DMSO Stock Solutions Research Articles

Discovery of a Plant 14-3-3 Inhibitor Possessing Isoform Selectivity and In Planta Activity.

Synthetic modulators of plant 14-3-3s are promising chemical tools both for understanding the 14-3-3-related signaling pathways and controlling plant physiology. Herein, we describe a novel small-molecule inhibitor for 14-3-3 proteins of Arabidopsis thaliana. The inhibitor was identified from unexpected products in a stock solution in dimethyl sulfoxide (DMSO) of an in-house chemical library. Mass spectroscopy, mutant-based analyses, fluorescence polarization assays, and thermal shift assays revealed that the inhibitor covalently binds to an allosteric site of 14-3-3 with isoform selectivity. Moreover, infiltration of the inhibitor to Arabidopsis leaves suppressed the stomatal aperture. The inhibitor should provide new insight into the design of potent and isoform-selective 14-3-3 modulators.

Discovery of a Plant 14‐3‐3 Inhibitor Possessing Isoform Selectivity and In Planta Activity

AbstractSynthetic modulators of plant 14‐3‐3s are promising chemical tools both for understanding the 14‐3‐3‐related signaling pathways and controlling plant physiology. Herein, we describe a novel small‐molecule inhibitor for 14‐3‐3 proteins of Arabidopsis thaliana. The inhibitor was identified from unexpected products in a stock solution in dimethyl sulfoxide (DMSO) of an in‐house chemical library. Mass spectroscopy, mutant‐based analyses, fluorescence polarization assays, and thermal shift assays revealed that the inhibitor covalently binds to an allosteric site of 14‐3‐3 with isoform selectivity. Moreover, infiltration of the inhibitor to Arabidopsis leaves suppressed the stomatal aperture. The inhibitor should provide new insight into the design of potent and isoform‐selective 14‐3‐3 modulators.

Application of rational enzyme engineering in a new route to etonogestrel and levonorgestrel: carbonyl reductase bioreduction of ethyl secodione.

Women in developing countries still face enormous challenges when accessing reproductive health care. Access to voluntary family planning empowers women allowing them to complete their education and join the paid workforce. This effectively helps to end poverty, hunger and promotes good health for all. According to the United Nations (UN) organization, in 2022, an estimated 257 million women still lacked access to safe and effective family planning methods globally. One of the main barriers is the associated cost of modern contraceptive methods. Funded by the Bill & Melinda Gates Foundation, Almac Group worked on the development of a novel biocatalytic route to etonogestrel and levonorgestrel, two modern contraceptive APIs, with the goal of substantially decreasing the cost of production and so enabling their use in developing nations. This present work combines the selection and engineering of a carbonyl reductase (CRED) enzyme from Almac's selectAZyme™ panel, with process development, to enable efficient and economically viable bioreduction of ethyl secodione to (13R,17S)-secol, the key chirality introducing intermediate en route to etonogestrel and levonorgestrel API. CRED library screening returned a good hit with an Almac CRED from Bacillus weidmannii, which allowed for highly stereoselective bioreduction at low enzyme loading of less than 1% w/w under screening assay conditions. However, the only co-solvent tolerated was DMSO up to ∼30% v/v, and it was impossible to achieve reaction completion with any enzyme loading at substrate titres of 20 g L-1 and above, due to the insolubility of the secodione. This triggered a rapid enzyme engineering program fully based on computational mutant selection. A small panel of 93 CRED mutants was rationally designed to increase the catalytic activity as well as thermal and solvent stability. The best mutant, Mutant-75, enabled a reaction at 45 °C to go to completion at 90 g L-1 substrate titre in a buffer/DMSO/heptane reaction medium fed over 6 h with substrate DMSO stock solution, with a low enzyme loading of 3.5% w/w wrt substrate. In screening assay conditions, Mutant-75 also showed a 2.2-fold activity increase. Our paper shows which computations and rational decisions enabled this outcome.

Application of Pluronics for Enhancing Aqueous Solubility of Lipophilic Microtubule Destabilizing Compounds on the Sea Urchin Embryo Model.

