Johannes Kornhuber; Markus Muehlbacher; Stefan Trapp; Stefanie Pechmann; Astrid Friedl; Martin Reichel; Christiane Mühle; Lothar Terfloth; Teja W. Groemer; Gudrun M. Spitzer; Klaus R. Liedl; Erich Gulbins; Philipp Tripal

“ Moreover, FIASMAs more frequently violate Lipinski's Rule-of-Five than compounds without effect on ASM. Inhibition of ASM appears to be associated with good permeability across the blood-brain barrier. In the present investigation, we developed a novel structure-property-activity relationship by using a random forest-based binary classification learner. Virtual screening revealed that only six out of 768 (0.78%) compounds of natural products functionally inhibit ASM, whereas this inhibitory activity occurs in 135 out of 2028 (6.66%) drugs licensed for medical use in humans. ”

“ Our results prove that functional inhibition of ASM does not depend on a specific molecular drug class. Instead of specific structural prerequisites, functional inhibition of ASM requires specific physicochemical characteristics of compounds, resulting in high intra-lysosomal concentrations and partitioning of the drug into the inner leaf of the lysosomal membrane. ”

“ Molecular descriptors were calculated by the ACD/LogD Suite, version 10 [76] (the two most basic pKa-values, the most acidic pKa-value, logP-value) , by visual inspection of the molecules [number of heavy atoms at the most basic nitrogen atom (k [37] ) ] and by MOE 2009.10 [82] . The lysosomal concentration of each drug was calculated by a previously developed numerical single cell model [34] (see above) and was also used as a molecular descriptor (CLys) . ”

“ Functional inhibitors of ASM (FIASMAs) are an important class of drugs with newly emerging broad clinical applications. In the present study we have identified 27 novel FIASMAs. Based on a large set of experimental data, we have developed a chemoinformatic model for the accurate prediction of functional inhibition of ASM. Furthermore, we describe important properties of FIASMAs, such as BBB permeability. ”

“ Inhibition of ASM results in anti-apoptotic, proliferative and anti-inflammatory effects. Furthermore, ASM could play a key role in the pathophysiology of depression and in the action of antidepressant drugs [17] . Inhibitors of ASM therefore hold promise for a number of new clinical therapies. Several examples are given here: Induction of stroke by experimental ischemia of the brain was shown to correlate with an activation of the ASM and a release of ceramide [54] , [55] . Importantly, ASM-deficient mice were protected from tissue damage caused by focal cerebral ischemia. ”

“ The experimental set investigated here is showing comparable divrel values to the ATC set. It is therefore appropriate to apply conclusions from the experimental set onto the larger ATC collection. The set of 72 FIASMAs is a subset of the experimental set, which in turn is largely a subset of the ATC drug library. Therefore, these three sets occupy a comparable region of the chemical space, as all these sets are mainly composed of small drug like molecules. The Pure Natural Products cannot easily be compared to the drug-like molecules as their chemicals space differs. ”

“ AC is a lysosomal enzyme degrading ceramide to sphingosine. Cationic amphiphilic agents like desipramine, chlorpromazine and chloroquine have been shown to result in both, a reduced level of AC protein and in an increased level of cellular ceramide [67] , [68] . Despite its potential importance, the influence of medically used drugs on AC has not yet been systematically studied. In a cell culture-based pilot study on more than 100 small drug-like compounds (CM, unpublished) , FIASMAs had a significantly stronger AC inhibitory effect than non-FIASMAs. ”

“ Missing values for the most basic pKa-values were substituted by 0, missing most acidic pKa-values were substituted by 15, missing values for k were substituted by 12. Otherwise, we used only molecular descriptors that were available for all compounds and thereby obtained a total of 708 molecular descriptors. The presence of irrelevant and redundant attributes may reduce the performance of learners such as decision tree and random forest [87] . Therefore, before applying random forest learners we reduced the dimensionality of the data by applying feature selection methods. ”

“ Strength: We identified 27 novel FIASMAs, many of them FDA-approved for clinical use in humans. Model construction was based on a large number of experimentally investigated compounds, and used standard Molecular Operating Environment (MOE) descriptors as well as a multitude of novel hypothesis-driven molecular descriptors, like k, n_pdN, CSA and calculated lysosomal drug concentration (Table 5) . ”

