Improved correction of F508del-CFTR biogenesis with a folding facilitator and an inhibitor of protein ubiquitination.

A growing number of diseases are linked to the misfolding of integral membrane proteins, and many of these proteins are targeted for ubiquitin-proteasome-dependent degradation. One such substrate is a mutant form of the Cystic Fibrosis Transmembrane Conductance Regulator (F508del-CFTR). Protein folding "correctors" that repair the F508del-CFTR folding defect have entered the clinic, but they are unlikely to protect the entire protein from degradation. To increase the pool of F508del-CFTR protein that is available for correction by existing treatments, we determined a structure-activity relationship to improve the efficacy and reduce the toxicity of an inhibitor of the E1 ubiquitin activating enzyme that facilitates F508del-CFTR maturation. A resulting lead compound lacked measurable toxicity and improved the ability of an FDA-approved corrector to augment F508del-CFTR folding, transport the protein to the plasma membrane, and maintain its activity. These data support a proof-of-concept that modest inhibition of substrate ubiquitination improves the activity of small molecule correctors to treat CF and potentially other protein conformational disorders.

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.

From CFTR biology toward combinatorial pharmacotherapy: expanded classification of cystic fibrosis mutations.

More than 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been described that confer a range of molecular cell biological and functional phenotypes. Most of these mutations lead to compromised anion conductance at the apical plasma membrane of secretory epithelia and cause cystic fibrosis (CF) with variable disease severity. Based on the molecular phenotypic complexity of CFTR mutants and their susceptibility to pharmacotherapy, it has been recognized that mutations may impose combinatorial defects in CFTR channel biology. This notion led to the conclusion that the combination of pharmacotherapies addressing single defects (e.g., transcription, translation, folding, and/or gating) may show improved clinical benefit over available low-efficacy monotherapies. Indeed, recent phase 3 clinical trials combining ivacaftor (a gating potentiator) and lumacaftor (a folding corrector) have proven efficacious in CF patients harboring the most common mutation (deletion of residue F508, ΔF508, or Phe508del). This drug combination was recently approved by the U.S. Food and Drug Administration for patients homozygous for ΔF508. Emerging studies of the structural, cell biological, and functional defects caused by rare mutations provide a new framework that reveals a mixture of deficiencies in different CFTR alleles. Establishment of a set of combinatorial categories of the previously defined basic defects in CF alleles will aid the design of even more efficacious therapeutic interventions for CF patients.

Expression of a malarial Hsp70 improves defects in chaperone-dependent activities in ssa1 mutant yeast.

Plasmodium falciparum causes the most virulent form of malaria and encodes a large number of molecular chaperones. Because the parasite encounters radically different environments during its lifecycle, many members of this chaperone ensemble may be essential for P. falciparum survival. Therefore, Plasmodium chaperones represent novel therapeutic targets, but to establish the mechanism of action of any developed therapeutics, it is critical to ascertain the functions of these chaperones. To this end, we report the development of a yeast expression system for PfHsp70-1, a P. falciparum cytoplasmic chaperone. We found that PfHsp70-1 repairs mutant growth phenotypes in yeast strains lacking the two primary cytosolic Hsp70s, SSA1 and SSA2, and in strains harboring a temperature sensitive SSA1 allele. PfHsp70-1 also supported chaperone-dependent processes such as protein translocation and ER associated degradation, and ameliorated the toxic effects of oxidative stress. By introducing engineered forms of PfHsp70-1 into the mutant strains, we discovered that rescue requires PfHsp70-1 ATPase activity. Together, we conclude that yeast can be co-opted to rapidly uncover specific cellular activities mediated by malarial chaperones.

Plasmodium falciparum encodes a single cytosolic type I Hsp40 that functionally interacts with Hsp70 and is upregulated by heat shock.

Heat shock protein 70 (Hsp70) and heat shock protein 40 (Hsp40) function as molecular chaperones during the folding and trafficking of proteins within most cell types. However, the Hsp70-Hsp40 chaperone partnerships within the malaria parasite, Plasmodium falciparum, have not been elucidated. Only one of the 43 P. falciparum Hsp40s is predicted to be a cytosolic, canonical Hsp40 (termed PfHsp40) capable of interacting with the major cytosolic P. falciparum-encoded Hsp70, PfHsp70. Consistent with this hypothesis, we found that PfHsp40 is upregulated under heat shock conditions in a similar pattern to PfHsp70. In addition, PfHsp70 and PfHsp40 reside mainly in the parasite cytosol, as assessed using indirect immunofluorescence microscopy. Recombinant PfHsp40 stimulated the ATP hydrolytic rates of both PfHsp70 and human Hsp70 similar to other canonical Hsp40s of yeast (Ydj1) and human (Hdj2) origin. In contrast, the Hsp40-stimulated plasmodial and human Hsp70 ATPase activities were differentially inhibited in the presence of pyrimidinone-based small molecule modulators. To further probe the chaperone properties of PfHsp40, protein aggregation suppression assays were conducted. PfHsp40 alone suppressed protein aggregation, and cooperated with PfHsp70 to suppress aggregation. Together, these data represent the first cellular and biochemical evidence for a PfHsp70-PfHsp40 partnership in the malaria parasite, and furthermore that the plasmodial and human Hsp70-Hsp40 chaperones possess unique attributes that are differentially modulated by small molecules.

Select pyrimidinones inhibit the propagation of the malarial parasite, Plasmodium falciparum.

Plasmodium falciparum, the Apicomplexan parasite that is responsible for the most lethal forms of human malaria, is exposed to radically different environments and stress factors during its complex lifecycle. In any organism, Hsp70 chaperones are typically associated with tolerance to stress. We therefore reasoned that inhibition of P. falciparum Hsp70 chaperones would adversely affect parasite homeostasis. To test this hypothesis, we measured whether pyrimidinone-amides, a new class of Hsp70 modulators, could inhibit the replication of the pathogenic P. falciparum stages in human red blood cells. Nine compounds with IC(50) values from 30 nM to 1.6 micrOM were identified. Each compound also altered the ATPase activity of purified P. falciparum Hsp70 in single-turnover assays, although higher concentrations of agents were required than was necessary to inhibit P. falciparum replication. Varying effects of these compounds on Hsp70s from other organisms were also observed. Together, our data indicate that pyrimidinone-amides constitute a novel class of anti-malarial agents.