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).