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Page 2 of 10 Raevsky et al. Neuroimmunol Neuroinflammation 2018;5:33 I http://dx.doi.org/10.20517/2347-8659.2018.34

the preliminary stage of the biological screening of newly designed inhibitors. Based on the structure-function
relationships discovered by recording the empirical score values generated for the screening model, a series of
subsequent in silico predictions of compound inhibitory activity were generated, which led to new structures with
increased inhibitory activity and selectivity towards cathepsin B.

Conclusion: The described molecular modeling strategy could be employed to design novel inhibitors of cathepsin B
enzymatic activity, which could be used to slow down neuroinflammation in neurodegenerative disorders including
Alzheimer’s disease.

Keywords: Anti-inflammatory drugs, auranofin, cathepsins, drug design, fragment-based docking, neuroinflammation,
microglia

INTRODUCTION
Alzheimer’s disease is the most common cause of dementia. It is characterized by abnormal brain protein
deposits including amyloid ß (Aß)-containing plaques and neurofibrillary tangles. In addition, chronic
neuroinflammation, driven by adverse activation of non-neuronal microglial cells, is believed to contrib-
ute to the pathogenesis of this neurodegenerative disease. Microglial immune functions can be regulated
[1,2]
by various cathepsin enzymes, many of which are the components of lysosomes . Cathepsins belong to
[3]
the papain superfamily and are synthesized as inactive pro-enzymes . Cathepsin B, a cysteine protease,
[4-7]
is expressed and can be secreted by activated microglia . Cathepsin B has been proposed as a potential
[8]
therapeutic target to reduce neuroinflammation in Alzheimer’s disease based on observations showing this
protein upregulated in brain tissues, serum and cerebrospinal fluid of Alzheimer’s patients [9-13] . Such high
levels of cathepsin B also correlate with decreased cognition, which could be caused by adverse microglia
activation [11,14] .

A series of pre-clinical studies support using cathepsin B inhibitors to slow the progression of Alzheimer’s
disease. Reduced Aß plaque load and improved memory were reported in a transgenic Alzheimer mice mod-
[15]
el after deleting the cathepsin B gene . In vitro studies showed that cathepsin B inhibitors downregulated
pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-18 [16-18] . Cathepsin
B has been linked to specific microglial functions including their secretion of cytokines, neurotoxicity, and
Aß degradation [5,17,19] . Currently available cathepsin B inhibitors are not specific since they inhibit enzymatic
activity of other cysteine proteases including calpains, as well as cathepsins S and L [3,20-23] . Therefore, novel
highly selective cathepsin B inhibitors should be developed and tested for their ability to ameliorate neuroin-
flammation in neurodegenerative diseases.

Cathepsin B enzymatic activity is inhibited by alpha-macroglobulin from the cystatin family of inhibitors of
[24]
papain-like cysteine peptidases, and by representatives of the equistatin family . There are three additional
groups of naturally occurring cathepsin B inhibitors: the aziridinyl peptides, peptide epoxysuccinyls, and
peptide aldehydes [25,26] . Known synthetic cathepsin B inhibitors can be divided into groups of compounds,
which contain either flavonoids, cyclic sulfates, or nitriles [27,28] . Cathepsins B and K are inhibited with rea-
sonable potency by gold(I)-based compounds such as auranofin (Ridaura), which is clinically used as an
[29]
anti-rheumatic agent, and its analogs . Structure-activity relationship (SAR) studies revealed that replace-
ment of ethyl substituent with a voluminous aryl substituent in auranofin, which is a clinically approved
[30]
triethylphosphine (PEt ) gold-containing drug, significantly increased its anti-inflammatory activity . A
3
triphenylphosphine gold compound was shown to be a more effective cathepsin B inhibitor (IC = 337 nmol/L)
50
[30]
than its parent compound auranofin (IC > 250 µmol/L) . Further studies confirmed that compounds with
50
more than one aryl group (e.g., triarylphosphines) were much stronger cathepsin B inhibitors than trieth-
[31]
ylphosphine Au(I)-containing derivatives .