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. Existing inhibitors target both kinases with essentially the same efficiency, and the unavailability of the DYRK1B crystal structure makes the discovery of DYRK1B-specific inhibitors even more challenging. Here, we propose a novel multi-stage compound discovery pipeline aimed at in silico identification of both potent and selective small molecules from a large set of initial candidates. The method uses structure-based docking and ligand-based quantitative structure-activity relationship modeling. This approach allowed us to identify lead and runner-up small-molecule compounds targeting DYRK1B with high efficiency and specificity.

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. However, both numbers of clones and their sizes are critical parameters for analysis of drug effects. Here we developed a modification of the barcoding approach for monitoring drug effects on tumors and metastasis that is quantitative, highly sensitive and highly reproducible. This novel cell double-barcoding system allows simultaneously following the fate of two or more cell variants or cell lines in xenograft models in vivo, and also following the fates of individual clones within each of these populations. This system allows comparing effects of drugs on different cell populations and thus normalizing drug effects by drug-resistant lines, which corrects for both biological and technical variabilities and significantly increases the reproducibility of results. Using this barcoding system, we uncovered that effects of a novel DYRK1B kinase inhibitor FX9847 on primary tumors and metastasis is clone-dependent, while a distinct drug osimertinib demonstrated clone-independent effects on cancer cell populations. Overall, a cell double-barcoding approach can significantly enrich our understanding of drug effects in basic research and preclinical studies.

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. Genetic and pharmacologic inhibition of DYRK1A decreased leukemic cell expansion and suppressed B-ALL development in vitro and in vivo. Furthermore, we found that FOXO1 and STAT3, transcription factors that are indispensable for B cell development, are critical substrates of DYRK1A. Loss of DYRK1A-mediated FOXO1 and STAT3 signaling disrupted DNA damage and ROS regulation, respectively, leading to preferential cell death in leukemic B cells. Thus, we reveal a DYRK1A/FOXO1/STAT3 axis that facilitates the development and maintenance of 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 G0 (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, GSK3ß, which phosphorylates an important regulator of cell cycle progression on the same residue as DYRK1B and is involved in multiple signaling pathways. In the current work, we performed a detailed comparative structural analysis of DYRK1B and GSK3ß ATP-binding sites and identified key regions responsible for selectivity. As the crystal structure of DYRK1B has never been reported, we built and optimized a homology model by comparative modeling and metadynamics simulations. Calculation of interaction energies between docked ligands in the ATP-binding sites of both kinases allowed us to pinpoint key residues responsible for potency and selectivity. Specifically, the role of the gatekeeper residues in DYRK1B and GSK3ß is discussed in detail, and two other residues are identified as key to selectivity of DYRK1B inhibition versus GSK3ß. The analysis presented in this work was used to support the design of potent and selective azaindole-quinoline-based DYRK1B inhibitors and can facilitate development of more selective inhibitors for DYRK kinases.

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. Mirk uses several mechanisms to block cell cycling, and Mirk increases expression of antioxidant genes that decrease reactive oxygen species (ROS) levels and increase quiescent cell viability. We now show that a novel small molecule Mirk kinase inhibitor blocked tumor cells from undergoing reversible arrest in a quiescent G(0) state and enabled some cells to exit quiescence. The inhibitor increased cycling in Panc1, AsPc1, and SW620 cells that expressed Mirk, but not in HCT116 cells that did not. Mirk kinase inhibition elevated ROS levels and DNA damage detected by increased phosphorylation of the histone protein H2AX and by S-phase checkpoints. The Mirk kinase inhibitor increased cleavage of the apoptotic proteins PARP and caspase 3, and increased tumor cell kill several-fold by gemcitabine and cisplatin. A phenocopy of these effects occurred following Mirk depletion, showing drug specificity. In previous studies Mirk knockout or depletion had no detectable effect on normal tissue, suggesting that the Mirk kinase inhibitor could have a selective effect on cancer cells expressing elevated levels of Mirk kinase.

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). RO4929097 inhibits Notch processing in tumor cells as measured by the reduction of intracellular Notch expression by Western blot. This leads to reduced expression of the Notch transcriptional target gene Hes1. RO4929097 does not block tumor cell proliferation or induce apoptosis but instead produces a less transformed, flattened, slower-growing phenotype. RO4929097 is active following oral dosing. Antitumor activity was shown in 7 of 8 xenografts tested on an intermittent or daily schedule in the absence of body weight loss or Notch-related toxicities. Importantly, efficacy is maintained after dosing is terminated. Angiogenesis reverse transcription-PCR array data show reduced expression of several key angiogenic genes. In addition, comparative microarray analysis suggests tumor cell differentiation as an additional mode of action. These preclinical results support evaluation of RO4929097 in clinical studies using an intermittent dosing schedule. A multicenter phase I dose escalation study in oncology is under way.

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. Unlike the well-characterized antiapoptotic chaperone Hsp70, Hsp90 is not a general inhibitor of apoptosis, but it assumes this role in systems such as small-cell lung carcinoma, in which apoptosis is uniquely dependent on and effected through the intrinsic pathway, without involvement of caspase elements upstream of mitochondria or alternate pathways that are not apoptosome-channeled. These results provide important evidence for a transformation-specific interplay between chaperones in regulating apoptosis in malignant cells.

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. This review will discuss several emerging classes of Hsp90 inhibitors and their modes of action.

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. Normal cells are 10- to 50-fold more resistant to these effects. Its Hsp90 inhibition results in multiple anti-tumor-specific effects, such as degradation of Hsp90-client proteins involved in cell growth, survival, and specific transformation, inhibition of cancer cell growth, delay of cell cycle progression, induction of morphological and functional changes, and apoptosis. In concordance with its higher affinity for tumor Hsp90, in vivo PU24FCl accumulates in tumors while being rapidly cleared from normal tissue. Concentrations achieved in vivo in tumors lead to single-agent anti-tumor activity at non-toxic doses.

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. The assay is useful in identifying small molecules that alter cellular levels of these kinases and in quantifying their effect. The method gives results comparable to Western blot, but it is faster, less labor intensive, and amenable to high throughput.

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. In the DU145 (and to a lesser extent the LNCaP) transfectants, phenotypic changes (versus mock-transfected cells) included an increase in doubling time (from 36 to 175 h) in the clone in which bcl-xL protein expression was 25% of control. The transfectants did not demonstrate characteristic apoptotic changes, as demonstrated by 4',6-diamidino-2-phenylindole staining, lack of either DNA laddering, caspase-3 activation, or poly(ADP)ribose and lamin cleavage, and the absence of a significant sub-G(0) population. Cell cycle analysis demonstrated an increase in a tetraploid population (from 28% to 66%), as well as the appearance of a hypertetraploid population. Levels of cIAP-1 protein were almost undetectable in the mock cells but increased at least 25-fold in the DU145 transfectants. The down-regulation of bcl-xL in both DU145 (and to a much lesser extent in LNCaP) cells led to their resistance to cytotoxic agents, including docetaxel, mitoxantrone, etoposide, vinblastine, and carboplatin. Reversion of bcl-xL expression in stable DU145 transfectants to nearly the levels found in the mock-transfected cells was accomplished by retroviral infection of the cells with a bcl-xL sense cDNA under control of a prolific promoter. This led to a dramatic increase in the growth rate and in BrdUrd incorporation, as well as a sharp decrease in the expression of cIAP-1 protein. Overall, these findings highlight the adaptability of prostate cancer cells to loss of bcl-xL and suggest that in addition to its prosurvival role, bcl-xL protein may also be involved in the regulation of the rate of cellular proliferation.