. Here, we establish that differential timing relies also on a mechanism that promotes mitosis onset preferentially in AB. The polo-like kinase PLK-1, a positive regulator of mitotic entry, is distributed in an asymmetric manner in two-cell stage embryos, with more protein present in AB than in P1. We find that PLK-1 asymmetry is regulated by A-P polarity cues through preferential protein retention in the embryo anterior. Importantly, mild inactivation of plk-1 by RNAi delays entry into mitosis in P1, but not in AB, in a manner that is independent of ATL-1/CHK-1. Together, our findings support a model in which differential timing of mitotic entry in C. elegans embryos relies on two complementary mechanisms: ATL-1/CHK-1-dependent preferential retardation in P1 and PLK-1-dependent preferential promotion in AB, which together couple polarity cues and cell cycle progression during early development.     

Biochemical and structural characterization of a novel class of inhibitors of the type 1 insulin-like growth factor and insulin receptor kinases

The type 1 insulin-like growth factor receptor (IGF-1R) is often overexpressed on tumor cells and is believed to play an important role in anchorage-independent proliferation. Additionally, cell culture studies have indicated that IGF-1R confers increased resistance to apoptosis caused by radiation or chemotherapeutic agents. Thus, inhibitors of the intracellular kinase domain of this receptor may have utility for the clinical treatment of cancer. As part of an effort to develop clinically useful inhibitors of IGF-1R kinase, a novel class of pyrrole-5-carboxaldehyde compounds was investigated. The compounds exhibited selectivity against the closely related insulin receptor kinase intrinsically and in cell-based assays. The inhibitors formed a reversible, covalent adduct at the kinase active site, and treatment of such adducts with sodium borohydride irreversibly inactivated the enzyme. Analysis of a tryptic digest of a covalently modified IGF-1R kinase fragment revealed that the active site Lys1003 had been reductively alkylated with the aldehyde inhibitor. Reductive alkylation of the insulin receptor kinase with one of these inhibitors led to a similarly inactivated enzyme which was examined by X-ray crystallography. The crystal structure confirmed the modification of the active site lysine side chain and revealed details of the key interactions between the inhibitor and enzyme.     

Highly potent macrocyclic BACE-1 inhibitors incorporating a hydroxyethylamine core: design, synthesis and X-ray crystal structures of enzyme inhibitor complexes

A series of P1-P3 linked macrocyclic BACE-1 inhibitors containing a hydroxyethylamine (HEA) isostere scaffold has been synthesized. All inhibitors comprise a toluene or N-phenylmethanesulfonamide P2 moiety. Excellent BACE-1 potencies, both in enzymatic and cell-based assays, were observed in this series of target compounds, with the best candidates displaying cell-based IC(50) values in the low nanomolar range. As an attempt to improve potency, a phenyl substituent aiming at the S3 subpocket was introduced in the macrocyclic ring. X-ray analyzes were performed on selected compounds, and enzyme-inhibitor interactions are discussed.     

Development of a Highly Protective Combination Monoclonal Antibody Therapy against Chikungunya Virus

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes global epidemics of a debilitating polyarthritis in humans. As there is a pressing need for the development of therapeutic agents, we screened 230 new mouse anti-CHIKV monoclonal antibodies (MAbs) for their ability to inhibit infection of all three CHIKV genotypes. Four of 36 neutralizing MAbs (CHK-102, CHK-152, CHK-166, and CHK-263) provided complete protection against lethality as prophylaxis in highly susceptible immunocompromised mice lacking the type I IFN receptor (Ifnar^−/−) and mapped to distinct epitopes on the E1 and E2 structural proteins. CHK-152, the most protective MAb, was humanized, shown to block viral fusion, and require Fc effector function for optimal activity in vivo. In post-exposure therapeutic trials, administration of a single dose of a combination of two neutralizing MAbs (CHK-102+CHK-152 or CHK-166+CHK-152) limited the development of resistance and protected immunocompromised mice against disease when given 24 to 36 hours before CHIKV-induced death. Selected pairs of highly neutralizing MAbs may be a promising treatment option for CHIKV in humans.     

The discovery of novel benzofuran-2-carboxylic acids as potent Pim-1 inhibitors

Novel benzofuran-2-carboxylic acids, exemplified by 29, 38 and 39, have been discovered as potent Pim-1 inhibitors using fragment based screening followed by X-ray structure guided medicinal chemistry optimization. The compounds demonstrate potent inhibition against Pim-1 and Pim-2 in enzyme assays. Compound 29 has been tested in the Ambit 442 kinase panel and demonstrates good selectivity for the Pim kinase family. X-ray structures of the inhibitor/Pim-1 binding complex reveal important salt-bridge and hydrogen bond interactions mediated by the compound’s carboxylic acid and amino groups.     

Design, synthesis, and biological activity of a novel series of 2,5-disubstituted furans/pyrroles as HIV-1 fusion inhibitors targeting gp41

Based on molecular docking analysis of earlier results, we designed a series of 2,5-disubstituted furans/pyrroles (5a-h) as HIV-1 entry inhibitors. Compounds were synthesized by Suzuki-Miyaura cross coupling, followed by a Knoevenagel condensation or Wittig reaction. Four of these compounds were found to be effective in inhibiting HIV-1 infection, with the best compounds being 5f and 5h, which exhibited significant inhibition on HIV-1(IIIB) infection at micromolar levels with low cytotoxicity. These compounds are also effective in blocking HIV-1 mediated cell-cell fusion and the gp41 six-helix bundle formation, suggesting that they are also HIV-1 fusion inhibitors targeting gp41 and have potential to be developed as a new class of anti-HIV-1 agents.     

2-Aminothiadiazole inhibitors of AKT1 as potential cancer therapeutics

A series of 2-aminothiadiazole of inhibitors of AKT1 is described. SAR relationships are discussed, along with selectivity for protein kinase A (PKA) and cyclin-dependent kinase 2 (CDK2). Moderate selectivity observed in several compounds for AKT1 versus PKA is rationalized by X-ray crystallographic analysis. Key compounds showed activity in cellular assays measuring phosphorylation of two AKT substrates, PRAS40 and FKHRL1. Compound 30 was advanced to a mouse liver PD assay, where it showed dose-dependent inhibition of AKT activity, as measured by the inhibition of phospho-PRAS40.     

Tricyclic azepine derivatives: pyrimido[4,5-b]-1,4-benzoxazepines as a novel class of epidermal growth factor receptor kinase inhibitors

A novel class of pyrimido[4,5-b]-1,4-benzoxazepines is described as inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase. Two compounds display potent EGFR inhibitory activity of less than 1 microM in cellular phosphorylation assays (IC(50) 0.47-0.69 microM) and are highly selective against a small kinase panel. Such compounds demonstrate anti-EGFR activity within a class that is different from any known EGFR inhibitor scaffolds. They also provide a basis for the design of kinase inhibitors with the desired selectivity profile.