In screening, the dilution of DMSO stock solution of a lipophilic molecule with an assay medium often causes compound precipitation. To overcome the issue, the application of Pluronics as cosolvents was examined using a phenotypic sea urchin embryo assay that allows for the quick and facile evaluation of the antiproliferative effect together with systemic toxicity. Maximum tolerated concentration values for Pluronics L121, P123, and F127 were 1.4 μM, 8.6 μM, and 39.7 μM, respectively, and correlated directly with their hydrophilicity. Pluronics L121 and P123 suppressed cleavage and blastomeres retained the round shape, unlike hydrophilic Pluronic F127, which induced fertilization envelope creasing and embryo deformation that could be associated with the interaction of hydrophilic PEO units with mucopolysaccharides at the surface of sea urchin embryos. The toxicity of P123, but not of L121 and F127, was temperature-dependent and markedly increased at lower temperatures. CMC values obtained at different temperatures confirmed that the toxic effect of P123 was associated with both unimers and micelles, whereas F127 toxicity was related mainly to micelles. Evaluation using phenotypic sea urchin embryo assay revealed that potent microtubule destabilizers, namely albendazole, diarylisoxazole, and two chalcones, retained antimitotic activity after the dilution of their DMSO or 2-pyrrolidone stock solutions with 1.25% w/v Pluronic P123 or 5% w/v Pluronic F127. It was suggested that Pluronic P123 and Pluronic F127 could be used as cosolvents to improve the solubility of lipophilic molecules in aqueous medium.

Avoid missing pKas: High-throughput workflow using solution pH-metric in tandem with UV-metric measurements

We describe a new high-throughput automated pKa workflow using potentiometry starting with 10 mM DMSO stock (solution pH-metric). Two approaches using either neat DMSO stock solution or removal of DMSO were evaluated with different sample amounts and cosolvent schemes. These were validated against traditional potentiometric measurements for optimal conditions. Further, we detail how high throughput solution pH-metric experiments are performed in tandem with established UV-metric measurements to capitalize on the advantages of both approaches. This new workflow maintains the sample and time savings required for measuring a large number of samples in a drug discovery setting, while avoiding “missing pKas” due to lack of sufficient UV chromophores. The combination of the two assays is key to tackle the challenges of low solubility, overlapping pKas, and preliminary assignment of pKas for Structure-Activity Relationship (SAR) understanding.

Integrated and automated high-throughput purification of libraries on microscale.

We herein report the development of an automation platform for rapid purification and quantification of chemical libraries including reformatting of chemical matter to 10mM DMSO stock solutions. This fully integrated workflow features tailored conditions for preparative reversed-phase (RP) HPLC-MS on microscale based on analytical data, online fraction QC and CAD-based quantification as well as automated reformatting to enable rapid purification of chemical libraries. This automated workflow is entirely solution-based, eliminating the need to weigh or handle solids. This increases process efficiency and creates a link between high-throughput synthesis and profiling of novel chemical matter with respect to biological and physicochemical properties in relevant assays.

Antibacterial and Anticandidal Activity of Ethanolic Extract of Immature Flower Buds of Syzygium aromaticum

Microbial resistance to antibiotics has been raised over time, creating a serious burden and an issue that requires a quick response. As a result, interest has shifted to medicinal plants as natural, effective, and cost-efficient alternatives. The goal of this study was to assess the antibacterial and antifungal activities of the dried buds of the clove’s flowers, which were extracted in 250 ml of ethanol using the Soxhlet apparatus. Subsequently, DMSO stock solutions were utilized to create concentrations of 100, 50, 25, 12.5, 6.25, and ultimately 3.125 mg/ml. Following that, the agar-well diffusion technique was utilized for the evaluation of the antibacterial activities of the clove extract against gram-positive bacteria (Staphylococcus aureus and Group A Streptococcus bacteria) and gram-negative bacteria (Escherichia coli), as well as Candida albicans, isolated and identified through the pediatric hospital's medical laboratory west of Gaza city. Lastly, the results of this study clearly showed that the ethanolic extract of dried immature flower buds of clove has anti-S. aureus and anti-group A Streptococcus effects, as well as for the C. albicans used in the present study, with the exception of E. coli, which demonstrated resistance to the plant extract at all concentrations tested. While the lowest concentrations that showed anti-bacterial activity were revealed to be 12.5 and 25 mg/ml for anti-S. aureus and anti-group A Streptococcus, respectively, with inhibition zone diameters of 2 and 1 mm, on the other hand, 25 mg/ml with a 2 mm inhibition zone was determined to have the lowest concentration that had anti-C. albicans activity. This study concluded that cloves can be employed as a plant with anti-bacterial and anti-fungal characteristics, but additional research is required.

