The phosphatidylinostitol-3-kinase (PI3K)/AKT/mammalian target of rapamycin signaling pathway is a vital regulator of cell proliferation, growth, and survival, which is frequently overactivated in many human cancers. To this effect, PI3K, which is an important mediator of this pathway, has been pinpointed as a crucial target in cancer therapy and hence the importance of PI3K inhibitors. It was recently reported that defluorination and pyridine-to-pyrimidine ring interconversion increase the potency of specific small-molecule inhibitors of PI3K. Compound 4, an inhibitor with the difluorinated pyrimidine motif, was found to be eight times more potent against PI3K than compound 1, an inhibitor with the trifluorinated pyridine motif. This observation presents the need to rationally resolve the differential inhibitory mechanisms exhibited by both compounds. In this present work, we employed multiple computational approaches to investigate and distinguish the binding modes of 1 and 4 in addition to the effects they mediate on the secondary structure of PI3K. Likewise, we evaluated two other derivatives, compounds 2 with the difluorinated pyridine motif and 3 with the trifluorinated pyrimidine motif, to investigate the cooperativity effect between the defluorination of CF3 and pyridine-to-pyrimidine ring interconversion. Findings revealed that PI3K, upon interaction with 4, exhibited a series of structural changes that favored the binding of the inhibitor at the active-site region. Furthermore, a positive (synergistic) cooperativity effect was observed between CF3 defluorination and pyridine-to-pyrimidine ring interconversion. Moreover, there was a good correlation between the binding free energy estimated and the biological activity reported experimentally. Energy decomposition analysis revealed that the major contributing force to binding affinity variations between 1 and 4 is the electrostatic energy. Per-residue energy-based hierarchical clustering analysis further identified four hot-spot residues ASP841, TYR867, ASP964, and LYS833 and four warm-spot residues ASP836, SER806, ASP837, and LYS808, which essentially mediate the optimal and higher-affinity binding of compound 4 to PI3K relative to 1. This study therefore provides rational insights into the mechanisms by which 4 exhibited superior PI3K-inhibitory activities over 1, which is vital for future structure-based drug discovery efforts in PI3K targeting.