Supplementary Tables 1-3 from Distinct Concentration-Dependent Effects of the Polo-like Kinase 1–Specific Inhibitor GSK461364A, Including Differential Effect on Apoptosis

Additions to Supplement Table 3 from Distinct Concentration-Dependent Effects of the Polo-like Kinase 1–Specific Inhibitor GSK461364A, Including Differential Effect on Apoptosis

Abstract Introduction: African Americans have higher incidence of, and mortality from, prostate cancer (PCa) compared to other racial/ethnic groups. Frequency of polymorphisms in genes involved in vitamin D metabolism, transport, and activity differ by race/ethnicity. Examining the association between polymorphisms in vitamin D-related genes and PCa aggressiveness may explain differing susceptibility to aggressive PCa among individuals and across different racial/ethnic groups. Methods: TagSNPs (n = 315) in 13 genes (VDR, GC, CYP24A1, CYP27A1, CYP27B1, CYP2R1, CYP3A4, DHCR7, CASR, NADSYN1, RXRA, RXRB, RXRG) were genotyped using Illumina GoldenGate or Sequenom assays in 524 African-American and 657 European-American men with newly-diagnosed PCa from PCaP. DNA extracted from blood samples collected at enrollment was stored at -80C prior to analyses. Research subjects were classified as high aggressive if Gleason sum ≥8, or Gleason score (4+3), or PSA >20 ng/ml, or Gleason score (3+4) AND clinical stage = T3-T4. The comparison group (low aggressive) included research subjects with Gleason sum <7 AND Stage T1-T2 AND PSA < 9 ng/ml. Odds ratios (OR) and 95% confidence intervals (95%CI) were calculated for high aggressive PCa for each SNP using logistic regression with adjustment for age and proportion of African ancestry. Associations were considered statistically significant at p<0.05. A polygenic score based on a previous study of SNP predictors of serum 25-hydroxy-vitamin D levels was calculated utilizing SNPs in the GC, CYP24A1, CYP2R1, and NADSYN1 genes. Results: Among African Americans, 21 SNPs were associated with PCa aggressiveness. The variant allele was associated negatively or positively with high aggressive PCa in eleven and ten SNPs, respectively. For example, two SNPs in the vitamin D binding protein gene known as GC, were inversely associated (rs222054: OR, 0.55, 95%CI, 0.38-0.80; and rs16847028: OR, 0.61, 95%CI, 0.39-0.94). Among European Americans, the variant allele was inversely associated with high aggressive PCa for four SNPs in three genes (CASRrs3863977; CYP24A1rs4809960; RXRArs1007971; and RXRArs3118526), and positively associated with high aggressive PCa for three SNPs in the CYP27B1 gene (rs703842, rs4646536, and rs10877013). There was no association between higher number of ‘low vitamin D’ alleles in the four SNPs that comprised the polygenic score and PCa aggressiveness for either race. Conclusions: Polymorphisms in genes involved in vitamin D etabolism and activity, the vitamin D binding protein and calcium sensing receptor were associated with PCa aggressiveness, and there was no overlap in SNPs between race groups. Our ongoing work will examine interaction between polymorphisms of vitamin D-related genes and vitamin D metabolite levels on PCa aggressiveness. Citation Format: Susan E. Steck, Anna Woloszynska-Read, Samuel Antwi, Hongmei Zhang, Lenore Arab, Elizabeth T.H. Fontham, James Mohler, L. Joseph Su, Feifei Xiao, Gary Smith, Donald Trump, Candace Johnson, Jeannette Bensen. SNPs in vitamin D-related genes are associated with prostate cancer aggressiveness in the North Carolina-Louisiana Prostate Cancer Project (PCaP). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 806.

A boronic acid moiety was found to be a critical pharmacophore for enhanced in vitro potency against wild-type hepatitis C replicons and known clinical polymorphic and resistant HCV mutant replicons. The synthesis, optimization, and structure-activity relationships associated with inhibition of HCV replication in a subgenomic replication system for a series of non-nucleoside boron-containing HCV RNA-dependent RNA polymerase (NS5B) inhibitors are described. A summary of the discovery of 3 (GSK5852), a molecule which entered clinical trials in subjects infected with HCV in 2011, is included.

The macrocyclic urea 2, a byproduct in the synthesis of benzoxaborole 1, was identified to be a novel and potent HCV protease inhibitor. We further explored this motif by synthesizing additional urea-based inhibitors and by characterizing them in replicase HCV protease-resistant mutants assay. Several compounds, exemplified by 12, were found to be more potent in HCV replicon assays than leading second generation inhibitors such as danoprevir and TMC-435350. Additionally, following oral administration, inhibitor 12 was found in rat liver in significantly higher concentrations than those reported for both danoprevir and TMC-435350, suggesting that inhibitor 12 has the combination of anti-HCV and pharmacokinetic properties that warrants further development of this series.

We have synthesized and evaluated a series of novel HCV NS3 protease inhibitors with various P4 capping groups, which include urea, carbamate, methoxy-carboxamide, cyclic carbamate and amide, pyruvic amide, oxamate, oxalamide and cyanoguanidine. Most of these compounds are remarkably potent, exhibiting single-digit to sub-nanomolar activity in the enzyme assay and cell-based replicon assay. Selected compounds were also evaluated in the protease-inhibitor-resistant mutant transient replicon assay, and they were found to show quite different potency profiles against a panel of HCV protease-inhibitor-resistant mutants.

