A Clerodane Diterpene from <i>Aquarius scaber</i> (Rataj) Christenh. & Byng Leaves as Inhibitor of <i>Leishmania mexicana</i> Trypanosomatid Enzymes

The clerodane diterpenoid 2-oxo-5α,8α-cleroda-3,13-dien-16,15-olide (1) isolated from the leaves of A. scaber was identified by IR, 1H and 13C NMR (1D and 2D) and HRESIMS spectrometric analysis. This is the first report of this diterpene in the Aquarius genus. The inhibitory potential of 1 has been determined against cysteine proteases from Leishmania mexicana rCPB2.8, rCPB3.0 and rH84Y. The inhibitory constants (Ki and αKi) as well as the mechanism of action were also investigated. Compound 1 was 7- and 18-fold more potent in the inhibition of rCPB3.0 (IC50 = 4.2 µM) than rCPB2.8 (IC50 = 20 µM) and rH84Y (IC50 = 75 µM). From the kinetic mechanisms of inhibitor binding, we found that compound 1 has a higher affinity to rCPB3.0 (Ki = 7.5 µM and αKi = 7.4 µM) than rCPB2.8 (Ki = 15.4 µM, αKi = 14.7 µM) and rH84Y (Ki = 81.4, and αKi = 27.9 μM and βKi = 41.5 µM). Compound 1 also presented a non-competitive linear simple inhibition mechanism at both enzymes rCPB3.0 and rCPB2.8. On the other hand, 1 presented a positive cooperativity mechanism of inhibition at rH84Y.从A. scaber叶片中分离得到的克罗丹二萜类化合物2-氧代-5α,8α-克罗丹-3,13-二烯-16,15-内酯（1），经红外光谱（IR）、一维（1D）与二维（2D）氢核磁共振谱（¹H NMR）、碳核磁共振谱（¹³C NMR）及高分辨电喷雾电离质谱（HRESIMS）分析鉴定。这是该二萜类化合物首次在Aquarius属中被报道。已测定化合物1对墨西哥利什曼原虫（Leishmania mexicana）来源的半胱氨酸蛋白酶（cysteine proteases）rCPB2.8、rCPB3.0及rH84Y的抑制活性，并对其抑制常数（Ki、αKi）与作用机制展开了研究。相较于rCPB2.8（半数抑制浓度（IC50）=20 μM）与rH84Y（IC50=75 μM），化合物1对rCPB3.0的抑制活性分别为其7倍与18倍，其对rCPB3.0的IC50为4.2 μM。通过抑制剂结合动力学机制分析发现，相较于rCPB2.8（Ki=15.4 μM，αKi=14.7 μM）与rH84Y（Ki=81.4 μM，αKi=27.9 μM，βKi=41.5 μM），化合物1对rCPB3.0具有更高的亲和力，其对rCPB3.0的Ki=7.5 μM且αKi=7.4 μM。化合物1对rCPB3.0与rCPB2.8均表现为非竞争性线性简单抑制（non-competitive linear simple inhibition）机制。而针对rH84Y，化合物1则呈现出正协同性抑制机制（positive cooperativity mechanism of inhibition）。