Parasitology assays to assess Trichomonas vaginalis nucleoside ribohydrolase inhibitors

Trichomonas vaginalis is a protozoan parasite that causes trichomoniasis, a sexually transmitted disease that affects an estimated 275 million people worldwide. The Centers for Disease Control and Prevention recognizes trichomoniasis as a neglected parasitic infection, with an estimated 2.6 million prevalent infections in the United States in 2018. Clinical manifestations of infections are typically mild, but the immune system can be compromised resulting in higher susceptibility to more serious conditions such as pelvic inflammatory disease, HIV‐1, and other infections. Trichomonal infection has also been associated with prostate cancer and benign prostatic hyperplasia. Traditionally, compounds such as metronidazole or tinidazole have been used to treat trichomoniasis, however, strains resistant to these drugs are becoming more widespread. T. vaginalis requires nucleoside salvage pathways for its survival. Nucleoside ribohydrolase inhibitors thus represent a possible target for the development of new treatments with different mechanisms of action compared to metronidazole. Toward this goal, fragment screening identified nucleoside ribohydrolase inhibitors as starting points for medicinal chemistry efforts. Newly synthesized compounds are routinely evaluated for potency against purified enzymes. However, a better test of potency is antitrichomonal activity. Metronidazole‐sensitive strain B7RC2 (ATCC 50167) of T. vaginalis was cultured anaerobically at 37 ℃ in TYM Diamond’s media supplemented with streptomycin, penicillin and iron solution. Compounds were prepared as 25 mM DMSO stock solutions and tested at 100 µM. DMSO and metronidazole were used as vehicle and positive controls respectively. Parasite cellular viability was measured after 24 hours of incubation with compounds or controls. Cells were counted using a hemocytometer. Active compounds were then tested in a dose‐dependent manner to determine antitrichomonal IC50 values. Compounds with the best combination of enzyme inhibition and T. vaginalis activity were then prioritized for guiding the design of compounds to be synthesized. It is expected that this process will simultaneously optimize enzyme inhibition and antitrichomonal activity resulting in compounds with nM activity in enzyme assays, against metronidazole‐sensitive T. vaginalis, and ultimately against metronidazole‐resistant T. vaginalis.

Search for the Active Ingredients from a 2-Aminothiazole DMSO Stock Solution with Antimalarial Activity.

Chemical decomposition of DMSO stock solutions is a common incident that can mislead biological screening campaigns. Here, we share our case study of 2‐aminothiazole 1, originating from an antimalarial class that undergoes chemical decomposition in DMSO at room temperature. As previously measured biological activities observed against Plasmodium falciparum NF54 and for the target enzyme PfIspE were not reproducible for a fresh batch, we tackled the challenge to understand where the activity originated from. Solvent‐ and temperature‐dependent studies using HRMS and NMR spectroscopy to monitor the decomposition led to the isolation and in vitro evaluation of several fractions against PfIspE. After four days of decomposition, we successfully isolated the oxygenated and dimerised compounds using SFC purification and correlated the observed activities to them. Due to the unstable nature of the two isolates, it is likely that they undergo further decomposition contributing to the overall instability of the compound.

Rapid and Selective Labeling of Endogenous Transmembrane Proteins in Living Cells with a Difluorophenyl Ester Affinity-Based Probe.

The long-term stability of affinity-based protein labeling probes is crucial to obtain reproducible protein labeling results. However, highly stable probes generally suffer from low protein labeling efficiency and pose significant challenges when labeling low abundance native proteins in living cells. In this paper, we report that protein labeling probes based on an ortho-difluorophenyl ester reactive module exhibit long-term stability in DMSO stock solution and aqueous buffer, yet they can undergo rapid and selective labeling of native proteins. This novel electrophile can be customized with a wide range of different protein ligands and is particularly well-suited for the labeling and imaging of transmembrane proteins. With this probe design, the identity and relative levels of basal and hypoxia-induced transmembrane carbonic anhydrases were revealed by live cell imaging and in-gel fluorescence analysis. We believe that the extension of this difluorophenyl ester reactive module would allow for the specific labeling of various endogenous membrane proteins, facilitating in-depth studies of their distribution and functions in biological processes.

Influences of Linker and Nucleoside for the Helical Self-Assembly of Perylene Along DNA Templates.

