The Pan-Canadian Chemical Library: A Mechanism to Open Academic Chemistry to High-Throughput Virtual Screening.

Computationally screening chemical libraries to discover molecules with desired properties is a common technique used in early-stage drug discovery. Recent progress in the field now enables the efficient exploration of billions of molecules within days or hours, but this exploration remains confined within the boundaries of the accessible chemistry space. While the number of commercially available compounds grows rapidly, it remains a limited subset of all druglike small molecules that could be synthesized. Here, we present a workflow where chemical reactions typically developed in academia and unconventional in drug discovery are exploited to dramatically expand the chemistry space accessible to virtual screening. We use this process to generate a first version of the Pan-Canadian Chemical Library, a collection of nearly 150 billion diverse compounds that does not overlap with other ultra-large libraries such as Enamine REAL or SAVI and could be a resource of choice for protein targets where other libraries have failed to deliver bioactive molecules.

Total Synthesis of the 2,5-Disubstituted γ-Pyrone E1 UAE Inhibitor Himeic Acid A.

The first total synthesis of the E1 ubiquitin-activating enzyme inhibitor, himeic acid A, is reported. A McCombie reaction was used to form the core γ-pyrone via a 6π-electrocyclization. A dioxenone ring-opening/acyl ketene trapping reaction with a primary amide provided the unusual unsymmetrical imide functionality. Other key steps include the use of an Evans auxiliary alkylation (d.r. ≥ 95:5) to install the (S)-2-methyl succinic acid fragment and a cross-metathesis to install the unsaturated side-chain.

Electrophoretic mobility, catalytic rate, and activation energy of catalysis of single molecules of the enzyme ß-glucuronidase from Escherichia coli.

Single molecule assays were performed on the enzyme E. coli ß-glucuronidase using a capillary electrophoresis-based protocol. Electrophoretic mobility, catalytic rate and activation energy of catalysis were all found to be heterogeneous. The average mobility at 22°C was -1.1×10-8±0.1m2V-1s-1 (N=49) with a total range of -0.6 to -1.3×10-8m2V-1s-1. The range in electrophoretic mobility suggests that the differences in shape or charge of the individual molecules underlying the heterogeneity are likely minimal. The average catalytic rate at 22°C was 37,000±19,000min-1 (N=49) with a total range of 14,000 to 130,000min-1. Both of these properties were measured simultaneously for each of the molecules. There was a weak correlation (r2=0.43) between mobility and rate with the molecules with a less negative mobility having a tendency to have a higher rate. The average activation energy of catalysis, as determined by comparing rates at 22 and 35°C, was found to be 48±18kJmol-1 (N=7) with a total range of 18-66kJmol-1.

Truce-Smiles rearrangement of substituted phenyl ethers.

The requirement of aryl ring activation by strong-electron withdrawing substituents in substrates for the intramolecular nucleophilic aromatic substitution reaction known as the Truce-Smiles rearrangement was examined. Preliminary mechanistic experiments support the SNAr mechanism, including (1)H and (13)C NMR spectra of a Meisenheimer intermediate formed in situ. The rearrangement was generally observed to be successful for substrates with strong electron withdrawing substituents, such as nitro-, cyano-, and benzoyl- functional groups, but also for those with multiple, weakly electron withdrawing substituents, such as chloro- and bromo-functional groups. These results lend further clarification to the effect of aryl substituents in this type of SNAr reaction. Additionally, the survey revealed several tandem cyclization and/or elimination reactions accessed by certain substrates.

An improved method for the synthesis of F-BODIPYs from dipyrrins and bis(dipyrrin)s.

An improved methodology for the synthesis of F-BODIPYs from dipyrrins and bis(dipyrrin)s is reported. This strategy employs lithium salts of dipyrrins as intermediates that are then treated with only 1 equiv of boron trifluoride diethyletherate to obtain the corresponding F-BODIPYs. This scalable route to F-BODIPYs renders high yields with a facile purification process involving merely filtration of the reaction mixture through Celite in many cases.

A small-molecule inhibitor of D-cyclin transactivation displays preclinical efficacy in myeloma and leukemia via phosphoinositide 3-kinase pathway.

D-cyclins are universally dysregulated in multiple myeloma and frequently overexpressed in leukemia. To better understand the role and impact of dysregulated D-cyclins in hematologic malignancies, we conducted a high-throughput screen for inhibitors of cyclin D2 transactivation and identified 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (S14161), which inhibited the expression of cyclins D1, D2, and D3 and arrested cells at the G(0)/G(1) phase. After D-cyclin suppression, S14161 induced apoptosis in myeloma and leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. In mouse models of leukemia, S14161 inhibited tumor growth without evidence of weight loss or gross organ toxicity. Mechanistically, S14161 inhibited the activity of phosphoinositide 3-kinase in intact cells and the activity of the phosphoinositide 3-kinases α, ß, δ, and γ in a cell-free enzymatic assay. In contrast, it did not inhibit the enzymatic activities of other related kinases, including the mammalian target of rapamycin, the DNA-dependent protein kinase catalytic subunit, and phosphoinositide-dependent kinase-1. Thus, we identified a novel chemical compound that inhibits D-cyclin transactivation via the phosphoinositide 3-kinase/protein kinase B signaling pathway. Given its potent antileukemia and antimyeloma activity and minimal toxicity, S14161 could be developed as a novel agent for blood cancer therapy.

