Malaria is an infectious disease caused by Plasmodium parasites. It results in 
an annual death-toll of ~ 600,000. Resistance to all medications currently in 
use exists, and novel antimalarial drugs are urgently needed. Plasmepsin V (PmV) 
is an essential Plasmodium protease and a highly promising antimalarial target, 
which still lacks molecular characterization and drug-like inhibitors. PmV, 
cleaving the PExEl motif, is the key enzyme for PExEl-secretion, an 
indispensable parasitic process for virulence and infection. Here, we describe 
the accessibility of PmV catalytic pockets to inhibitors and propose a novel 
strategy for PmV inhibition. We also provide molecular and structural data 
suitable for future drug development. Using high-throughput platforms, we 
identified a novel scaffold that interferes with PmV in-vitro at picomolar 
ranges (~ 1,000-fold more active than available compounds). Via systematic 
replacement of P and P' regions, we assayed the physico-chemical requirements 
for PmV inhibition, achieving an unprecedented IC50 of ~20 pM. The 
hydroxyethylamine moiety, the hydrogen acceptor group in P2', the lipophilic 
groups upstream to P3, the arginine and other possible substitutions in position 
P3 proved to be critically important elements in achieving potent inhibition. 
In-silico analyses provided essential QSAR information and model validation. Our 
inhibitors act 'on-target', confirmed by cellular interference of PmV function 
and biochemical interaction with inhibitors. Our inhibitors are poorly 
performing against parasite growth, possibly due to poor stability of their 
peptidic component and trans-membrane permeability. The lowest IC50 for parasite 
growth inhibition was ~ 15 μM. Analysis of inhibitor internalization revealed 
important pharmacokinetic features for PExEl-based molecules. Our work disclosed 
novel pursuable drug design strategies for highly efficient PmV inhibition 
highlighting novel molecular elements necessary for picomolar activity against 
PmV. All the presented data are discussed in respect to human aspartic proteases 
and previously reported inhibitors, highlighting differences and proposing new 
strategies for drug development.