Design,Synthesized And Evaluation Of Novel Anti-apoptotic Proteins Bcl-2/Mcl-1 Dual Inhibitors

The Bcl-2 family proteins play fundamental roles in the regulation of endogenous apoptosis pathway,and determine the fate of cells through the complex interaction network between anti-apoptotic and pro-apoptotic proteins.Overexpression of Bcl-2 anti-apoptotic proteins is prevalent in cancer cells.Overexpressed anti-apoptotic proteins bind to pro-apoptotic proteins and sequester their physiological functions,leading to the block of apoptosis.Thus,Bcl-2 protein,as a therapeutic target for cancer,has been paid for more than 30 years.The selective Bcl-2 inhibitor Venetoclax was subject to approval by the FDA in 2016 for the treatment of 17p-deleted chronic lymphocytic leukemia.However,the high selectivity towards Bcl-2 protein limited efficacy of Venetoclax in some cancers depending on Mcl-1 or Bcl-xL.Moreover,overexpressed Mcl-1/Bcl-xL make cancer cells resistant to Venetoclax.Considering the fact that inhibition of Bcl-xL leads to the inevitable thrombocytopenia,we ventured the opinion that co-inhibition of Bcl-2/Mcl-1 can synergistically induce apoptosis of cancer cells and avoid thrombocytopenia caused by Bcl-xL inhibition.In this work,Bcl-2/Mcl-1 dual target inhibitors were designed based on pharmacophore mode integration strategy and structure-based drug design.Guided by pharmacophore mode integration strategy,the distinctive pharmacophores of Venetoclax and artesunate were combined through various linkers containing chain/cyclic aliphatic amines and 12 compounds(LSL-A1～A12)were designed and synthesized.All the novel structures were confirmed by 1H-NMR,13C-NMR,LC-MS and HR-MS spectrums.Tested by fluorescence polarization assay,compounds LSL-A1～A7 showed similar inhibitory activities to Venetoclax,whose inhibition constants Ki was lower than the detection limits(Ki<1 nM).In addition,the selectivity against Bcl-2 protein of these compounds was enhanced,all the 12 compounds showed no binding affinity to Bcl-xL and Mcl-1(IC50>200μM).In cell-based tests,LSL-A1-A12 showed more potent antiproliferative activity than DHA and Venetoclax in THP-1 and Molm-13 cell lines(which expressed high level Bcl-2/Mcl-1),with sub-micromole level GI50s(0.02～0.50 μM).Flow cytometry showed that LSL-A2/A3/A5/A6 could efficaciously induce apoptosis of THP-1 cells in a dose-dependent manner,showing much more potent activity than DHA and Venetoclax.Typical dual inhibitor LSL-A5 exhibited excellent antiproliferative activity against THP-1/Molm-13 cell lines,with GI50 value were 0.090 μM and 0.063 μM,respectively.Western blot confirmed that LSL-A5 could indirectly inhibit Mcl-1 protein by significantly up-regulation of Noxa in THP-1 cells,whose mechanism is similar to artemisinin angloues.Moreover,LSL-A5 could potently induce apoptosis of THP-1 cells at the concentration of 0.2μM,exhibiting higher activity than Venetoclax and DHA.The transmembrane permeability,liver microsomal stability and plasma stability of compound LSL-A5 were preliminarily evaluated by in vitro models.We speculated that the extremely low permeability and the instability in plasma(T1/2～20 min)should be held responsible for the bad in vivo antitumor activity of LSL-A5.Based on the structure-based strategy,the characteristics of BH3 binding pockets of three anti-apoptotic proteins(Bcl-2/Mcl-1/Bcl-xL)were analyzed and concluded.P2 pocket,the most different sub-pocket in Mcl-1/Bcl-xL,was exploited for designing the dual inhibitors,considering the special characteristics such as distinct flexibility and ability to accommodate polar groups.Built on the structure of a reported Mcl-1 selective inhibitor 1-(3-(naphthalen-1-yloxy)propyl)-1H-indole-2-carboxylic acid(Indole B),optimizations were focused on increasing the flexibility of acidic moiety and inducting polar 