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The clinical practice of oncology is being transformed by molecular diagnostics that will enable predictive and personalized medicine. Current technologies for quantitation of the cancer proteome are either qualitative (e.g., immunohistochemistry) or require large sample sizes (e.g., flow cytometry). Here, we report a microfluidic platform—microfluidic image cytometry (MIC)—capable of quantitative, single-cell proteomic analysis of multiple signaling molecules using only 1,000 to 2,800 cells. Using cultured cell lines, we show simultaneous measurement of four critical signaling proteins (EGFR, PTEN, phospho-Akt, and phospho-S6) within the oncogenic phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway. To show the clinical application of the MIC platform to solid tumors, we analyzed a panel of 19 human brain tumor biopsies, including glioblastomas. Our MIC measurements were validated by clinical immunohistochemistry and confirmed the striking intertumoral and intratumoral heterogeneity characteristic of glioblastoma. To interpret the multiparameter, single-cell MIC measurements, we adapted bioinformatic methods including self-organizing maps that stratify patients into clusters that predict tumor progression and patient survival. Together with bioinformatic analysis, the MIC platform represents a robust, enabling in vitro molecular diagnostic technology for systems pathology analysis and personalized medicine. Cancer Res; 70(15); 6128–38. ©2010 AACR.

Determination of impurity level in weight percent against the claimed active pharmaceutical ingredient (API) in a drug product is of critical importance in drug development. However, authentic material used to determine relative UV response factor (RRF) for impurity quantitation is sometimes not available or not stable. In a particular drug product liquid formulation, three degradants, including one unstable impurity, are consistently observed in stability studies. Here we describe the application of NMR spectroscopy to assist in the determination of RRFs, simultaneously, for all three degradants directly from degraded drug product extracts. This technique was not only effective in determining RRF of an unstable substance, but also in defining RRFs of multiple degradants from a single drug product sample. Overall, this study continues to demonstrate the capabilities of NMR for impurity quantitation in drug development.

We report the synthesis and photochemical and biological characterization of the first selective and potent metal-based inhibitors of cytochrome P450 3A4 (CYP3A4), the major human drug metabolizing enzyme. Five Ru(II)-based derivatives were prepared from two analogs of the CYP3A4 inhibitor ritonavir, 4 and 6: [Ru(tpy)(L)(6)]Cl(2) (tpy = 2,2′:6′,2″-terpyridine) with L = 6,6′-dimethyl-2,2′-bipyridine (Me(2)bpy; 8), dimethylbenzo[i]dipyrido[3,2-a:2′,3′-c]phenazine (Me(2)dppn; 10) and 3,6-dimethyl-10,15-diphenylbenzo[i]dipyrido[3,2-a:2′,3′-c]phenazine (Me(2)Ph(2)dppn; 11), [Ru(tpy)(Me(2)bpy)(4)]Cl(2) (7) and [Ru(tpy)(Me(2)dppn)(4)]Cl(2) (9). Photochemical release of 4 or 6 from 7–11 was demonstrated, and the spectrophotometric evaluation of 7 showed that it behaves similarly to free 4 (type II heme ligation) after irradiation with visible light but not in the dark. Unexpectedly, the intact Ru(II) complexes 7 and 8 were found to inhibit CYP3A4 potently and specifically through direct binding to the active site without heme ligation. Caged inhibitors 9–11 showed dual action properties by combining photoactivated dissociation of 4 or 6 with efficient (1)O(2) production. In prostate adenocarcinoma DU-145 cells, compound 9 had the best synergistic effect with vinblastine, the anticancer drug primarily metabolized by CYP3A4 in vivo. Thus, our study establishes a new paradigm in CYP inhibition using metalated complexes and suggests possible utilization of photoactive CYP3A4 inhibitory compounds in clinical applications, such as enhancement of therapeutic efficacy of anticancer drugs.

Identification of structural determinants required for potent inhibition of drug-metabolizing cytochrome P450 3A4 (CYP3A4) could help develop safer drugs and more effective pharmacoenhancers. We utilize a rational inhibitor design to decipher structure-activity relationships in analogues of ritonavir, a highly potent CYP3A4 inhibitor marketed as pharmacoenhancer. Analysis of compounds with the R(1) side-group as phenyl or naphthalene and R(2) as indole or naphthalene in different stereo configuration showed that (i) analogues with the R(2)-naphthalene tend to bind tighter and inhibit CYP3A4 more potently than the R(2)-phenyl/indole containing counterparts; (ii) stereochemistry becomes a more important contributing factor, as the bulky side-groups limit the ability to optimize protein-ligand interactions; (iii) the relationship between the R(1)/R(2) configuration and preferential binding to CYP3A4 is complex and depends on the side-group functionality/interplay and backbone spacing; and (iv) three inhibitors, 5a-b and 7d, were superior to ritonavir (IC(50) of 0.055–0.085 μM vs. 0.130 μM, respectively).

