Potential Alzheimer’s Disease Therapeutics Among Weak Cysteine Protease Inhibitors Exhibit Mechanistic Differences Regarding Extent of Cathepsin B Up-Regulation and Ability to Block Calpain

Abstract

Cysteine protease inhibitors have long been part of drug discovery programs for Alzheimer’s disease (AD), traumatic brain injury (TBI), and other disorders. Select inhibitors reduce accumulating proteins and AD pathology in mouse models. One such compound, Z-Phe-Aladiazomethylketone (PADK), exhibits a very weak IC50 (9–11 μM) towards cathepsin B (CatB), but curiously PADK causes marked up-regulation of the Aβ-degrading CatB and improves spatial memory. Potential therapeutic and weak inhibitor E64d (14 μM IC50) also up-regulates CatB. PADK and E64d were compared regarding the blockage of calcium-induced cytoskeletal deterioration in brain samples, monitoring the 150-kDa spectrin breakdown product (SBDP) known to be produced by calpain. PADK had little to no effect on SBDP production at 10–100 μM. In contrast, E64d caused a dose-dependent decline in SBDP levels with an IC50 of 3–6 μM, closely matching its reported potency for inhibiting μ-calpain. Calpain also cleaves the cytoskeletal organizing protein gephyrin, producing 49-kDa (GnBDP49) and 18-kDa (GnBDP18) breakdown products. PADK had no apparent effect on calcium-induced gephyrin fragments whereas E64d blocked their production. E64d also protected the parent gephyrin in correspondence with reduced BDP levels. The findings of this study indicate that PADK’s positive and selective effects on CatB are consistent with human studies showing exercise elevates CatB and such elevation correlates with improved memory. On the other hand, E64d exhibits both marginal CatB enhancement and potent calpain inhibition. This dual effect may be beneficial for treating AD. Alternatively, the potent action on calpain-related pathology may explain E64d’s protection in AD and TBI models.

The Role of Lysosomes in a Broad Disease-Modifying Approach Evaluated across Transgenic Mouse Models of Alzheimer’s Disease and Parkinson’s Disease and Models of Mild Cognitive Impairment

“…No difference in exploratory mobility in the maze was evident among the groups of mice (lower graph in Figure 5A). In addition to PADK being linked to cognitive repair, SD1002 from Table 1 also exhibited 83% functional recovery in the AD mouse model (Mann–Whitney test: p = 0.033), and E64d is a compound shown by several studies to improve cognition [27]. From the PADK results, it is noteworthy that a within-animal analysis between percentage increase in hippocampal CatB and the extent of functional recovery found a positive relationship as determined by linear regression ( R = 0.860; p < 0.001).…”

“…CatB enzymatic activity was assessed in brain sample aliquots using the InnoZyme Cathepsin B Activity Assay Kit (Millipore) as previously described [14,27]. Equal amounts of brain homogenates (10 μg of protein) were assessed in duplicate per sample for proteolytic activity using the Z-Arg-Arg-7-amido-4-methylcoumarin (AMC) substrate and a SpectraMax M3 microplate reader.…”

“…Note that plasma CatB measures increased with exercise in humans and monkeys [25], and the resulting CatB levels correlated with improved memory. While previous studies suggested some neuroprotective agents acted by inhibiting CatB, evidence points to such protectants as acting through a non-CatB pathway [26,27], in contrast to CatB-enhancing properties found linked to synaptic and neuronal repair. The CatB proteolytic responses support early and prolonged signaling events that work to maintain proteostasis and reduce pathogenic protein deposition and, when effective, they perhaps explain the extended nature of AD in many individuals.…”

Cathepsins in the Pathophysiology of Mucopolysaccharidoses: New Perspectives for Therapy

“…The inhibition of CTSs has been widely explored over the last decades in the field of chronic inflammatory diseases [27,37,204,205], cardiovascular diseases [10,19,181], osteoporosis [70][71][72][73], arthritis [28,206], kidney diseases [30][31][32]84], pancreatitis [207], obesity [208][209][210], cancer [25,34,48,74,82,211], neurodegenerative diseases [39,41,184,185,212,213], and many other pathological states. Multiple inhibitors are currently available, ranging from reversible covalent inhibitors to irreversible inhibitors [214][215][216][217][218] (Table 4).…”

Engulfment and cell motility protein 1 potentiates diabetic cardiomyopathy via Rac-dependent and Rac-independent ROS production

“…Moreover, previous studies reported the involvement of a calcium-activated protease, calpain, in the development of diabetic cardiomyopathy via disrupting ATP synthase and inducing ROS generation (27,28). Calpain activity, measured as ratios of degradation product (BDP145) of αII-spectrin to intact αII-spectrin with and without calcium (29), was enhanced as the Elmo1 expression increased (Supplemental Figure 8D), suggesting some role for calpain in the ELMO1-linked enhancement of cardiomyopathy.…”