The antiparasitic agent ivermectin induces chloride-dependent membrane hyperpolarization and cell death in leukemia cells.

To identify known drugs with previously unrecognized anticancer activity, we compiled and screened a library of such compounds to identify agents cytotoxic to leukemia cells. From these screens, we identified ivermectin, a derivative of avermectin B1 that is licensed for the treatment of the parasitic infections, strongyloidiasis and onchocerciasis, but is also effective against other worm infestations. As a potential antileukemic agent, ivermectin induced cell death at low micromolar concentrations in acute myeloid leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. Ivermectin also delayed tumor growth in 3 independent mouse models of leukemia at concentrations that appear pharmacologically achievable. As an antiparasitic, ivermectin binds and activates chloride ion channels in nematodes, so we tested the effects of ivermectin on chloride flux in leukemia cells. Ivermectin increased intracellular chloride ion concentrations and cell size in leukemia cells. Chloride influx was accompanied by plasma membrane hyperpolarization, but did not change mitochondrial membrane potential. Ivermectin also increased reactive oxygen species generation that was functionally important for ivermectin-induced cell death. Finally, ivermectin synergized with cytarabine and daunorubicin that also increase reactive oxygen species production. Thus, given its known toxicology and pharmacology, ivermectin could be rapidly advanced into clinical trial for leukemia.

Effect of noncompetitive proteasome inhibition on bortezomib resistance.

BACKGROUND: Bortezomib and the other proteasome inhibitors that are currently under clinical investigation bind to the catalytic sites of proteasomes and are competitive inhibitors. We hypothesized that proteasome inhibitors that act through a noncompetitive mechanism might overcome some forms of bortezomib resistance. METHODS: 5-amino-8-hydroxyquinoline (5AHQ) was identified through a screen of a 27-compound chemical library based on the quinoline pharmacophore to identify proteasome inhibitors. Inhibition of proteasome activity by 5AHQ was tested by measuring 7-amino-4-methylcoumarin (AMC) release from the proteasome substrate Suc-LLVY-AMC in intact human and mouse leukemia and myeloma cells and in tumor cell protein extracts. Cytotoxicity was assessed in 5AHQ-treated cell lines and primary cells from myeloma and leukemia patients using AlamarBlue fluorescence and MTS assays, trypan blue staining, and annexin V staining. 5AHQ-proteasome interaction was assessed by nuclear magnetic resonance. 5AHQ efficacy was evaluated in three leukemia xenograft mouse models (9-10 mice per group per model). All statistical tests were two-sided. RESULTS: 5AHQ inhibited the proteasome when added to cell extracts and intact cells (the mean concentration inhibiting 50% [IC(50)] of AMC release in intact cells ranged from 0.57 to 5.03 microM), induced cell death in intact cells from leukemia and myeloma cell lines (mean IC(50) values for cell growth ranged from 0.94 to 3.85 microM), and preferentially induced cell death in primary myeloma and leukemia cells compared with normal hematopoietic cells. 5AHQ was equally cytotoxic to human myelomonocytic THP1 cells and to THP1/BTZ500 cells, which are 237-fold more resistant to bortezomib than wild-type THP1 cells because of their overexpression and mutation of the bortezomib-binding beta5 proteasome subunit (mean IC(50) for cell death in the absence of bortezomib, wild-type THP1: 3.7 microM, 95% confidence interval = 3.4 to 4.0 microM; THP1/BTZ500: 6.6 microM, 95% confidence interval = 5.9 to 7.5 microM). 5AHQ interacted with the alpha subunits of the 20S proteasome at noncatalytic sites. Orally administered 5AHQ inhibited tumor growth in all three mouse models of leukemia without overt toxicity (eg, OCI-AML2 model, median tumor weight [interquartile range], 5AHQ vs control: 95.7 mg [61.4-163.5 mg] vs 247.2 mg [189.4-296.2 mg], P = .002). CONCLUSIONS: 5AHQ is a noncompetitive proteasome inhibitor that is cytotoxic to myeloma and leukemia cells in vitro and inhibits xenograft tumor growth in vivo. 5AHQ can overcome some forms of bortezomib resistance in vitro.

