Novel Efficient Multistage Lead Optimization Pipeline Experimentally Validated for DYRK1B Selective Inhibitors.

In addition to general challenges in drug discovery such as the identification of lead compounds in time- and cost-effective ways, specific challenges also exist. Particularly, it is necessary to develop pharmacological inhibitors that effectively discriminate between closely related molecular targets. DYRK1B kinase is considered a valuable target for cancer-specific mono- or combination chemotherapy; however, the inhibition of its closely related DYRK1A kinase is not beneficial.

A Cell Double-Barcoding System for Quantitative Evaluation of Primary Tumors and Metastasis in Animals That Uncovers Clonal-Specific Anti-Cancer Drug Effects.

Imaging in monitoring metastasis in mouse models has low sensitivity and is not quantitative. Cell DNA barcoding, demonstrating high sensitivity and resolution, allows monitoring effects of drugs on the number of tumor and metastatic clones. However, this technology is not suitable for comparison of sizes of metastatic clones in different animals, for example, drug treated and untreated, due to high biological and technical variability upon tumor and metastatic growth and isolation of barcodes from tissue DNA.

DYRK1A regulates B cell acute lymphoblastic leukemia through phosphorylation of FOXO1 and STAT3.

DYRK1A is a serine/threonine kinase encoded on human chromosome 21 (HSA21) that has been implicated in several pathologies of Down syndrome (DS), including cognitive deficits and Alzheimer's disease. Although children with DS are predisposed to developing leukemia, especially B cell acute lymphoblastic leukemia (B-ALL), the HSA21 genes that contribute to malignancies remain largely undefined. Here, we report that DYRK1A is overexpressed and required for B-ALL.

How to design potent and selective DYRK1B inhibitors? Molecular modeling study.

DYRK1B protein kinase is an emerging anticancer target due to its overexpression in a variety of cancers and its role in cancer chemoresistance through maintaining cancer cells in the G (quiescent) state. Consequently, there is a growing interest in the development of potent and selective DYRK1B inhibitors for anticancer therapy. One of the major off-targets is another protein kinase, GSK3β, which phosphorylates an important regulator of cell cycle progression on the same residue as DYRK1B and is involved in multiple signaling pathways.

Pyrido[2,3-d]pyrimidines: discovery and preliminary SAR of a novel series of DYRK1B and DYRK1A inhibitors.

DYRK1B is a kinase over-expressed in certain cancer cells (including colon, ovarian, pancreatic, etc.). Recent publications have demonstrated inhibition of DYRK1B could be an attractive target for cancer therapy.

Inactivation of mirk/dyrk1b kinase targets quiescent pancreatic cancer cells.

A major problem in the treatment of cancer arises from quiescent cancer cells that are relatively insensitive to most chemotherapeutic drugs and radiation. Such residual cancer cells can cause tumor regrowth or recurrence when they reenter the cell cycle. Earlier studies showed that levels of the serine/theronine kinase Mirk/dyrk1B are elevated up to 10-fold in quiescent G(0) tumor cells.

Preclinical profile of a potent gamma-secretase inhibitor targeting notch signaling with in vivo efficacy and pharmacodynamic properties.

Notch signaling is an area of great interest in oncology. RO4929097 is a potent and selective inhibitor of gamma-secretase, producing inhibitory activity of Notch signaling in tumor cells. The RO4929097 IC50 in cell-free and cellular assays is in the low nanomolar range with >100-fold selectivity with respect to 75 other proteins of various types (receptors, ion channels, and enzymes).

Selective compounds define Hsp90 as a major inhibitor of apoptosis in small-cell lung cancer.

The heat shock protein 90 (Hsp90) has a critical role in malignant transformation. Whereas its ability to maintain the functional conformations of mutant and aberrant oncoproteins is established, a transformation-specific regulation of the antiapoptotic phenotype by Hsp90 is poorly understood. By using selective compounds, we have discovered that small-cell lung carcinoma is a distinctive cellular system in which apoptosis is mainly regulated by Hsp90.

Radiation dose-rate effects, endogenous DNA damage, and signaling resonance.

We previously concluded, from our analysis of the published data of other investigators, that the yield of germ-line and somatic mutations after exposure to ionizing radiation is parabolically related to the logarithm of the dose-rate at which a given dose is administered. Here we show that other data reveal a similarly parabolic relationship for other ionizing radiation-associated phenomena, namely, genetic recombination, chromosomal translocation, cell inactivation and lethality, and human leukemogenesis. Furthermore, the minima for all effects fall in a relatively narrow range of the dose-rate logarithms.

Hsp90: the vulnerable chaperone.

The molecular chaperone Hsp90 has emerged as an important target in cancer treatment because of its roles in maintaining transformation and regulating the function of proteins involved in apoptotic, survival and growth pathways. Many Hsp90 inhibitors function by binding to the N-terminal ATP pocket, but the chaperone has many other vulnerable points. Agents that interact with its C-terminus or modify its post-translational status represent additional ways of interfering with chaperone activity.

Targeting wide-range oncogenic transformation via PU24FCl, a specific inhibitor of tumor Hsp90.

Agents that inhibit Hsp90 function hold significant promise in cancer therapy. Here we present PU24FCl, a representative of the first class of designed Hsp90 inhibitors. By specifically and potently inhibiting tumor Hsp90, PU24FCl exhibits wide-ranging anti-cancer activities that occur at similar doses in all tested tumor types.

Endogenous DNA double-strand breaks: production, fidelity of repair, and induction of cancer.

