Potent memapsin 2 (β-secretase) inhibitors: Design, synthesis, protein-ligand X-ray structure, and in vivo evaluation

Abstract

Structure-based design, synthesis and biological evaluation of a series of peptidomimetic β-secretase inhibitors incorporating hydroxyethylamine isosteres are described. We have identified inhibitor 24 which has shown exceedingly potent activity in memapsin 2 enzyme inhibitory (K i 1.8 nM) and cellular (IC 50 = 1 nM in Chinese hamster cells) assays. Inhibitor 24 has also shown very impressive in vivo properties (up to 65% reduction of plasma Aβ) in transgenic mice. The X-ray structure of protein-ligand complexes of memapsin 2 revealed critical interactions in the memapsin 2 active site. KeywordsAlzheimer; design; synthesis; inhibitor; memapsin 2; secretase Memapsin 2 (β-secretase, BACE 1) has become a major target for the development of inhibitor drugs for the treatment of Alzheimers's Disease (AD). 1 Memapsin 2 is the first protease that cleaves β-amyloid precursor protein (APP) in the pathway leading to the generation of amyloid-β peptide (Aβ) in the brain. 2,3 The excess level of Aβ results in the formation of amyloid plaques and neurofibrillary tangles. The neurotoxicity of Aβ ultimately leads to neuronal death, brain inflammation, dementia and AD. 4 Consequently, inhibition of memapsin 2 has emerged as an excellent therapeutic target for the intervention of AD. On the basis of initial kinetics and substrate specificity information, we designed a number of potent inhibitors incorporating a nonhydrolyzable Leu-Ala hydroxyethylene dipeptide isostere. 5, 6 One of the early key inhibitors was OM99-2 (1 , Figure 1). It has shown potent inhibitory activity (K i = 1.6 nM) for human memapsin 2. 6a An X-ray crystal structure of 1-bound memapsin 2 provided critical molecular insight and drug-design templates for the memapsin 2 active site. 7 Based upon the crystallographic information and preliminary structure-activity relationship studies, we then designed a number of highly potent and selective inhibitors. 8 Inhibitor 2 incorporating Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. methylsulfonyl alanine as the P 1 -ligand and oxazolylmethyl urethane as the P 3 -ligand exhibited excellent potency (K i = 0.12 nM against memapsin 2) and selectivity over BACE-2 (3800-fold) and cathepsin D (2500-fold). 8 However, its cellular inhibitory potency was in the low micromolar range (IC 50 of 1.4 μM) in Chinese hamster ovary cells. NIH Public AccessTo further improve inhibitor properties, we subsequently developed inhibitors incorporating substituted isophthalamides as P 2 -P 3 ligands in combination with the Leu-Ala hydroxyethylene dipeptide isostere. 9 As shown in Figure 1, inh...

Discovery of potent β-secretase (bace-1) inhibitors by the synthesis of isophthalamide-containing hybrids

“…Coburn provided a possible explanation for the unexpected phenomena [23] that the α-methyl of (4-fluorophenyl)ethyl of 3 at the 3-position packs firmly against Ile110 of BACE-1, which orients the 4-fluorophenyl ring toward S 3 and creates a novel S 3 subpocket (S 3 SP ). The subpocket is unique, different from other reported BACE-1 complexed structures, which may make (4-fluorophenyl) ethyl as a suitable substituent as R 3 , even for HE-based inhibitors [14,15] , although it is relatively large for the S 3 pocket. The special orientation of the molecules in the active pockets of BACE-1 is closely associated with the groups of the 5-position of N-terminal isophthalamide, which may result in N-methyl(methylsulfonyl)amino being more preferable than the nitro group at this site when (4-fluorophenyl)ethyl group is selected as R 3 .…”

“…Compounds 10 and 11, which contain the substituent, did show nanomolar activities. The unexpected results pushed us to carefully study the complex crystal structures of 3 and 4 [14,15,23,24] . Coburn provided a possible explanation for the unexpected phenomena [23] that the α-methyl of (4-fluorophenyl)ethyl of 3 at the 3-position packs firmly against Ile110 of BACE-1, which orients the 4-fluorophenyl ring toward S 3 and creates a novel S 3 subpocket (S 3 SP ).…”

“…Moreover, BACE-1 gene knockout mice showed no Aβ production and appeared healthy, which offered further powerful in vivo evidence for the opinion that BACE-1 was an attractive AD target and inhibition of the protease could effectively reduce Aβ formation, thereby halting the progression of AD [11,12] . Since first discovered in 1999, numerous BACE-1 inhibitors have been designed, synthesized and evaluated the biological activities, in which some molecules caused an in vivo decrease of amyloids when used in animal models (eg, 1, 2) [13][14][15] , especially compound GRL-7234 bearing the HE scaffold, which has been shown to induce a reduction of Aβ 40 production in transgenic mice after a single intraperitoneal administration ( Figure 1) [14] . However, the majority of these compounds are peptide-based analogs [16][17][18][19][20][21] that, if used as drug agents, will face formidable difficulties due to their vulnerability to degradative enzymes, rapid biliary clearance and poor oral absorption [22] .…”

Structure and modeling in the design of β‐ and γ‐secretase inhibitors

“…Several examples are shown in Figure 10. These can be roughly classified by their transition state mimic and include: (1) peptidomimetic designs incorporating statine Hu et al, 2003Hu et al, , 2004Lamar et al, 2004;Tung et al, 2002], hydroxymethylcarbonyl [Hamada et al, 2006[Hamada et al, , 2008aShuto et al, 2003], or hydroxyethylene [Brady et al, 2004;Ghosh et al, 2006;Hanessian et al, 2005;Hom et al, 2004;Xiao et al, 2006]; (2) nonpeptidic designs incorporating a reduced amide , hydroxymethylcarbonyl [Hamada et al, 2008a,b] hydroxyethylene [Ghosh et al, 2007], hydroxyethylamine Freskos et al, 2007a,b;Ghosh et al, 2008;Kortum et al, 2007;Maillard et al, 2007;Park et al, 2008;Stachel et al, 2004Stachel et al, , 2006Stauffer et al, 2007], aminoethylene , or tertiary carbinamine [Lindsley et al, 2007;Rajapakse et al, 2006]; (3) arylpiperazines [Garino et al, 2006]; (4) acylguanidines Fobare et al, 2007;Jennings et al, 2008]; (5) dihydroquinazolines [Baxter et al, 2007]; (6) isocytosines Geschwindner et al, 2007]; (7) pyrrolidine/piperidines [Iserloh et al,Fig. 9.…”

Estudos de QSAR 3D para um conjunto de inibidores de butirilcolinesterase humana

“…[8][9][10] Segundo a hipótese da cascata amiloide, os níveis elevados de proteínas insolúveis b-Amiloides (Ab) são responsáveis pela formação de placas amiloides e emaranhados neurofibrilares que estão relacionados com morte celular, processos inflamatórios, demência e DA. 11 Embora o desenvolvimento de fárma-cos que bloqueiem ou revertam a formação de proteínas insolúveis bAmiloides seja uma área de grande interesse, 12,13 as terapias disponíveis atualmente para o tratamento de DA visam manter e facilitar a atividade dos sistemas colinérgicos sobreviventes. 14 Nesse sentido, a principal estratégia terapêutica disponível se baseia na inibição das colinesterases (ChEIs), uma vez que a modulação dos níveis de ACh é feita por duas enzimas, a acetilcolinesterase (AChE -EC 3.1.1.7) e butirilcolinesterase (BuChE -EC 3.1.1.8).…”