3.6 Identifying BCG ‘mimics’
In order to identify experimentally validated upstream regulators that cause transcriptional changes similar to those induced by BCG, we queried the Connectivity Map (CMap) database of the Broad Institute with BCG-CGS and identified proteins and small-molecule drugs that have strong connectivity scores with BCG (Figure 1). The CMap approach enabled us to compare BCG-CGS with ‘experimentally’ predefined signatures of therapeutic compounds and genetic perturbations (i.e., over expression or knockdown) included in the CMap and ranked according to a connectivity scores (ranging from +100 to −100), representing relative similarity to BCG-CGS. The connectivity score itself is derived using a nonparametric, rank-based, pattern-matching strategy based on the Kolmogorov-Smirnov statistic. All instances in the database are then ranked according to their connectivity scores with BCG-CGS; those at the top (+) are most strongly correlated to the query signature and looked at as BCG mimics, and those at the bottom (−) are most strongly anticorrelated and can reverse BCG’s gene signature.
Our analysis identified three highly enriched classes of genetic knockdown (KD) perturbagens and one pharmacological class of drugs that have positive connectivity scores in alveolar A549 cells (i.e., caused similar transcriptional changes to those induced by BCG in alveolar A549 cells). These hits can be considered as BCG mimics capable of inducing transcriptional changes similar to those caused by BCG vaccine. Therefore, we suggest that BCG mimics can be used as alternatives to BCG vaccination to promote long-lasting beneficial effects on immune cells. The three enriched protein classes are: protein phosphatases (with best positive connection for PPP4C KD), histone deacetylases (with best positive connection for HDAC10 KD followed by HDAC11 KD), and mediator complex proteins (with best positive connection for MED6 KD followed by MED7 KD). Additionally, protein kinase C (PKC) activators were enriched as a drug class; and top 3 PKC activators with highest CMap connectivity scores to BCG-CGS prostratin, phorbol-12-myristate-13-acetate, and ingenol. It is evident that all of the above 4 classes of proteins share one common feature: they participate in the transcriptional and metabolic regulation of immune cells in response to environmental cues including responses to pathogens. All top-scoring PKC activators from the CMap, are also known to have antiviral effects or affect T cell activation.
Remarkably, analyzing top ten CMap positive connections with BCG-CGS obtained from nine cell lines indicated that two compounds are approved antiviral drugs: raltegravir (top 3rd positive connection, an HIV integrase inhibitor) and lopinavir (top 6th positive connection, an HIV protease inhibitor). More interestingly, emetine (top 4th positive connection) and lopinavir were recently validated to inhibit SARS-CoV-2 replication in vitro. We also found evidence in the biomedical literature indicating that MST-312, narciclasine and verrucarin-a possess antiviral activities. All CMap hits are provided in tables S5 and S8 (Supporting Information).
In order to prioritize high confidence BCG genetic mimics, we integrated hypotheses derived independently from the CMap with those predicted by causal reasoning, and accepted common hits only (i.e., CMap positive connections with BCG-CGS that are also predicted as beneficial drug targets by causal reasoning). This analysis resulted 30 high confidence common hits reported in table S9 (Supporting Information).