Six different conjugates of perylene with 2′-deoxyuridine and with 2-amino-2′-deoxyadenosine were synthesized and applied for DNA-templated assembly in aqueous buffer solutions. They differ by the linkers ethynylene, phenylene, and phenylene–ethynylene between nucleoside and chromophore. The nucleosides were investigated as monomers in CHCl3 and dimethyl sulfoxide by optical spectroscopy. The properties of the four phenylene-linked conjugates are similar to that of perylene as reference because these linkers separate both aromatic parts. The ethynylene linker electronically couples the chromophore with the respective nucleoside and thus red shifts the absorbance. The DNA-templated assembly properties were elucidated by mixing the templates in aqueous buffer with the perylene–nucleoside conjugates from a dimethyl sulfoxide stock solution. Specific binding of the nucleosides was probed by comparing the results with dA20 and T20 as single-stranded DNA templates. Our studies reveal the structural parameters that are important for the DNA-templated assembly of perylenes. First, perylene-2′-deoxyuridine conjugates do not form DNA-templated helical assemblies, regardless of the choice of linker. Second, the ethynylene linker is crucial for successful DNA-templated chromophore assemblies of perylene-2-amino-2′-deoxyadenosine conjugates. Third, in contrast, the phenylene linker inhibits self-assembly along single-stranded DNA templates. In conclusion, the 2-amino-2′-deoxyadenosin in combination with the ethynylene linker provides the best structural feature for specific and helical DNA-templated assembly of perylenes. This result is important for the design of future DNA-based supramolecular architectures with chromophores, in particular DNA-based light-harvesting systems and DNA systems for emitting or sensing circularly polarized luminescence.

Investigation and Mathematical Description of the Real Driving Force of Passive Transport of Drug Molecules from Supersaturated Solutions.

The aim of this study was to investigate the impact of formulation excipients and solubilizing additives on dissolution, supersaturation, and membrane transport of an active pharmaceutical ingredient (API). When a poorly water-soluble API is formulated to enhance its dissolution, additives, such as surfactants, polymers, and cyclodextrins, have an effect not only on dissolution profile but also on the measured physicochemical properties (solubility, pKa, permeability) of the drug while the excipient is present, therefore also affecting the driving force of membrane transport. Meloxicam, a nonsteroidal anti-inflammatory drug, was chosen as a poorly water-soluble model drug and formulated in order to enhance its dissolution using solvent-based electrospinning. Three polyvinylpyrrolidone (PVP) derivatives (K30, K90, and VA 64), Soluplus, and (2-hydroxypropyl)-β-cyclodextrin were used to create five different amorphous solid dispersions of meloxicam. Through experimental design, the various formulation additives that could influence the characteristics of dissolution and permeation through artificial membrane were observed by carrying out a simultaneous dissolution-permeation study with a side-by-side diffusion cell, μFLUX. Although the dissolution profiles of the formulations were found to be very similar, in the case of Soluplus containing formulation the flux was superior, showing that the driving force of membrane transport cannot be simplified to the concentration gradient. Supersaturation gradient, the difference in degree of supersaturation (defined as the ratio of dissolved amount of the drug to its thermodynamic solubility) between the donor and acceptor side, was found to be the driving force of membrane transport. It was mathematically derived from Fick's first law, and experimentally proved to be universal on several meloxicam containing ASDs and DMSO stock solution.

Combining 'dry' co-crystallization and in situ diffraction to facilitate ligand screening by X-ray crystallography.

X-ray crystallography is an established technique for ligand screening in fragment-based drug-design projects, but the required manual handling steps - soaking crystals with ligand and the subsequent harvesting - are tedious and limit the throughput of the process. Here, an alternative approach is reported: crystallization plates are pre-coated with potential binders prior to protein crystallization and X-ray diffraction is performed directly 'in situ' (or in-plate). Its performance is demonstrated on distinct and relevant therapeutic targets currently being studied for ligand screening by X-ray crystallography using either a bending-magnet beamline or a rotating-anode generator. The possibility of using DMSO stock solutions of the ligands to be coated opens up a route to screening most chemical libraries.

Permeapad™ for investigation of passive drug permeability: The effect of surfactants, co-solvents and simulated intestinal fluids (FaSSIF and FeSSIF)

The aim of the present work was to investigate the potential of the new and innovative artificial barrier, Permeapad™, when exposed to surfactants and co-solvents, often employed for poorly water soluble compounds. The barrier was in addition also exposed to fasted and fed state simulated intestinal fluids versions 1 and 2 (FaSSIF and FeSSIF), all of which the Permeapad™ barrier was compatible with based upon relative comparison of the permeability of the hydrophilic marker calcein in phosphate buffer. The new barrier therefore holds a huge potential due to its functional stability and robustness. It can be used as a standard tool to investigate permeability of drugs in the presence of different surfactants and co-solvents, from DMSO stock solutions at even high concentrations and for the evaluation of permeability in the presence of biomimetic media (BMM).11Biomimetic media=BMM.