Modified amino acids are useful synthetic components in both chemistry and biology. Here we describe a simple, scalable two-step procedure to generate α-thio aromatic acids from aromatic amino acids with yields of up to 96%. Diazotization and α-lactone mediated bromination efficiently form the α-bromo acid with retention of configuration. Thiol substitution with mild reagents such as sodium hydrosulfide or sodium trithiocarbonate provides the inverted, free α-thio acid. The mildly acidic soft nucleophile can then be utilized in many synthetic applications.

In a continuing effort to identify structural attributes required for strong binding and potent inhibition of human drug-metabolizing CYP3A4, we designed ten ritonavir-like analogs differing in the side-group stereochemistry, backbone atomic composition, and head-group spacing. All analogs had pyridine and tert-butyloxycarbonyl (Boc) as the heme-ligating and tail groups, respectively, phenyl side-groups, and either a methyl- or ethyl-pyridyl linker. Each linker subseries had S/R, R/S, R/R and S/S side-group conformers (4a-d and 4e-h, respectively), and one S/S stereoisomer with the backbone S-to-N-heteroatom substitution (6a and 6b). To elucidate structure-activity relationships, ligand-dependent changes in optical spectra, dissociation constant (K(s)), inhibitory potency (IC(50)), thermostability, and heme ligation and reduction kinetics were analyzed. Comparison of the subseries and individual compounds showed that CYP3A4 only weakly discriminates between side-group configurations, associates more tightly with the pyridyl-ethyl-linker analogs, and strongly disfavors the N-containing backbone. K(s) and IC(50) for the pyridyl-ethyl R/R conformer, 4g, were the lowest and close to those for ritonavir: 0.04 and 0.31 µM versus 0.02 and 0.13 µM, respectively. Determination of the X-ray structures of the inhibitory complexes was critical for experimental data interpretation, especially for the uniquely oriented 4a and 4e. Based on structural analysis we conclude that, for this series of analogs, the ligand-mediated interactions near the heme are dominant and define the binding mode, and that fine-tuning of these interactions, as well as the backbone spacing could further improve the affinity and inhibitory strength.

Inhibition of the major human drug-metabolizing cytochrome P450 3A4 (CYP3A4) by pharmaceuticals and other xenobiotics could lead to toxicity, drug&ndash, drug interactions and other adverse effects, as well as pharmacoenhancement. Despite serious clinical implications, the structural basis and attributes required for the potent inhibition of CYP3A4 remain to be established. We utilized a rational inhibitor design to investigate the structure&ndash, activity relationships in the analogues of ritonavir, the most potent CYP3A4 inhibitor in clinical use. This study elucidated the optimal length of the head-group spacer using eleven (series V) analogues with the R1/R2 side-groups as phenyls or R1&ndash, phenyl/R2&ndash, indole/naphthalene in various stereo configurations. Spectral, functional and structural characterization of the inhibitory complexes showed that a one-atom head-group linker elongation, from pyridyl&ndash, ethyl to pyridyl&ndash, propyl, was beneficial and markedly improved Ks, IC50 and thermostability of CYP3A4. In contrast, a two-atom linker extension led to a multi-fold decrease in the binding and inhibitory strength, possibly due to spatial and/or conformational constraints. The lead compound, 3h, was among the best inhibitors designed so far and overall, the strongest binder (Ks and IC50 of 0.007 and 0.090 &micro, M, respectively). 3h was the fourth structurally simpler inhibitor superior to ritonavir, which further demonstrates the power of our approach.

