Discovery of Piplartine as a Potent Multitargeting Anti‐HIV and Anti‐TB Lead Molecule: Network Pharmacology, In silico, and In vitro Studies to Analyze Its Role in HIV/TB Co‐infection

ABSTRACT Piplartine (PL) modulates key cancer‐related signalling pathways. Though these pathways also influence immune responses during human immunodeficiency virus (HIV) and Mycobacterium tuberculosis (Mtb) infection, PL's effects on these infections remain unexplored. This study aims to investigate PL's therapeutic potential against HIV, Mtb, and co‐infection using network pharmacology, in silico, and in vitro approaches. Gene targets associated with HIV, Mtb, and PL were retrieved from public databases, and a protein‐protein interaction (PPI) network was constructed to identify 16 hub genes among 53 common targets. Gene Ontology and KEGG pathway enrichment analyses highlighted TP53, STAT3, HSP90AA1, IL1B, and CASP3 as key regulators. in silico studies revealed PL's strong affinity for host, HIV, and Mtb targets. Isolated PL exhibited potent anti‐HIV activity with an EC 50 of 0.735 µg/mL in TZM‐bl cells, indicating strong efficacy at sub‐cytotoxic levels (CC 50 5.24 µg/mL). It inhibited HIV‐1 replication in strains HIV‐1 VB028 and HIV‐1 UG070 . PL inhibited RTase and Protease activity by 75% and 58.6%, respectively, and lowered p24 antigen levels by ∼84% at day 5 postinfection. Viral load dropped to 66,180 copies/mL by day 11, compared to >1,000,000 copies/mL in controls. Additionally, PL showed antimicrobial activity against Mtb H37Rv with an MIC of 37.5 µg/mL, highlighting its potential as a promising lead candidate against HIV/Mtb co‐infections.