Carrier Mediated Distribution System (CAMDIS): A new approach for the measurement of octanol/water distribution coefficients

Here we present a miniaturized assay, referred to as Carrier-Mediated Distribution System (CAMDIS) for fast and reliable measurement of octanol/water distribution coefficients, logDoct. By introducing a filter support for octanol, phase separation from water is facilitated and the tendency of emulsion formation (emulsification) at the interface is reduced. A guideline for the best practice of CAMDIS is given, describing a strategy to manage drug adsorption at the filter-supported octanol/buffer interface. We validated the assay on a set of 52 structurally diverse drugs with known shake flask logDoct values. Excellent agreement with literature data (r2=0.996, standard error of estimate, SEE=0.111), high reproducibility (standard deviation, SD<0.1 logDoct units), minimal sample consumption (10μL of 100μM DMSO stock solution) and a broad analytical range (logDoct range=−0.5 to 4.2) make CAMDIS a valuable tool for the high-throughput assessment of logDoct.

Discovery of potent KIFC1 inhibitors using a method of integrated high-throughput synthesis and screening.

KIFC1 (HSET), a member of the kinesin-14 family of motor proteins, plays an essential role in centrosomal bundling in cancer cells, but its function is not required for normal diploid cell division. To explore the potential of KIFC1 as a therapeutic target for human cancers, a series of potent KIFC1 inhibitors featuring a phenylalanine scaffold was developed from hits identified through high-throughput screening (HTS). Optimization of the initial hits combined both design-synthesis-test cycles and an integrated high-throughput synthesis and biochemical screening method. An important aspect of this integrated method was the utilization of DMSO stock solutions of compounds registered in the corporate compound collection as synthetic reactants. Using this method, over 1500 compounds selected for structural diversity were quickly assembled in assay-ready 384-well plates and were directly tested after the necessary dilutions. Our efforts led to the discovery of a potent KIFC1 inhibitor, AZ82, which demonstrated the desired centrosome declustering mode of action in cell studies.

Decrease in phosphatidylinositol 4,5‐bisphosphate levels mediates desensitization of the cold sensor TRPM8 channels

The activity of the cold- and menthol-activated transient receptor potential melastatin 8 (TRPM8) channels diminishes over time in the presence of extracellular Ca(2+), a phenomenon referred to as desensitization or adaptation. Here we show that activation of TRPM8 by cold or menthol evokes a decrease in cellular phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] levels. The decrease in PtdIns(4,5)P(2) levels was accompanied by increased inositol 1,4,5 trisphosphate (InsP(3)) production, and was inhibited by loading the cells with the Ca(2+) chelator BAPTA-AM, showing that it was the consequence of the activation of phospholipase C (PLC) by increased intracellular Ca(2+) concentrations. PtdIns(4,5)P(2) hydrolysis showed excellent temporal correlation with current desensitization in simultaneous patch clamp and fluorescence-based PtdIns(4,5)P(2) level measurements. Intracellular dialysis of PtdIns(4,5)P(2) inhibited desensitization both in native neuronal and recombinant TRPM8 channels. PtdIns(4)P, the precursor of PtdIns(4,5)P(2), did not inhibit desensitization, consistent with its minimal effect in excised patches. Omission of MgATP from the intracellular solution accelerated desensitization, and MgATP reactivated TRPM8 channels in excised patches in a phosphatidylinositol 4-kinase (PI4K)-dependent manner. PLC-independent depletion of PtdIns(4,5)P(2) using a voltage-sensitive phosphatase (ci-VSP) inhibited TRPM8 currents, and omission of ATP from the intracellular solution inhibited recovery from this inhibition. Inhibitors of PKC had no effect on the kinetics of desensitization. We conclude that Ca(2+) influx through TRPM8 activates a Ca(2+)-sensitive PLC isoform, and the resulting depletion of PtdIns(4,5)P(2) plays a major role in desensitization of both cold and menthol responses.

Translation Initiator EIF4G1 Mutations in Familial Parkinson Disease

Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.

