Gustavo R. Rosania

Myoseverin, a microtubule-binding molecule with novel cellular effects

A new microtubule-binding molecule, myoseverin, was identified from a library of 2,6,9-trisubstituted purines in a morphological differentiation screen. Myoseverin induces the reversible fission of multinucleated myotubes into mononucleated fragments. Myotube fission promotes DNA synthesis and cell proliferation after removal of the compound and transfer of the cells to fresh growth medium. Transcriptional profiling and biochemical analysis indicate that myoseverin alone does not reverse the biochemical differentiation process. Instead, myoseverin affects the expression of a variety of growth factor, immunomodulatory, extracellular matrix-remodeling, and stress response genes, consistent with the activation of pathways involved in wound healing and tissue regeneration.

Data mining the NCI60 to predict generalized cytotoxicity

Elimination of cytotoxic compounds in the early and later stages of drug discovery can help reduce the costs of research and development. Through the application of principal components analysis (PCA), we were able to data mine and prove that approximately 89% of the total log GI 50 variance is due to the nonspecific cytotoxic nature of substances. Furthermore, PCA led to the identification of groups of structurally unrelated substances showing very specific toxicity profiles, such as a set of 45 substances toxic only to the Leukemia_SR cancer cell line. In an effort to predict nonspecific cytotoxicity on the basis of the mean log GI 50, we created a decision tree using MACCS keys that can correctly classify over 83% of the substances as cytotoxic/noncytotoxic in silico, on the basis of the cutoff of mean log GI 50 = -5.0. Finally, we have established a linear model using least-squares in which nine of the 59 available NCI60 cancer cell lines can be used to predict the mean log GI 50. The model has R (2) = 0.99 and a root-mean-square deviation between the observed and calculated mean log GI 50 (RMSE) = 0.09. Our predictive models can be applied to flag generally cytotoxic molecules in virtual and real chemical libraries, thus saving time and effort.

Targeting hyperproliferative disorders with cyclin dependent kinase inhibitors

Deregulated cell proliferation is associated with a variety of diseases including dysplasias and metaplasias of different tissues, psoriasis, atherosclerosis, restenosis after angioplastic surgery, and cancer [1-3]. Until recently, treatments for hyperproliferative disorders have revolved around chemical agents that kill actively dividing cells [4]. These agents include drugs that block DNA synthesis, such as DNA alkylators, topoisomerase, polymerase and nucleotide biosynthesis inhibitors. In addition, microtubule depolymerising and stabilising compounds have had widespread use as agents that interfere with spindle function essential for chromosome segregation and the process of cell division. Over the last seven years, understanding the mechanism regulating entry and exit from the cell cycle has led to the identification of signal transduction, transcriptional and cell cycle regulatory pathways orchestrating cell proliferation. Abnormalities in these regulatory mechanisms have been implicated in a variety of hyperproliferative disorders, prompting the development of cyclindependent kinase (CDK) inhibitors as therapeutic agents [5-9]. Unlike drugs that are meant to kill actively dividing cells, CDK inhibitors can be used to re-establish order in the biochemical mechanisms governing a cell’s decision to grow and divide - a departure from the traditional chemotherapeutic approach to the treatment of hyperproliferative disorders.

MICROTUBULES CAN MODULATE PSEUDOPOD ACTIVITY FROM A DISTANCE INSIDE MACROPHAGES

Microtubules are thought to influence cell shape as structural components of an integrated cytoskeletal matrix. Here we show that microtubules can affect the dynamics of macrophage pseudopodia without being integrated into their structure. Macrophages landing on glass surfaces spread within 15 min into flattened circular cells with radial symmetry, and the radial distribution of microtubules reflected this symmetry. Depolymerization of microtubules using nocodazole, colchicine, or vinblastine did not inhibit macrophage spreading or the early establishment of radial symmetry. Shortly after spreading, however, macrophages without microtubules gradually became asymmetric, assuming irregular, lobed profiles. The asymmetry resulted from exaggerated protrusion and retraction of pseudopodia, with net retraction overall. This loss of radial symmetry could be inhibited by treatment of initially spread cells with cytochalasin D, indicating that the change in cell shape was mediated by the actin cytoskeleton. Intact microtubules suppressed the exaggerated pseudopod movements, even when they were separated by a distance from the cell margin. In cells treated with taxol, microtubules remained clustered near the cell center after spreading, yet the dynamics of pseudopodia at the cell margin were reduced and cells maintained a circular profile. Similarly, in cells treated with low concentrations of nocodazole, a much reduced microtubule cytoskeleton nonetheless suppressed pseudopod dynamics. We propose that microtubules act to stabilize cell shape at a distance from the cell edge via a biochemical intermediate that affects the structure or function of the microfilament system.

Development of novel cell-permeable DNA sensitive dyes using combinatorial synthesis and cell-based screening.

A novel cell-permeable DNA fluorescence sensor was developed based on combinatorially-created styryl dyes and cell-based localization screening.

Chemoinformatic analysis of a supertargeted combinatorial library of styryl molecules.

