HSP60/10 chaperonin systems are inhibited by a variety of approved drugs,natural products,and known bioactive molecules

All living organisms contain a unique class of molecular chaperones called 60?kDa heat shock proteins (HSP60 – also known as GroEL in bacteria). While some organisms contain more than one HSP60 or GroEL isoform, at least one isoform has always proven to be essential. Because of this, we have been investigating targeting HSP60 and GroEL chaperonin systems as an antibiotic strategy. Our initial studies focused on applying this antibiotic strategy for treating African sleeping sickness (caused by Trypanosoma brucei parasites) and drug-resistant bacterial infections (in particular Methicillin-resistant Staphylococcus aureus – MRSA). Intriguingly, during our studies we found that three known antibiotics – suramin, closantel, and rafoxanide – were potent inhibitors of bacterial GroEL and human HSP60 chaperonin systems. These findings prompted us to explore what other approved drugs, natural products, and known bioactive molecules might also inhibit HSP60 and GroEL chaperonin systems. Initial high-throughput screening of 3680 approved drugs, natural products, and known bioactives identified 161 hit inhibitors of the Escherichia coli GroEL chaperonin system (4.3% hit rate). From a purchased subset of 60 hits, 29 compounds (48%) re-confirmed as selective GroEL inhibitors in our assays, all of which were nearly equipotent against human HSP60. These findings illuminate the notion that targeting chaperonin systems might be a more common occurrence than we previously appreciated. Future studies are needed to determine if the in vivo modes of action of these approved drugs, natural products, and known bioactive molecules are related to GroEL and HSP60 inhibition.     

Analogs of nitrofuran antibiotics are potent GroEL/ES inhibitor pro-drugs

In two previous studies, we identified compound 1 as a moderate GroEL/ES inhibitor with weak to moderate antibacterial activity against Gram-positive and Gram-negative bacteria including Bacillus subtilis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, and SM101 Escherichia coli (which has a compromised lipopolysaccharide biosynthetic pathway making bacteria more permeable to drugs). Extending from those studies, we developed two series of analogs with key substructures resembling those of known antibacterials, nitroxoline (hydroxyquinoline moiety) and nifuroxazide/nitrofurantoin (bis-cyclic-N-acylhydrazone scaffolds). Through biochemical and cell-based assays, we identified potent GroEL/ES inhibitors that selectively blocked E. faecium, S. aureus, and E. coli proliferation with low cytotoxicity to human colon and intestine cells in vitro. Initially, only the hydroxyquinoline-bearing analogs were found to be potent inhibitors in our GroEL/ES-mediated substrate refolding assays; however, subsequent testing in the presence of an E. coli nitroreductase (NfsB) in situ indicated that metabolites of the nitrofuran-bearing analogs were potent GroEL/ES inhibitor pro-drugs. Consequently, this study has identified a new target of nitrofuran-containing drugs, and is the first reported instance of such a unique class of GroEL/ES chaperonin inhibitors. The intriguing results presented herein provide impetus for expanded studies to validate inhibitor mechanisms and optimize this antibacterial class using the respective GroEL/ES chaperonin systems and nitroreductases from E. coli and the ESKAPE bacteria.     

A new Drosophila model of Ubiquilin knockdown shows the effect of impaired proteostasis on locomotive and learning abilities

Ubiquilin (UBQLN) plays a crucial role in cellular proteostasis through its involvement in the ubiquitin proteasome system and autophagy. Mutations in the UBQLN2 gene have been implicated in amyotrophic lateral sclerosis (ALS) and ALS with frontotemporal lobar dementia (ALS/FTLD). Previous studies reported a key role for UBQLN in Alzheimer's disease (AD); however, the mechanistic involvement of UBQLN in other neurodegenerative diseases remains unclear. The genome of Drosophila contains a single UBQLN homolog (dUbqn) that shows high similarity to UBQLN1 and UBQLN2; therefore, the fly is a useful model for characterizing the role of UBQLN in vivo in neurological disorders affecting locomotion and learning abilities. We herein performed a phenotypic and molecular characterization of diverse dUbqn RNAi lines. We found that the depletion of dUbqn induced the accumulation of polyubiquitinated proteins and caused morphological defects in various tissues. Our results showed that structural defects in larval neuromuscular junctions, abdominal neuromeres, and mushroom bodies correlated with limited abilities in locomotion, learning, and memory. These results contribute to our understanding of the impact of impaired proteostasis in neurodegenerative diseases and provide a useful Drosophila model for the development of promising therapies for ALS and FTLD.     

Synthesis and evaluation of esterified Hsp70 agonists in cellular models of protein aggregation and folding

Over-expression of the Hsp70 molecular chaperone prevents protein aggregation and ameliorates neurodegenerative disease phenotypes in model systems. We identified an Hsp70 activator, MAL1-271, that reduces α-synuclein aggregation in a Parkinson’s Disease model. We now report that MAL1-271 directly increases the ATPase activity of a eukaryotic Hsp70. Next, twelve MAL1-271 derivatives were synthesized and examined in a refined α-synuclein aggregation model as well as in an assay that monitors maturation of a disease-causing Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mutant, which is also linked to Hsp70 function. Compared to the control, MAL1-271 significantly increased the number of cells lacking α-synuclein inclusions and increased the steady-state levels of the CFTR mutant. We also found that a nitrile-containing MAL1-271 analog exhibited similar effects in both assays. None of the derivatives exhibited cellular toxicity at concentrations up to 100?μm, nor were cellular stress response pathways induced. These data serve as a gateway for the continued development of a new class of Hsp70 agonists with efficacy in these and potentially other disease models.     