Summary Ubiquitin-like (Ubl) post-translational modifications are potential targets for therapeutics. However, the only known mechanism for inhibiting a Ubl-activating enzyme is through targeting its ATP-binding site. Here we identify an allosteric inhibitory site in the small ubiquitin-like modifier (SUMO)-activating enzyme (E1). This site was unexpected because both it and analogous sites are deeply buried in all previously solved structures of E1s of ubiquitin-like modifiers (Ubl). The inhibitor not only suppresses SUMO E1 activity, but also enhances its degradation in vivo, presumably due to a conformational change induced by the compound. In addition, the lead compound increased the expression of miR-34b and reduced c-Myc levels in lymphoma and colorectal cancer cell lines and a colorectal cancer xenograft mouse model. Identification of this first-in-class inhibitor of SUMO E1 is a major advance in modulating Ubl modifications for therapeutic aims.

Structure-function relationships of nine rationally designed ritonavir-like compounds were investigated to better understand the ligand binding and inhibitory mechanism in human drug-metabolizing cytochrome P450 3A4 (CYP3A4). The analogs had a similar backbone and pyridine and tert-butyloxycarbonyl (Boc) as the heme-ligating and terminal groups, respectively. N-Isopropyl, N-cyclopentyl, or N-phenyl were the R1-side group substituents alone (compounds 5a-c) or in combination with phenyl or indole at the R2 position (8a-c and 8d-f subseries, respectively). Our experimental and structural data indicate that (i) for all analogs, a decrease in the dissociation constant (Ks) coincides with a decrease in IC50, but no relation with other derived parameters is observed; (ii) an increase in the R1 volume, hydrophobicity, and aromaticity markedly lowers Ks and IC50, whereas the addition of aromatic R2 has a more pronounced positive effect on the inhibitory potency than the binding strength; (iii) the ligands' association mode is strongly influenced by the mutually dependent R1-R2 interplay, but the R1-mediated interactions are dominant and define the overall conformation in the active site; (iv) formation of a strong H-bond with Ser119 is a prerequisite for potent CYP3A4 inhibition; and (v) the strongest inhibitor in the series, the R1-phenyl/R2-indole containing 8f (Ks and IC50 of 0.08 and 0.43 μM, respectively), is still less potent than ritonavir, even under conditions that prevent the mechanism based inactivation of CYP3A4. Crystallographic data were essential for better understanding and interpretation of the experimental results, and suggested how the inhibitor design could be further optimized.

Two synthetic routes to 3'-deoxy-3'-azido nucleosides are described, one toward the synthesis of 3'-deoxy-3'-azidouridine and a second toward 3'-deoxy-3'-azidocytidine. The target compounds may serve as precursors to provide building blocks for use in automated synthesis of guanidine-linked RNA analogs (RNG) or oligonucleotide N3'→P5' phosphoramidates. Moreover, the synthetic approaches are adaptable to the general synthesis of 3'-substituted 3'-deoxynucleosides for development of new antiviral drugs.

Two synthetic routes to 3'-deoxy-3'-azido nucleosides are described, one toward the synthesis of 3'-deoxy-3'-azidouridine and a second toward 3'-deoxy-3'-azidocytidine. The target compounds may serve as precursors to provide building blocks for use in automated synthesis of guanidine-linked RNA analogs (RNG) or oligonucleotide N3'→P5' phosphoramidates. Moreover, the synthetic approaches are adaptable to the general synthesis of 3'-substituted 3'-deoxynucleosides for development of new antiviral drugs.

The clinical practice of oncology is being transformed by molecular diagnostics that will enable predictive and personalized medicine. Current technologies for quantitation of the cancer proteome are either qualitative (e.g., immunohistochemistry) or require large sample sizes (e.g., flow cytometry). Here, we report a microfluidic platform—microfluidic image cytometry (MIC)—capable of quantitative, single-cell proteomic analysis of multiple signaling molecules using only 1,000 to 2,800 cells. Using cultured cell lines, we show simultaneous measurement of four critical signaling proteins (EGFR, PTEN, phospho-Akt, and phospho-S6) within the oncogenic phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway. To show the clinical application of the MIC platform to solid tumors, we analyzed a panel of 19 human brain tumor biopsies, including glioblastomas. Our MIC measurements were validated by clinical immunohistochemistry and confirmed the striking intertumoral and intratumoral heterogeneity characteristic of glioblastoma. To interpret the multiparameter, single-cell MIC measurements, we adapted bioinformatic methods including self-organizing maps that stratify patients into clusters that predict tumor progression and patient survival. Together with bioinformatic analysis, the MIC platform represents a robust, enabling in vitro molecular diagnostic technology for systems pathology analysis and personalized medicine. Cancer Res; 70(15); 6128–38. ©2010 AACR.