Water Bridges Play a Key Role in Affinity and Selectivity for Malarial Protease Falcipain‑2

Falcipain-2
(FP-2) is hemoglobinase considered an attractive drug
target of Plasmodium falciparum. Recently,
it has been shown that peptidomimetic nitriles containing a 3-pyridyl
(3Pyr) moiety at P2 display high affinity and selectivity for FP-2
with respect to human cysteine cathepsins (hCats), outperforming other
P2-Pyr isomers and analogs. Further characterization demonstrated
that certain P3 variants of these compounds possess micromolar inhibition
of parasite growth in vitro and no cytotoxicity against
human cell lines. However, the structural determinants underlying
the selectivity of the 3Pyr-containing nitriles for FP-2 remain unknown.
In this work, we conduct a thorough computational study combining
MD simulations and free energy calculations to decipher the bases
of the selectivity of the aforementioned nitriles. Our results reveal
that water bridges involving the nitrogen and one carboxyl oxygen
of I85 and D234 of FP-2, respectively, and the nitrogen of the neutral
3Pyr moiety, which are either less prevalent or nonexistent in the
other complexes, explain the experimental activity profiles. The presence
of crystallographic waters close to the bridging water positions in
the studied proteases strongly supports the occurrence of such interactions.
Overall, our findings suggest that selective FP-2 inhibitors can be
designed by promoting water bridge formation at the bottom of the
S2 subsite and/or by introducing complementary groups that displace
the bridging water.

Falcipain-2（FP-2）作为一种被寄希望于的恶性疟原虫药物靶点，其血红素酶的特性备受关注。近期研究表明，含有3-吡啶（3Pyr）基团的肽类模拟物对FP-2表现出极高的亲和力和选择性，相对于人类半胱氨酸组织蛋白酶（hCats），其性能优于其他P2-Pyr同系物和类似物。进一步的表征分析显示，这些化合物中的某些P3变异体在体外对寄生虫生长具有微摩尔级的抑制作用，同时对人类细胞系无细胞毒性。然而，3Pyr含氮化合物对FP-2选择性的结构决定因素尚不明确。在本研究中，我们通过结合分子动力学模拟和自由能计算，对上述化合物的选择性基础进行了深入研究。我们的结果表明，涉及FP-2中I85和D234的氮及其一个羧基氧的水桥，以及中性3Pyr基团的氮，这些在其它复合物中要么较少见，要么不存在，正是解释实验活性谱的原因。研究中的蛋白酶在桥接水位置附近存在晶体水，这强烈支持了此类相互作用的产生。总体而言，我们的发现表明，通过促进S2亚位点底部的水桥形成或引入互补基团以取代桥接水，可以设计出针对FP-2的选择性抑制剂。