Pneumocytes are distinguished by highly elevated expression of the ER stress biomarker GRP78, a co-receptor for SARS-CoV-2, in COVID-19 autopsies

Vaccinations are widely credited with reducing death rates from COVID-19, but the underlying host-viral mechanisms/interactions for morbidity and mortality of SARS-CoV-2 infection remain poorly understood. Acute respiratory distress syndrome (ARDS) describes the severe lung injury, which is pathologically associated with alveolar damage, inflammation, non-cardiogenic edema, and hyaline membrane formation. Because proteostatic pathways play central roles in cellular protection, immune modulation, protein degradation, and tissue repair, we examined the pathological features for the unfolded protein response (UPR) using the surrogate biomarker glucose-regulated protein 78 (GRP78) and co-receptor for SARS-CoV-2. At autopsy, immunostaining of COVID-19 lungs showed highly elevated expression of GRP78 in both pneumocytes and macrophages compared with that of non-COVID control lungs. GRP78 expression was detected in both SARS-CoV-2-infected and un-infected pneumocytes as determined by multiplexed immunostaining for nucleocapsid protein. In macrophages, immunohistochemical staining for GRP78 from deceased COVID-19 patients was increased but overlapped with GRP78 expression taken from surgical resections of non-COVID-19 controls. In contrast, the robust in situ GRP78 immunostaining of pneumocytes from COVID-19 autopsies exhibited no overlap and was independent of age, race/ethnicity, and gender compared with that from non-COVID-19 controls. Our findings bring new insights for stress-response pathways involving the proteostatic network implicated for host resilience and suggest that targeting of GRP78 expression with existing therapeutics might afford an alternative therapeutic strategy to modulate host-viral interactions during SARS-CoV-2 infections.     

The intrinsic proteostasis network of stem cells

The proteostasis network adjusts protein composition and maintains protein integrity, which are essential processes for cell function and viability. Current efforts, given their intrinsic characteristics, regenerative potential and fundamental biological functions, have been directed to define proteostasis of stem cells. These insights demonstrate that embryonic stem cells and induced pluripotent stem cells exhibit an endogenous proteostasis network that not only modulates their pluripotency and differentiation but also provides a striking ability to suppress aggregation of disease-related proteins. Moreover, recent findings establish a central role of enhanced proteostasis to prevent the aging of somatic stem cells in adult organisms. Notably, proteostasis is also required for the biological purpose of adult germline stem cells, that is to be passed from one generation to the next. Beyond these links between proteostasis and stem cell function, we also discuss the implications of these findings for disease, aging, and reproduction.     

Targeting of the unfolded protein response (UPR) as therapy for Parkinson's disease

Parkinson's disease is the second most common neurodegenerative disorder, leading to the progressive decline of motor control due to the loss of dopaminergic neurons in the substantia nigra pars compacta. At the molecular level, Parkinson's disease share common molecular signatures with most neurodegenerative diseases including the accumulation of misfolded proteins in the brain. Alteration in the buffering capacity of the proteostasis network during aging is proposed as one of the triggering steps leading to abnormal protein aggregation in this disease, highlighting disturbances in the function of the endoplasmic reticulum (ER). The ER is the main subcellular compartment involved in protein folding and quality control. ER stress triggers a signalling reaction known as the unfolded protein response (UPR), which aims restoring proteostasis through the induction of adaptive programs or the activation of cell death pathways when damage is chronic and cannot be repaired. Here, we overview most evidence linking ER stress to Parkinson's disease. Strategies to alleviate ER stress by targeting specific components of the UPR using small molecules and gene therapy are highlighted.     

Inflammasome Activation by Altered Proteostasis

The association between altered proteostasis and inflammatory disorders has been increasingly recognized, but the underlying mechanisms are not well understood. In this study, we show that deficiency of either autophagy or sequestosome 1 (p62 or SQSTM) led to inflammasome hyperactivation in response to LPS and ATP in primary macrophages and in mice in vivo. Importantly, induction of protein misfolding by puromycin, thapsigargin, or geldanamycin resulted in inflammasome activation that was more pronounced in autophagy- or p62-deficient macrophages. Accumulation of misfolded proteins caused inflammasome activation by inducing generation of nonmitochondrial reactive oxygen species and lysosomal damage, leading to release of cathepsin B. Our results suggest that altered proteostasis results in inflammasome activation and thus provide mechanisms for the association of altered proteostasis with inflammatory disorders.     

The Mechanistic Links Between Proteasome Activity,Aging and Age-related Diseases

Damaged and misfolded proteins accumulate during the aging process, impairing cell function and tissue homeostasis. These perturbations to protein homeostasis (proteostasis) are hallmarks of age-related neurodegenerative disorders such as Alzheimer’s, Parkinson’s or Huntington’s disease. Damaged proteins are degraded by cellular clearance mechanisms such as the proteasome, a key component of the proteostasis network. Proteasome activity declines during aging, and proteasomal dysfunction is associated with late-onset disorders. Modulation of proteasome activity extends lifespan and protects organisms from symptoms associated with proteostasis disorders. Here we review the links between proteasome activity, aging and neurodegeneration. Additionally, strategies to modulate proteasome activity and delay the onset of diseases associated to proteasomal dysfunction are discussed herein.