2-carbamoyl groups,37 Bcl-2/Mcl-1 dual inhibitors(LSL-B1～B37)bearing 1,6-disbustituted indole skeleton were designed and synthesized.FPA assay reported that most of them could inhibit Bcl-2/Mcl-1 simultaneously.Preliminary SAR was concluded:1.The hydrophobic chain is an essential moiety for binding,the 4-atom linkers were optimum.The 4-t-Bu and 4-Cl-3,5-dimethyl were the better substituted groups of the distal phenyl;2.The acidic moiety is rather tolerated for modifications.Replacement of acetic acid with benzoic acid(LSL-B37)improved the activity remarkably.The cell-based evaluation showed weak antiproliferative activity in HL-60 cells(GI50～50 μM),which is probably related to the weak Bcl-2/Mcl-1 binding affinity of these compounds.Designed to improve the binding affinity to Bcl-2/Mcl-1,13 compounds(LSL-B38～B50)bearing N-substituted indoles were designed and synthesized based on the structure of LSL-B37.Optimizations were focused on acidic moiety,such as the positions of carboxylic group and acidic side chain.The significant increase of binding affinity was achieved,LSL-B38～B50 showed similar inhibitory activity to positive control AT-101,a pan Bcl-2 inhibitor.Among them,LSL-B45 was the most potent molecule against Bcl-2 and Mcl-1(Bcl-2,Ki=0.15 μM;Mcl-1,Ki=0.14 μM).The SAR was summarized:1.The position of acidic chain on indole core has impact on activity,the site 4 was seemed as a optimal point;2.The substituted position of carboxylic group also showed correlation with activity(o/m>p);3.The necessity of hydrophobic chain was confirmed again,compounds without that moiety showed no binding affinity to Bcl-2/Mcl-1 proteins.Interestingly,some compounds such as LSL-B44～B46 showed decreased selectivity,which could bind to Bcl-xL.The deficiency of 2-carbomayl may be the point.Cell-based assay indicated that some compounds showed moderate antiproliferative activity in HL-60 cells(GI50～10 μM).The typical compound LSL-B45 had better inhibitory activity(GI50=0.89 μM)than positive control(GI50=5.45 μM).To regulate the selectivity of Bcl-2/Mcl-1 and confirm the relationship between the 2-carbamoyl group with selectivity,27 compounds(LSL-B56～B82)bearing 2-substituted carbamoyl indoles were drawn up and synthesized.Cell-free assay showed that all of these compounds showed inhibitory activity against Bcl-2/Mcl-1 and merely no binding affinity to Bcl-xL(IC50>200μM),indicating the important role of 2-carbamoyl group in selectivity and the different capacity of accommodating polar groups of P2 pockets in Mcl-1 and Bcl-xL.Despite of many efforts we made on the substitution of 2-carbamoyl,activity of LSL-B56～B82 achieved no noticeable improvement.Interestingly,compounds bearing 1,4-disubstituted indoles(LSL-B65)showed Mcl-1 selective inhibition.We considered that there are too many flexible bonds in molecular to bind target pocket tightly.Thus,we reduced the rotatable bonds of the linker between benzoic acid and indole,LSL-B83～B89 were designed and synthesized.Besides,carbazole was used as an alternative skeleton for Bcl-2/Mccl-1 dual inhibitors investigation,which produced compounds LSL-B90-B93.The primary research on these compounds indicated that modification on linkers of acidic chain and induction of carbazole was tolerated,all the compounds showed comparable activity as LSL-B37.Among them,compound LSL-B88 showed better selectivity against Mcl-1 protein,which was chosen as a promising lead compound for subsequent studies.In conclusion,105 compounds were designed and synthesized as Bcl-2/Mcl-1 dual inhibitors based on the strategies of pharmacophore integration and structure-based design.The analysis of protein structural difference of Bcl-2/Mcl-1/Bcl-xL and the rational design methods provided a theoretical basis for the design of selective inhibitors of specific subtypes of Bcl-2 anti-apoptotic protein.