Selective inhibition of histone deacetylases sensitizes malignant cells to death receptor ligands.

Evasion of death receptor ligand-induced apoptosis represents an important contributor to cancer development and progression. Therefore, molecules that restore sensitivity to death receptor stimuli would be important tools to better understand this biological pathway and potential leads for therapeutic adjuncts. Previously, the small-molecule 4-(4-chloro-2-methylphenoxy)-N-hydroxybutanamide (that we propose be named droxinostat) was identified as a chemical sensitizer to death receptor stimuli, decreasing the expression of the caspase-8 inhibitor FLIP. However, the direct targets of droxinostat were unknown. To better understand the mechanism of action of droxinostat and highlight new strategies to restore sensitivity to death receptor ligands, we analyzed changes in gene expression using the Connectivity Map after treating cells with droxinostat. Changes in gene expression after droxinostat treatment resembled changes observed after treatment with histone deacetylase (HDAC) inhibitors. Therefore, we examined the effects of droxinostat on HDAC activity and showed that it selectively inhibited HDAC3, HDAC6, and HDAC8 and that inhibition of these HDACs was functionally important for its ability to sensitize cells to death ligands. Thus, we have identified a selective HDAC inhibitor and showed that selective HDAC inhibition sensitizes cells to death ligands, thereby highlighting a new mechanism to overcome resistance to death receptor ligands.

The ubiquitin-activating enzyme E1 as a therapeutic target for the treatment of leukemia and multiple myeloma.

The proteasomal pathway of protein degradation involves 2 discrete steps: ubiquitination and degradation. Here, we evaluated the effects of inhibiting the ubiquitination pathway at the level of the ubiquitin-activating enzyme UBA1 (E1). By immunoblotting, leukemia cell lines and primary patient samples had increased protein ubiquitination. Therefore, we examined the effects of genetic and chemical inhibition of the E1 enzyme. Knockdown of E1 decreased the abundance of ubiquitinated proteins in leukemia and myeloma cells and induced cell death. To further investigate effects of E1 inhibition in malignancy, we discovered a novel small molecule inhibitor, 3,5-dioxopyrazolidine compound, 1-(3-chloro-4-fluorophenyl)-4-[(5-nitro-2-furyl)methylene]-3,5-pyrazolidinedione (PYZD-4409). PYZD-4409 induced cell death in malignant cells and preferentially inhibited the clonogenic growth of primary acute myeloid leukemia cells compared with normal hematopoietic cells. Mechanistically, genetic or chemical inhibition of E1 increased expression of E1 stress markers. Moreover, BI-1 overexpression blocked cell death after E1 inhibition, suggesting ER stress is functionally important for cell death after E1 inhibition. Finally, in a mouse model of leukemia, intraperitoneal administration of PYZD-4409 decreased tumor weight and volume compared with control without untoward toxicity. Thus, our work highlights the E1 enzyme as a novel target for the treatment of hematologic malignancies.

Potential use of cetrimonium bromide as an apoptosis-promoting anticancer agent for head and neck cancer.

A potential therapeutic agent for human head and neck cancer (HNC), cetrimonium bromide (CTAB), was identified through a cell-based phenotype-driven high-throughput screen (HTS) of 2000 biologically active or clinically used compounds, followed by in vitro and in vivo characterization of its antitumor efficacy. The preliminary and secondary screens were performed on FaDu (hypopharyngeal squamous cancer) and GM05757 (primary normal fibroblasts), respectively. Potential hit compounds were further evaluated for their anticancer specificity and efficacy in combination with standard therapeutics on a panel of normal and cancer cell lines. Mechanism of action, in vivo antitumor efficacy, and potential lead compound optimizations were also investigated. In vitro, CTAB interacted additively with gamma radiation and cisplatin, two standard HNC therapeutic agents. CTAB exhibited anticancer cytotoxicity against several HNC cell lines, with minimal effects on normal fibroblasts; a selectivity that exploits cancer-specific metabolic aberrations. The central mode of cytotoxicity was mitochondria-mediated apoptosis via inhibition of H(+)-ATP synthase activity and mitochondrial membrane potential depolarization, which in turn was associated with reduced intracellular ATP levels, caspase activation, elevated sub-G(1) cell population, and chromatin condensation. In vivo, CTAB ablated tumor-forming capacity of FaDu cells and delayed growth of established tumors. Thus, using an HTS approach, CTAB was identified as a potential apoptogenic quaternary ammonium compound possessing in vitro and in vivo efficacy against HNC models.

Chelation of intracellular iron with the antifungal agent ciclopirox olamine induces cell death in leukemia and myeloma cells.