This article extends our previous quantitative analysis of the relationship between the dynamics of the primary structure of DNA and mutagenesis associated with single-strand lesions to an analysis of the production and processing of endogenous double-strand breaks (EDSBs) and to their implications for oncogenesis. We estimate that in normal human cells approximately 1% of single-strand lesions are converted to approximately 50 EDSBs per cell per cell cycle. This number is similar to that for EDSBs produced by 1.

Microtiter cell-based assay for detection of agents that alter cellular levels of Her2 and EGFR.

Overexpression of the transmembrane tyrosine kinases Her2 and EGFR is associated with aggressive malignancies, and several therapeutic strategies targeting the two receptors are now in various stages of clinical development. Most of the known agents block the activation or inhibit the activity of the kinases; however, a more significant therapeutic outcome may result from degrading these oncoproteins. Here, we report the development of a microtiter cell-based assay that sensitively detects cellular levels of Her2 and EGFR.

Antisense RNA down-regulation of bcl-xL Expression in prostate cancer cells leads to diminished rates of cellular proliferation and resistance to cytotoxic chemotherapeutic agents.

bcl-xL is a M(r) 26,000 bcl-2 homologue that is highly expressed in prostate cancer cells. In previous studies, the down-regulation of its expression by antisense oligonucleotides led to resistance. In this work, the 445-bp 5' terminus of the bcl-xL cDNA was cloned in the antisense orientation and stably transfected into DU145 and LNCaP prostate cancer cells.

Evidence for higher-order structure formation by the c-myb 18-mer phosphorothioate antisense (codons 2-7) oligodeoxynucleotide: potential relationship to antisense c-myb inhibition.

We have demonstrated the formation of higher-order structures (presumably tetraplexes) by an 18-mer phosphorothioate antisense c-myb oligodeoxyribonucleotide that has been shown to have activity in the treatment of leukemia xenograft models. Although not observable by conventionally employed techniques, such as PAGE and dimethyl sulfate (DMS) protection, the formation of such higher-order structures by this oligonucleotide was revealed by several techniques. These included capillary gel electrophoresis (CGE), which demonstrated the presence of molecules with greatly increased retention time compared with the monomer; magnetic circular dichroism spectroscopy, which demonstrated a band at 290 nm, a characteristic of antiparallel tetraplexes; and fluorescence energy transfer measurements.

Cellular delivery of antisense oligonucleotides.

Antisense oligonucleotides can be successfully employed to inhibit specifically gene expression. However, many oligonucleotide classes are polyanions and cannot passively transit the cell membrane. Thus, the use of naked oligonucleotides for antisense purposes poses some rather stringent challenges, and it is not a trivial task to appropriately interpret the data derived from experiments in which they have been used.

Inverse radiation dose-rate effects on somatic and germ-line mutations and DNA damage rates.

The mutagenic effect of low linear energy transfer ionizing radiation is reduced for a given dose as the dose rate (DR) is reduced to a low level, a phenomenon known as the direct DR effect. Our reanalysis of published data shows that for both somatic and germ-line mutations there is an opposite, inverse DR effect, with reduction from low to very low DR, the overall dependence of induced mutations being parabolically related to DR, with a minimum in the range of 0.1 to 1.

Cationic porphyrins: novel delivery vehicles for antisense oligodeoxynucleotides.

Cationic porphyrins form stable complexes with oligodeoxynucleotides. To evaluate delivery, we used a 20mer phosphorothioate oligomer (Isis 3521) targeted to the 3'-untranslated region of the PKC-alpha mRNA, and complexed it with porphyrin. The expression of PKC-alpha protein and mRNA in T24 bladder carcinoma cells was reduced by approximately 80 +/- 10% at a concentration of oligomer of 3 microM, and 9 microM porphyrin.

Induction and repair of DNA double-strand breaks in the same dose range as the shoulder of the survival curve.

We have used pulsed-field gel electrophoresis (PFGE) to test two hypotheses that have been proposed to explain the survival curves with shoulders which are characteristic of low-LET ionizing radiation: (1) Neutral elution studies of the induction of double-strand breaks (DSBs) have suggested that ionizing radiation might induce DSBs in a nonlinear fashion at low doses. (2) Based on analogies to enzyme kinetics, DSB repair might be saturating in the shoulder region. We quantified DSB induction and survival resulting from doses between 0 and 5 Gy spanning the shoulder region of the survival curve.

Separation of DNA fragments by capillary electrophoresis using replaceable linear polyacrylamide matrices.

The use of low percent (1.5-6% T) replaceable linear polyacrylamide (LPA) network matrices for rapid separation of double-stranded DNA fragments was explored. Separations of fragments ranging from 20 to 23,000 base pairs were readily achieved.

Induction and repair of DNA double-strand breaks.

Induction and repair of DNA double-strand breaks (DSBs) was measured using a pulsed-field gel electrophoresis system. A cell line of methotrexate-resistant EMT-6 cells that contain numerous double-minutes (DMs) 3 million base pairs in size was employed. The electrophoretic mobility of these DMs depends on whether they have zero, one, or more than one DSB.

[The carcinogenic effect of 5-bromodeoxyuridine and its amplification by x-rays in rats].

5-bromodeoxyuridine (BUdR) injected to rats in the neonatal period is shown to produce a pronounced carcinogenic effect which brings about the appearance of various benign and malignant tumours. Exposure of females to X-ray total-body irradiation (1.5 Gy) was followed by intensification of the carcinogenic effect of BUdR.

[Effects of beta-carotene on genotoxic lesions in the liver under formation and action of carcinogenic N-nitrosodimethylamine].

beta-carotene pretreatment of rats decreased the methylation and formation of single-strand breaks in DNA and also decreased activity of alanine-aminotransferase, sorbitol dehydrogenase, gamma-glutamyl transpeptidase activities, caused by action of N-nitrosodimethylamine or synthesis of this carcinogen from precursors.