Development of Cathepsin B Positive Modulators to Treat Multi‐Proteinopathic Dementias: Exploiting the Lysosomal Avenue of Protein Clearance to Promote Synaptic Recovery
Authors: Pait Morgan C , Elliott Lyndsie , Hafner‐Ruiz Sarah , Mundell Cary , Patterson Walter , Romine Heather , Ikonne Uzoma , Bahr Ben A. 
Published: November 2023

Alzheimer's disease (AD) is characterized by pathological assembly states of Aβ42, tau aggregates, and other age‐related protein accumulations. Inefficient clearance contributes to AD, thus our drug discovery efforts have targeted the protein clearing role of lysosomes. The compound Z‐Phe‐Ala‐diazomethylketone (PADK; or Z‐FA‐DMK) positively modulates the lysosomal pathway by upregulating the active form of cathepsin B (CatB), an enzyme which degrades Aβ42, reducing disease parameters and synaptic decline in transgenic mouse models of early‐onset familial AD (Mueller‐Steiner et al. 2006: Neuron 51:703; Butler et al. 2011: PLoS One 6:e20501; Viswanathan et al. 2012: ACS Med Chem Lett 3:920) as well as AD lacking familial mutations (Wang et al. 2012: JBC 287:39834). Similarly, PADK promotes both PHF‐tau clearance and synaptic maintenance in the hippocampal slice model of protein accumulation pathology (Bendiske & Bahr 2003: JNEN 62:451; Ryzhikov & Bahr 2008: J Mol Neurosci 34:131; Bahr et al. 2012: Rejuvenation Res 15:189) Here, further development of synthesized PADK derivatives and related small molecules tested for enhanced levels of 25‐ and 30‐kDa CatB species in rat brain slice cultures and human iPSC‐derived neurons. PADK primarily modulates the 30 kDa form, whereas select derivatives appear to modulate the two CatB bands equally. In addition, a 2‐D similarity method is currently being assessed for distinguishing between at least two classes of modulators, using molecular mechanics data and Tanimoto coefficients, and may improve SAR determination. Protein clearance and synaptic protection were tested in 3xTg‐AD mice and other models, compounds administered ip (18 mg/kg/d) or orally (18 mg/kg/0.5 d) for 9–11 days and compared to mice treated with 20 mg/kg/d of inactive compound. Significant 50–60% reductions in Aβ, βCTF, αCTF, and pTau corresponded with 3–5‐fold CatB increases as well as GluR1 recovery. Oral PADK dosing in adult rats was confirmed to produce measurable PADK levels in plasma, and a dose‐dependent and selective increase in CatB protein and activity in hippocampus and other brain regions (p<0.001). A higher molecular weight derivative with similar CatB modulation as PADK in the slice model was less effective by oral dosing. In 22‐month WT mice (C57BL/6) which exhibit 26–37% age‐related declines in pre‐ and postsynaptic markers (compared to 3‐month mice), oral PADK resulted in 2–4‐fold CatB modulation in the hippocampus and frontal cortex. Correspondingly, these regions showed a 97% recovery of synaptophysin (p=0.0176) and a 65–100% recovery in GluR1 and GluR2/3 (p<0.05), respectively. These results indicate that CatB modulatory compounds enhance protein clearance mechanisms to reduce multi‐proteinopathy and promote recovery of synaptic markers. Thus, such modulators have the potential to slow the synaptic decline and associated cognitive deficits in AD and perhaps earlier syndromes including mild cognitive impairment (MCI).Support or Funding InformationCART Fund Organization, Alzheimer's Research Trust 2014–2016; NIH grant 5R25GM077634‐04 2012–2016; University of North Carolina President's Strategic Initiative Award 2014–2015