Perlara and Mission: Cure – a nonprofit organization with a mission to find a cure for those affected by pancreatitis – recently launched a Pancreatitis PerlQuest partnership. Ethan covers the backstory of this partnership – from the initial intro to developing a Research Plan – in an accompanying post. This post delves into the science behind this partnership, our plans for developing and screening pancreatitis yeast models, and the new scientific ground we are breaking together.

27 Variations of Pancreatitis Yeast Models

Chronic pancreatitis – the inflammation of the pancreas – can lead to serious complications including infection, kidney failure, malnutrition, and pancreatic cancer. There are many causes of pancreatitis, but a common genetic cause are mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. For this PerlQuest, we are modeling 27 patient alleles of CFTR in budding yeast. Modeling more than two dozen variants is ambitious, but it is well suited for the yeast system. It would require significantly more time and resources if done in human cell culture. Therefore, we are excited to use yeast to quickly characterize, identify, and prioritize which patient allele models would be most suitable to advance to a high-throughput drug screen. Furthermore, Mission: Cure has also established a collaboration with Cedars-Sinai Medical Center so that small molecules that we discover in our yeast screen can be advanced directly to testing in an artificial pancreas system using cells derived from pancreatitis patients!

CFTR deficiency and pancreatitis

The CFTR gene codes for an ion channel transporter protein that functions in epithelial cell membranes to regulate fluid transport. Its deficiency can lead to buildup of thick mucus in the lungs, pancreas, or other organs and compromises the function of the organ. Severe mutations in both copies of CFTR resulting in diminished ion channel function leads to cystic fibrosis (CF). However, heterozygous carriers of CF-causing mutations or loss-of-function mutations in CFTR that do not cause lung disease – for example, by reducing bicarbonate ion transport – are also associated with increased risk for recurrent acute and chronic pancreatitis.

CFTR mutations are grouped into six classes. Class I mutations are caused by a premature stop codon that result in lack of protein synthesis. Class II mutants are defective in protein processing that arise from protein mis-folding and mis-trafficking. Class III mutants are defective in channel regulation or gating, while class IV are defective in chloride conductance. Class V mutants have reduced protein level due to errors in splicing and class VI proteins are highly unstable and quickly turn over at the cell surface. Mutations in CFTR associated with pancreatitis cover all classes and varying mechanisms for compromised CFTR protein function.

The yeast ortholog of CFTR is YOR1, which encodes a member of the ABC transporter superfamily. YOR1 is 21% identical to CFTR at the amino acid level. Higher homology exists within the conserved nucleotide binding domains. The predicted structure of YOR1 resembles the solved structure of CFTR (Figure 1). The most common CF causing CFTR mutation, ∆F508, has been modeled in yeast YOR1 at the orthologous site ∆F670, and has the same molecular defects in trafficking, folding and conductance.  In fact, a genetic suppressor of ∆F670, RLP12, that was identified in yeast, rescues ∆F508 in a human bronchial cell line, indicating a highly conserved module and further evidence that yeast YOR1 protein is a good model for CFTR-opathies. Ethan wrote a blog post about this in 2016.

YOR1-CFTR comparison 700x365pxFigure 1. A predicted model of yeast YOR1 compared to the solved structure of CFTR. NBD = nucleotide-binding domain; TMD = transmembrane domain. Left: Molecular Structure of CFTR (Liu et al., 2017 Cell), right: https://swissmodel.expasy.org/repository/uniprot/P53049

Natural History Study

We will use yeast to identify novel small-molecule therapeutic options for CFTR mutations that are associated with pancreatitis. From a list of CFTR mutations reported in chronic pancreatitis studies that are conserved in YOR1 (Table 1), we will build the yeast allelic series containing the CFTR variants listed. This list includes CFTR variants across different mutational classes: I, II, III, and IV. Modeling mutations across different classes will allow us to screen for compounds that can rescue CFTR mutations by different but potentially synergizing mechanisms. We will include ∆F508 as a control because the mechanism of ∆F508 is well studied in yeast and in human cells.

We will use gene engineering technology to introduce individual CFTR mutations at the orthologous sites in YOR1. The resultant yeast strains will be characterized for resistance to the toxin oligomycin, which is extruded from yeast cells by YOR1 at the plasma membrane, which is analogous to ion transport by CFTR at the plasma membrane. Loss of YOR1 or expression of the orthologous CFTR ∆F508 mutation in yeast causes increased sensitivity to oligomycin. Suppressors of YOR1 mutations would rescue YOR1 function, which can be assayed in a simple, robust screen as resistance to oligomycin and restoration of cellular viability and growth. This can be achieved in a high-throughput manner in 384-well plates and assayed with an automated plate reader.

Table 1. CFTR variants reported in chronic pancreatitis studies that share homology with yeast YOR1. (Modified from J. LaRusch et al., 2014 PLoS Genet.)

Drug Discovery Screen

Once the pancreatitis yeast models have been validated and their phenotypes confirmed, we will advance the most promising model along with the well-characterized ∆F670 variant as a control to a 20,000-compound drug discovery campaign to identify novel chemical compounds that would serve as the starting point for a preclinical development candidate. We will screen for compounds that rescue growth of YOR1 variants in media containing oligomycin.

Stay tuned for more updates as our work in developing CFTR-related pancreatitis yeast models begins. In the meantime, if you haven’t had a chance, catch up on Ethan’s accompanying post about this partnership. 

References

1. P. Hegyi et al., CFTR: A New Horizon in the Pathomechanism and Treatment of Pancreatitis. Rev. Physiol. Biochem. Pharmacol. 170, 37–66 (2016). DOI: 10.1007/112_2015_5002

2. J. LaRusch et al., Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis. PLoS Genet. 10, e1004376 (2014). DOI: 10.1371/journal.pgen.1004376

3. F. Liu, Z. Zhang, L. Csanády, D. C. Gadsby, J. Chen, Molecular Structure of the Human CFTR Ion Channel. Cell. 169, 85–95.e8 (2017). DOI: 10.1016/j.cell.2017.02.024

4. D. J. Katzmann, E. A. Epping, W. S. Moye-Rowley, Mutational disruption of plasma membrane trafficking of Saccharomyces cerevisiae Yor1p, a homologue of mammalian multidrug resistance protein. Mol. Cell. Biol. 19, 2998–3009 (1999). DOI: 10.1128/MCB.19.4.2998

5. R. J. Louie et al., A yeast phenomic model for the gene interaction network modulating CFTR-ΔF508 protein biogenesis. Genome Med. 4, 103 (2012). DOI: 10.1186/gm404

6. G. Veit et al., Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect. PLOS Biol. 14, e1002462 (2016). DOI: 10.1371/journal.pbio.1002462

Image: FLICKR, [email protected] / CC BY 2.0, modified from the original 

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