“ In addition to the set of 101 compounds previously experimentally investigated with regard to functional inhibition of ASM [37] , we selected 175 small organic, drug-like compounds or natural products. The selection aimed at obtaining compounds with high structural diversity, with diversity regarding strength and number of acidic and basic functional groups, and diversity regarding logP values. Basic lipophilic compounds were overrepresented in order to obtain a sufficient number of FIASMAs and in order to avoid a greatly unbalanced class distribution for classification tasks. ”

“ All compounds were used in the highest purity available.
Cell culture
Human brain neuroglioma H4-cells were purchased from Promochem (Wesel, Germany) . H4-cells were cultured as described previously [37] . Only cells tested negative for mycoplasma infection were used in the experiments.
Kinetics of lysosomal accumulation
Usually, 30 min of incubation of H4-cells with the test drug was sufficient to induce functional inhibition of ASM. Due to slow accumulaion kinetics some compounds required a longer incubation time. ”

“ Because of the unbalanced class distribution, the Youden-Index [97] – [98] (sensitivity + specificity−1) was chosen as primary performance measure evaluating models based on two-class learners. We used a random forest learner [99] for binary classification. In contrast to a simple decision tree learner, random forest learners are more resistant against overfitting and result in more stable models, meaning that small changes in the training set do not lead to completely different models [100] , [101] , and lead to better predictivity on unknown compounds. ”

“ The severity of Niemann-Pick disease correlates with the decrease of ASM activity [15] . However, studies using cells derived from Niemann-Pick disease patients or from ASM knock-out mice revealed that the deficiency of this enzyme might also have beneficial consequences, including anti-apoptotic and cytoprotective effcts. In fact, there is increasing evidence that ASM activation and ceramide accumulation play a central role in the development of common human diseases (reviewed in Smith & Schuchman [16] ) . ”

“ We term this measure “relative structural diversity” (divrel) .
File:Pone.0023852.e001 Higher values for divrel indicate higher structural diversity. The values for divrel can vary from just above zero (when there is only one cluster) to 1 (when each compound defines a separate cluster) . For our analysis, we have used the structural TGT keys [103] , [104] consisting of 2D pharmacophore features with atom types and distances.
Violation of Lipinski's Rule-of-Five
Lipinski's Rule-of-Five [49] identifies compounds with properties that would likely make them an orally active drug in humans. ”

“ Our results demonstrate that it is important to consider slow lysosomal accumulation kinetics in compounds with very high pKa- and/or very high logP-values and in compounds with two basic nitrogen atoms. ”

“ Blocking of this cascade by ASM inhibitors should be preventive and/or therapeutic against the endotoxic shock syndrome. Finally, an imbalance between ASM and acid ceramidase followed by ceramide accumulation has been demonstrated in the hereditary disease cystic fibrosis [62] . In experimental animals, FIASMAs normalize pulmonary ceramide and inflammation [63] . In a clinical pilot study, amitriptyline 6 led to an improved respiratory function in affected patients [64] . Further examples include the treatment of e.g. ”

“ It has previously been suggested that the lowest cross-sectional area of a molecule perpendicular to the amphiphilic axis determines the insertion of a molecule into a biomembrane and thereby its passive diffusion across the BBB [83] , [84] . ”

“ Cheng, G & Schuchman, EH (2000) , “Pivotal role for acidic sphingomyelinase in cerebral ischemia-induced ceramide and cytokine production, and neuronal apoptosis.”, J Mol Neurosci 15: 85–97, 11220788, pmid:11220788 ↑ Altura, BM; Gebrewold, A; Zheng, T & Altura, BT (2002) , “Sphingomyelinase and ceramide analogs induce vasoconstriction and leukocyte-endothelial interactions in cerebral venules in the intact rat brain: Insight into mechanisms and possible relation to brain injury and stroke.”, Brain Res Bull 58: 271–278, 12128152, pmid:12128152 ↑ Pascual, M ”