Plasmodium falciparum HSP90 inhibitors show divergent resistance despite a shared ATP-binding site.

Drug resistance poses a major challenge across therapeutic areas including malaria, yet factors governing resistance propensity remain poorly understood. We demonstrate that two HSP90 inhibitors targeting the identical ATP-binding site exhibit dramatically different resistance profiles in P. falciparum. Geldanamycin readily selected 10 distinct resistance mutations conferring up to 22-fold resistance, while AUY-922 required 44 weeks to yield a single A41S mutation with only 2-fold resistance. Resistance mutations mapped throughout geldanamycin's binding pocket but localized near the ATP-binding site for AUY-922. Strikingly, A41S enhanced AUY-922 binding affinity via additional hydrogen bonding, yet stronger binding paradoxically correlated with resistance. Conditional HSP90 knockdown increased geldanamycin sensitivity but not AUY-922 activity, indicating distinct target dependencies despite shared binding sites. We propose AUY-922's lower resistance risk reflects engagement of multiple HSP90 family members. These findings demonstrate that resistance risk cannot be predicted from binding site identity alone, informing development of more durable therapeutics.