Hähnke V Rupp M Hartmann AK Schneider G 2013 Previously, we proposed a ligand-based virtual screening technique (PhAST) based on global alignment of linearized interaction patterns. Here, we applied techniques developed for similarity assessment in local sequence alignments to our method resulting in p-values for chemical similarity. We compared two sampling strategies, a simple sampling strategy and a Markov Chain Monte Carlo (MCMC) method, and investigated the similarity of sampled distributions to Gaussian, Gumbel, modified Gumbel, and Gamma distributions. The Gumbel distribution with a Gaussian correction term was identified as the most similar to the observed empirical distributions. These techniques were applied in retrospective screenings on a drug-like dataset. Obtained p-values were adjusted to the size of the screening library with four different methods. Evaluation of E-value thresholds corroborated the Bonferroni correction as a preferred means to identify significant chemical similarity with PhAST. An online version of PhAST with significance estimation is available at http://modlab-cadd.ethz.ch/. https://sonar.ch/global/documents/57700 eng info:eu-repo/semantics/altIdentifier/doi/10.1002/minf.201300021 info:eu-repo/semantics/altIdentifier/pmid/27481770 Molecular informatics. - 2013 Chemical similarity Pharmacophore Sequence alignment Significance Virtual screening Pharmacophore Alignment Search Tool (PhAST): Significance Assessment of Chemical Similarity. http://purl.org/coar/resource_type/c_6501