Abstract 1347: BAL101553: An optimized prodrug of the microtubule destabilizer BAL27862 with superior antitumor activity

Abstract Background: BAL27862 is a novel small molecule, inducing apoptosis in cancer cells through microtubule destabilization. A series of amino acid-derived BAL27862 prodrugs was evaluated for solubility and in vivo conversion into drug. The Lys-prodrug BAL101553 was further compared with BAL27862 in animal models of human cancer. Methods: Kinetic solubility of compounds was determined by diluting DMSO stock solutions with aqueous buffer. Pharmacokinetics and prodrug conversion were evaluated in mice. In vivo efficacy was analyzed in colon carcinoma SW480 and patient-derived mammary MaCa4151 xenografts. MTD dosing was used in all cases, adapted for each mouse strain. Results: All amino acid-derived prodrugs showed significantly increased aqueous solubility compared to BAL27862, most pronounced at pH 3. At pH 5 and 6.5, the dibasic Lys-prodrug BAL101553 proved to be the most soluble compound (&amp;gt;200 μM). In vivo conversion rates differed significantly between the prodrugs. Highest exposure to the parent drug was obtained with the Lys, Ala and Gly derivatives, whereas the exposure achieved with other amino acid prodrugs (e.g. Phe, Asn, Ser, Trp) was more than twofold lower. Combining high solubility, good conversion and oral bioavailability, BAL101553 was selected for further evaluation. In tumor models, BAL27862 was rapidly distributed into SW480 tumors after i.v. administration of drug or BAL101553. Strikingly, BAL27862 was retained in tumor ∼1.5 times longer after BAL101553 (T1/2: 8.3h) vs. BAL27862 (T1/2: 5.4h) administration. In all models, a higher MTD was reached with the prodrug, related to ∼60% prodrug conversion in vivo. In MaCa4151 xenografts, twice-weekly i.v. administration of BAL101553 (17 mg/kg BAL27862 equivalents [BE]) elicited superior antitumor effects (final T/C=30%; p&amp;lt;0.05 vs. controls) as compared to BAL27862 (10 mg/kg; T/C=66%). Once-weekly dosing of BAL101553 (14 mg/kg BE) and BAL27862 (8 mg/kg) in the SW480 model resulted in final T/C's of 34% and 49% (both p&amp;lt;0.001), resp. Strikingly, fractionation of the same total weekly dose did not significantly affect outcome for either BAL101553 (3x per day once-weekly: T/C=40%; 3x per week: T/C=26%; both p&amp;lt;0.001) or BAL27862 (3x per day once-weekly: T/C=54%; 3x per week: T/C=54%; p=0.001 and p=0.002, resp.), suggesting antitumor response is related to AUC. Again, indications of superior antitumor responses were observed with all prodrug schedules in this model. Conclusions: BAL101553 has been identified as a highly soluble prodrug of BAL27862, which can be administered p.o. or i.v. in the absence of solubilizing excipients known to be associated with adverse side-effects. Its administration facilitates higher tumor exposure to the active agent, with more profound responses in some tumor models. These data, together with a flexible dosing potential, support profiling of BAL101553 in cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1347. doi:10.1158/1538-7445.AM2011-1347

Disease-causing Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator Determine the Functional Responses of Alveolar Macrophages

Alveolar macrophages (AMs) play a major role in host defense against microbial infections in the lung. To perform this function, these cells must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Recently, we demonstrated that murine alveolar macrophages employ the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel as a determinant in lysosomal acidification (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Lysosomes and phagosomes in murine cftr(-/-) AMs failed to acidify, and the cells were deficient in bacterial killing compared with wild type controls. Cystic fibrosis is caused by mutations in CFTR and is characterized by chronic lung infections. The information about relationships between the CFTR genotype and the disease phenotype is scarce both on the organismal and cellular level. The most common disease-causing mutation, DeltaF508, is found in 70% of patients with cystic fibrosis. The mutant protein fails to fold properly and is targeted for proteosomal degradation. G551D, the second most common mutation, causes loss of function of the protein at the plasma membrane. In this study, we have investigated the impact of CFTR DeltaF508 and G551D on a set of core intracellular functions, including organellar acidification, granule secretion, and microbicidal activity in the AM. Utilizing primary AMs from wild type, cftr(-/-), as well as mutant mice, we show a tight correlation between CFTR genotype and levels of lysosomal acidification, bacterial killing, and agonist-induced secretory responses, all of which would be expected to contribute to a significant impact on microbial clearance in the lung.