Creek Darren, Giannangelo Carlo, Challis Matthew, Siddiqui Ghizal, Edgar Rebecca, Malcolm Tess, Webb Chaille, Drinkwater Nyssa, Vinh Natalie, MacRaild Christopher, Counihan Natalie, Duffy Sandra, Wittlin Sergio, Devine Shane, Avery Vicky, de Koning-Ward Tania, Scammells Peter, McGowan Sheena
Res Sq. 2024 Apr 26:rs.3.rs-3251230. doi: 10.21203/rs.3.rs-3251230/v3.
New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant ( A-M1) and ( A-M1) M1 metalloaminopeptidases, with selectivity over other and human aminopeptidases, and displayed excellent antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets A-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on A-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of A-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy.
迫切需要通过新机制发挥作用的新型抗疟药物候选物来对抗疟疾耐药性。在此，我们描述了使用选择性抑制剂MIPS2673对M1丙氨酰金属氨肽酶作为有吸引力的药物靶点进行的多组学化学验证。MIPS2673对重组（A-M1）和（A-M1）M1金属氨肽酶表现出强效抑制作用，对其他和人类氨肽酶具有选择性，并显示出优异的抗疟活性且对宿主无明显细胞毒性。基于全寄生虫裂解物的热稳定性和有限蛋白水解的正交无标记化学蛋白质组学方法表明，MIPS2673仅靶向寄生虫中的A-M1，有限蛋白水解还能够将A-M1上的结合位点估计在X射线晶体学确定的约5 Å范围内。最后，通过非靶向代谢组学进行的功能研究表明，MIPS2673抑制A-M1在血红蛋白消化中的关键作用。综合来看，我们无偏的多组学靶点反卷积方法证实了MIPS2673的靶向活性，并验证了对M1丙氨酰金属氨肽酶的选择性抑制是一种有前景的抗疟策略。