The “Kinome” describes the protein kinase component of the human genome, exhaustively compiled in 2002 by Manning et al.1 A search of available human sequence sources using a hidden Markov model identified 478 ePK (eukaryotic protein kinase) genes, 491 ePK domains, and 40 “atypical” protein kinases....

We have synthesized and evaluated a new series of acyclic P4-benzoxaborole-based HCV NS3 protease inhibitors. Structure–activity relationships were investigated, leading to the identification of compounds 5g and 17 with low nanomolar potency in the enzymatic and cell-based replicon assay. The linker-truncated compound 5j was found to exhibit improved absorption and oral bioavailability in rats, suggesting that further reduction of molecular weight and polar surface area could result in improved drug-like properties of this novel series.

Interactions between cancer cells and fibroblasts are crucial in cancer progression. We have previously shown that the aspartic protease cathepsin D (cath-D), a marker of poor prognosis in breast cancer that is overexpressed and highly secreted by breast cancer cells, triggers mouse embryonic fibroblast outgrowth via a paracrine loop. Here, we show the requirement of secreted cath-D for human mammary fibroblast outgrowth using a three-dimensional co-culture assay with breast cancer cells that do or do not secrete pro-cath-D. Interestingly, proteolytically-inactive pro-cath-D remains mitogenic, indicating a mechanism involving protein-protein interaction. We identify the low-density lipoprotein (LDL) receptor-related protein-1, LRP1, as a novel binding partner for pro-cath-D in fibroblasts. Pro-cath-D binds to residues 349-394 of the β chain of LRP1, and is the first ligand of the extracellular domain of LRP1β to be identified. We show that pro-cath-D interacts with LRP1β in cellulo. Interaction occurs at the cell surface, and overexpressed LRP1β directs pro-cath-D to the lipid rafts. Our results reveal that the ability of secreted pro-cath-D to promote human mammary fibroblast outgrowth depends on LRP1 expression, suggesting that pro-cath-D-LRP1β interaction plays a functional role in the outgrowth of fibroblasts. Overall, our findings strongly suggest that pro-cath-D secreted by epithelial cancer cells promotes fibroblast outgrowth in a paracrine LRP1-dependent manner in the breast tumor microenvironment.

We disclose here a series of P4-benzoxaborole-substituted macrocyclic HCV protease inhibitors. These inhibitors are potent against HCV NS3 protease, their anti-HCV replicon potencies are largely impacted by substitutions on benzoxaborole ring system and P2∗ groups. P2∗ 2-thiazole-isoquinoline provides best replicon potency. The in vitro SAR studies and in vivo PK evaluations of selected compounds are described herein.

HCV NS3/4A serine protease is essential for the replication of the HCV virus and has been a clinically validated target. A series of HCV NS3/4A protease inhibitors containing a novel acylsulfamoyl benzoxaborole moiety at the P1′ region was synthesized and evaluated. The resulting P1–P3 and P2–P4 macrocyclic inhibitors exhibited sub-nanomolar potency in the enzymatic assay and low nanomolar activity in the cell-based replicon assay. The in vivo PK evaluations of selected compounds are also described.

Kinases are important therapeutic targets. Potent in vitro inhibitors are readily produced, but these compounds are not always potent in cells. Because cell potency is crucial for efficacy, cell-based assays are important for successful inhibitor development. In this review kinase cell assays are divided into four types. Strengths and weaknesses of each and the value of combining types are discussed. With the current growth of kinase-based therapeutics, review of this field is important and timely.

We have designed and synthesized a novel series of α-amino cyclic boronates and incorporated them successfully in several acyclic templates at the P1 position. These compounds are inhibitors of the HCV NS3 serine protease, and structural studies show that they inhibit the NS3 protease by trapping the Ser-139 hydroxyl group in the active site. Synthetic methodologies and SARs of this series of compounds are described.

A novel series of P2–P4 macrocyclic HCV NS3/4A protease inhibitors with α-amino cyclic boronates as warheads at the P1 site was designed and synthesized. When compared to their linear analogs, these macrocyclic inhibitors exhibited a remarkable improvement in cell-based replicon activities, with compounds 9a and 9e reaching sub-micromolar potency in replicon assay. The SAR around α-amino cyclic boronates clearly established the influence of ring size, chirality and of the substitution pattern. Furthermore, X-ray structure of the co-crystal of inhibitor 9a and NS3 protease revealed that Ser-139 in the enzyme active site traps boron in the warhead region of 9a, thus establishing its mode of action.

Insulin-like growth factor 1 receptor (IGF-1R) is an attractive target for anti-cancer therapy due to its anti-apoptotic effect on tumor cells, but inhibition of insulin receptor (IR) may have undesired metabolic consequences. The primary sequences of the ATP substrate-binding sites of these receptors are identical and the crystal structures of the activated kinase domains are correspondingly similar. Thus, most small-molecule inhibitors described to date are equally potent against the activated kinase domains of IGF-1R and IR. In contrast, the non-phosphorylated kinase domains of these receptors have several structural features that may accommodate differences in binding affinity for kinase inhibitors. We used a cell-based assay measuring IGF-1R autophosphorylation as an inhibitor screen, and identified a potent purine derivative that is selective compared to IR. Surprisingly, the compound is a weak inhibitor of the activated IGF-1R tyrosine kinase domain. Biochemical and structural studies are presented that indicate the compound preferentially binds to the ATP site of non-phosphorylated IGF-1R compared to phosphorylated IGF-1R. The potential selectivity and potency advantages of this binding mode are discussed.