Styryl dyes are fluorescent, lipophilic cations that have been used as specific labeling probes of mitochondria in living cells. For specific applications such as epifluorescence microscopy or flow cytometry, it is often desirable to synthesize fluorescent derivatives with optimized excitation, emission, and localization properties. Here, we present a chemoinformatic strategy suitable for multiparameter analysis of a combinatorial library of styryl molecules supertargeted to mitochondria. The strategy is based on a simple additive model relating the spectral and subcellular localization characteristics of styryl compounds to the two chemical building blocks that are used to synthesize the molecules. Using a cross-validation approach, the additive model predicts with a high degree of confidence the subcellular localization and spectral properties of the styryl product, from numerical scores that are independently associated with the individual building blocks of the molecule. The fit of the data indicates that more complex, nonadditive interactions between the two building blocks play a minor role in determining the molecules optical or biological properties. Moreover, the observed additive relationship allows mechanistic inferences to be made regarding the structure-property relationship observed for this particular class of molecules. It points to testable, mechanistic hypotheses about how chemical structure, fluorescence, and localization properties are related.

Functional expression and characterization of a sodium-dependent nucleoside transporter hCNT2 cloned from human duodenum☆

We have cloned and functionally expressed a sodium-dependent human nucleoside transporter, hCNT2, from a CNS cancer cell line U251. Our cDNA clone of hCNT2 had the same predicted amino acid sequence as the previously cloned hCNT2 transporter. Of the several cell lines studied, the best hCNT2 transport function was obtained when transiently expressed in U251 cells. Na(+)-dependent uptake of [3H]inosine in U251 cells transiently expressing hCNT2 was 50-fold greater than that in non-transfected cells, and uptake in Na(+)-containing medium was approximately 30-fold higher than that at Na(+)-free condition. The hCNT2 displayed saturable uptake of [3H]inosine with K(m) of 12.8 microM and V(max) of 6.66 pmol/mg protein/5 min. Uptake of [3H]inosine was significantly inhibited by the purine nucleoside drugs dideoxyinosine and cladribine, but not by acyclic nucleosides including acyclovir, ganciclovir, and their prodrugs valacyclovir and valganciclovir. This indicates that the closed ribose ring is important for binding of nucleoside drugs to hCNT2. Among several pyrimidine nucleosides, hCNT2 favorably interacted with the uridine analogue floxuridine. Interestingly, we found that benzimidazole analogues, including maribavir, 5,6-dichloro-2-bromo-1-beta-D-ribofuranosylbenzimidazole (BDCRB), and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), were strong inhibitors of inosine transport, even though they have a significantly different heterocycle structure compared to a typical purine ring. As measured by GeneChip arrays, mRNA expression of hCNT2 in human duodenum was 15-fold greater than that of hCNT1 or hENT2. Further, the rCNT2 expression in rat duodenum was 20-fold higher than rCNT1, rENT1 or rENT2. This suggests that hCNT2 (and rCNT2) may have a significant role in uptake of nucleoside drugs from the intestine and is a potential transporter target for the development of nucleoside and nucleoside-mimetic drugs.

A Rational Approach to Personalized Anticancer Therapy: Chemoinformatic Analysis Reveals Mechanistic Gene-Drug Associations

AbstractPurpose. To predict the response of cells to chemotherapeutic agents based on gene expression profiles, we performed a chemoinformatic study of a set of standard anticancer agents assayed for activity against a panel of 60 human tumor-derived cell lines from the Developmental Therapeutics Program (DTP) at the National Cancer Institute (NCI). Methods. Mechanistically-relevant gene:drug activity associations were identified in the scientific literature. The correlations between expression levels of drug target genes and the activity of the drugs against the NCIs 60 cell line panel were calculated across and within each tumor tissue type, using published drug activity and gene expression data. Results. Compared to other mechanistically-relevant gene-drug associations, that of triciribine phosphate (TCN-P) and adenosine kinase (ADK) was exceptionally strong—overall and within tumor tissue types—across the 60 cell lines profiled for chemosensitivity (1) and gene expression (2). Conclusion. The results suggest ADK expression may be useful for stratifying TCN-P-responsive vs. non-responsive tumors. Based on TCN-Ps mechanism of action and the observed TCN-P:ADK association, we contend that catalytic drug activation provides a rational, mechanistic basis for personalizing cancer treatment based on tumor-specific differences in the expression of drug-activating enzymes.

PROSPECTING FOR LIVE CELL BIOIMAGING PROBES WITH CHEMINFORMATIC ASSISTED IMAGE ARRAY (CAIA)

Cheminformatic assisted image array (CAIA) is a data mining and visualization tool linking chemical structures to microscope images of cells incubated with prospective bioimaging probes. In a CAIA, machine vision can be used to calculate the quantitative contribution of the chemical features of candidate probes to the apparent image features. In turn, image arrays can be constructed and sorted based on the features of molecules or images, so that the contribution of chemical features on visual features of the images can be readily discerned, across a large data set. By enabling visualization of complex multidimensional relationships between chemical structures and visual signals, CAIA can facilitate the search for new classes of fluorescent probes of cell structure and function.

Gene expression associations with the growth inhibitory effects of small molecules on live cells: Specificity of effects and uniformity of mechanisms

An alternator pulley includes a driving member driven and rotated via a belt from an output shaft of an engine. A driving member is disposed on an inner surface of the driving member and a one-way clutch is interposed between the driving and driven member. The one-way clutch includes rollers capable of rolling in a locked side direction along which a rotating power of the driving member is transmitted to the driven member or a free side direction along which the rotating powder is interrupted. Depending on a relative speed difference between the driving member and the driven member, the rollers are biased for pressing in the locked side direction and a torque value of the pressing is set preferably to less than 4 Nm.