Off-patent drugs with previously unrecognized anticancer activity could be rapidly repurposed for this new indication. To identify such compounds, we conducted 2 independent cell-based chemical screens and identified the antimicrobial ciclopirox olamine (CPX) in both screens. CPX decreased cell growth and viability of malignant leukemia, myeloma, and solid tumor cell lines as well as primary AML patient samples at low-micromolar concentrations that appear pharmacologically achievable. Furthermore, oral CPX decreased tumor weight and volume in 3 mouse models of leukemia by up to 65% compared with control without evidence of weight loss or gross organ toxicity. In addition, oral CPX prevented the engraftment of primary AML cells in nonobese diabetic/severe combined immunodeficiency mouse models, thereby establishing its ability to target leukemia stem cells. Mechanistically, CPX bound intracellular iron, and this intracellular iron chelation was functionally important for its cytotoxicity. By electron paramagnetic resonance, CPX inhibited the iron-dependent enzyme ribonucleotide reductase at concentrations associated with cell death. Thus, in summary, CPX has previously unrecognized anticancer activity at concentrations that are pharmacologically achievable. Therefore, CPX could be rapidly repurposed for the treatment of malignancies, including leukemia and myeloma.

A novel inhibitor of glucose uptake sensitizes cells to FAS-induced cell death.

Evasion of death receptor ligand-induced apoptosis is an important contributor to cancer development and progression. Therefore, molecules that restore sensitivity to death receptor stimuli would be important tools to better understand this biological pathway and potential leads for therapeutic adjuncts. Previously, the small-molecule N-[4-chloro-3-(trifluoromethyl)phenyl]-3-oxobutanamide (fasentin) was identified as a chemical sensitizer to the death receptor stimuli FAS and tumor necrosis factor apoptosis-inducing ligand, but its mechanism of action was unknown. Here, we determined that fasentin alters expression of genes associated with nutrient and glucose deprivation. Consistent with this finding, culturing cells in low-glucose medium recapitulated the effects of fasentin and sensitized cells to FAS. Moreover, we showed that fasentin inhibited glucose uptake. Using virtual docking studies with a homology model of the glucose transport protein GLUT1, fasentin interacted with a unique site in the intracellular channel of this protein. Additional chemical studies with other GLUT inhibitors and analogues of fasentin supported a role for partial inhibition of glucose transport as a mechanism to sensitize cells to death receptor stimuli. Thus, fasentin is a novel inhibitor of glucose transport that blocks glucose uptake and highlights a new mechanism to sensitize cells to death ligands.

15N NMR chemical shifts for the identification of dipyrrolic structures.

A variety of dipyrromethanes and dipyrromethenes have been prepared, and their 15N NMR chemical shifts have been measured by two-dimensional correlation to 1H NMR signals. The nitrogen atoms in five examples of dipyrromethanes consistently exhibit chemical shifts around -231 ppm, relative to nitromethane. Seven examples of hydrobromide salts of meso-unsubstituted dipyrromethenes consistently display 15N chemical shifts around -210 ppm, while their corresponding zinc(II) complexes exhibit chemical shifts around -170 ppm. The presence of electron-withdrawing substituents on one of the pyrrolic rings of dipyrromethenes affects the chemical shifts of both of the nitrogen nuclei in the molecule. Boron difluoride complexes of meso-unsubstituted dipyrromethenes display 15N chemical shifts around -190 ppm. Two examples of free-base dipyrromethenes bearing substituents at the meso-position exhibit 15N chemical shifts at approximately -156 ppm, and for the zinc complexes of these compounds at -162 ppm. One-bond nitrogen-hydrogen coupling constants, when measurable, were consistently in the range of -96 Hz. Since the measured 15N chemical shifts have such a high regularity correlated to structure, they can be used as diagnostic indications for identifying the structure of dipyrrolic compounds.

Dinuclear zinc(II) double-helicates of homochirally substituted bis(dipyrromethene)s.

[structure: see text] A series of bis(dipyrromethene)s substituted with aromatic amide and aliphatic ester homochiral auxiliaries have been prepared and complexed with zinc(II) ions to form double-helical dinuclear complexes. CD analysis of the crude complexes revealed that the helicates formed in a diastereoselective manner. The helicates have been resolved into their constituent M and P helices by HPLC, indicating that the helical sense of the complexes is stable to racemization.

Stereochemically stable double-helicate dinuclear complexes of bis(dipyrromethene)s: a chiroptical study.

Helical zinc(II) complexes of bis(dipyrromethene)s bearing homochiral amide substituents were synthesized. Analysis of the products by chiral HPLC showed two diastereomeric major products and showed that dipyrromethene double-nuclear helicates are stereochemically stable and do not interconvert. Circular dichroism (CD) studies showed that the complexation reactions had proceeded with modest diastereomeric excesses. Analysis of an analogous symmetric zinc(II) bis(dipyrromethene) lacking homochiral substituents that could act as chromophores discounted induced CD by the chiral auxiliaries.