Biophysical Approaches Facilitate Computational Drug Discovery for ATP-Binding Cassette Proteins

Although membrane proteins represent most therapeutically relevant drug targets, the availability of atomic resolution structures for this class of proteins has been limited. Structural characterization has been hampered by the biophysical nature of these polytopic transporters, receptors, and channels, and recent innovations to in vitro techniques aim to mitigate these challenges. One such class of membrane proteins, the ATP-binding cassette (ABC) superfamily, are broadly expressed throughout the human body, required for normal physiology and disease-causing when mutated, yet lacks sufficient structural representation in the Protein Data Bank. However, recent improvements to biophysical techniques (e.g., cryo-electron microscopy) have allowed for previously “hard-to-study” ABC proteins to be characterized at high resolution, providing insight into molecular mechanisms-of-action as well as revealing novel druggable sites for therapy design. These new advances provide ample opportunity for computational methods (e.g., virtual screening, molecular dynamics simulations, and structure-based drug design) to catalyze the discovery of novel small molecule therapeutics that can be easily translated from computer to bench and subsequently to the patient’s bedside. In this review, we explore the utility of recent advances in biophysical methods coupled with well-established in silico techniques towards drug development for diseases caused by dysfunctional ABC proteins

Biochemical Interrogation of Rare Cystic Fibrosis Mutations Informs Strategies for Future Therapeutic Intervention

There are over 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene associated with Cystic Fibrosis (CF) disease, and to date, the two recently FDA-approved drugs KalydecoTM and OrkambiTM have been effective in rescuing functional expression of the two most common CFTR mutations, G551D and ΔF508, respectively, representing approximately 90% of the patient population worldwide. However, potential efficacy of these therapies for the remaining ~2000 mutations is unknown. Thus, biochemical characterization of each rare variant is needed to assess potential clinical benefit. My thesis attempts to understand molecular defects associated with less common CFTR mutations, mainly c.3700A>G (causing abberant splicing and subsequent translation of ΔI1234_R1239-CFTR) and c.2052_2053insA (Q685TfsX4-CFTR), with the goal of determining whether current CF therapies (specifically designed for G551D and ΔF508) could be used for these rare variants. Additionally, my work aims to identify unique molecular aberrations for these rare, mutant CFTR proteins in order to facilitate development of mutation-specific therapeutics. Through a collaborative initiative involving clinicians, biochemists and biophysicists, we found that ΔI1234_R1239-CFTR shared certain biochemical attributes with ΔF508-CFTR, however contained intrinsic defects which differed significantly (i.e. poorer interdomain assembly between the amino- and carboxy-termini, and decreased functional responses to cAMP agonists, CFTR correctors and potentiators). When CFTR activity was evaluated in nasal epithelial cultures from siblings homozygous for c.3700A>G, we likewise found that ΔI1234_R1239-CFTR responded poorly to small molecules. For Q685TfsX4-CFTR, we found that this variant was prematurely truncated, and therefore did not respond to CFTR-specific therapies. However, a proof-of-concept in vitro strategy to ‘skip’ the exon containing the c.2052_2053insA mutation led to an incomplete, yet partially mature CFTR molecule which exhibited channel activity, albeit this function was unregulated. In parallel to these studies, we generated and functionally characterized several other rare CFTR mutations using novel mutagenesis and high-throughput functional screening methods, allowing for rapid identification and stratification of genotypes that could potentially benefit from current CF therapies. Furthermore, our efforts described here aim to facilitate future mutation-specific CF drug discovery, as well as a ‘personalized medicine’ approach to assist in the development of novel strategies to combat this fatal genetic disease.Ph.D.2017-07-08 00:00:0

Correctors of the major cystic fibrosis mutant interact through membrane-spanning domainsS

The most common cystic fibrosis causing mutation is deletion of phenylalanine at position 508 (F508del), a mutation that leads to protein misassembly with defective processing. Small molecule corrector compounds: VX-809 or Corr-4a (C4) partially restores processing of the major mutant. These two prototypical corrector compounds cause an additive effect on F508del/cystic fibrosis transmembrane conductance regulator (CFTR) processing, and hence were proposed to act through distinct mechanisms: VX-809 stabilizing the first membrane-spanning domain (MSD) 1, and C4 acting on the second half of the molecule [consisting of MSD2 and/or nucleotide binding domain (NBD) 2]. We confirmed the effect of VX-809 in enhancing the stability of MSD1 and showed that it also allosterically modulates MSD2 when coexpressed with MSD1. We showed for the first time that C4 stabilizes the second half of the CFTR protein through its action on MSD2. Given the allosteric effect of VX-809 on MSD2, we were prompted to test the hypothesis that the two correctors interact in the full-length mutant protein. We did see evidence supporting their interaction in the full-length F508del-CFTR protein bearing secondary mutations targeting domain:domain interfaces. Disruption of the MSD1:F508del-NBD1 interaction (R170G) prevented correction by both compounds, pointing to the importance of this interface in processing. On the other hand, stabilization of the MSD2: F508del-NBD1 interface (by introducing R1070W) led to a synergistic effect of the compound combination on the total abundance of both the immature and mature forms of the protein. Together, these findings suggest that the two correctors interact in stabilizing the complex of MSDs in F508del-CFTR

Sphingosine-1-Phosphate Is a Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) attenuates sphingosine-1-phosphate (S1P) signaling in resistance arteries and has emerged as a prominent regulator of myogenic vasoconstriction. This investigation demonstrates that S1P inhibits CFTR activity via adenosine monophosphate-activated kinase (AMPK), establishing a potential feedback link. In Baby Hamster Kidney (BHK) cells expressing wild-type human CFTR, S1P (1μmol/L) attenuates forskolin-stimulated, CFTR-dependent iodide efflux. S1P's inhibitory effect is rapid (within 30 seconds), transient and correlates with CFTR serine residue 737 (S737) phosphorylation. Both S1P receptor antagonism (4μmol/L VPC 23019) and AMPK inhibition (80μmol/L Compound C or AMPK siRNA) attenuate S1P-stimluated (i) AMPK phosphorylation, (ii) CFTR S737 phosphorylation and (iii) CFTR activity inhibition. In BHK cells expressing the ΔF508 CFTR mutant (CFTRΔF508), the most common mutation causing cystic fibrosis, both S1P receptor antagonism and AMPK inhibition enhance CFTR activity, without instigating discernable correction. In summary, we demonstrate that S1P/AMPK signaling transiently attenuates CFTR activity. Since our previous work positions CFTR as a negative S1P signaling regulator, this signaling link may positively reinforce S1P signals. This discovery has clinical ramifications for the treatment of disease states associated with enhanced S1P signaling and/or deficient CFTR activity (e.g. cystic fibrosis, heart failure). S1P receptor/AMPK inhibition could synergistically enhance the efficacy of therapeutic strategies aiming to correct aberrant CFTR trafficking

Giving Drugs a Second Chance: Overcoming Regulatory and Financial Hurdles in Repurposing Approved Drugs As Cancer Therapeutics

The repositioning or “repurposing” of existing therapies for alternative disease indications is an attractive approach that can save significant investments of time and money during drug development. For cancer indications, the primary goal of repurposed therapies is on efficacy, with less restriction on safety due to the immediate need to treat this patient population. This report provides a high-level overview of how drug developers pursuing repurposed assets have previously navigated funding efforts, regulatory affairs, and intellectual property laws to commercialize these “new” medicines in oncology. This article provides insight into funding programs (e.g., government grants and philanthropic organizations) that academic and corporate initiatives can leverage to repurpose drugs for cancer. In addition, we highlight previous examples where secondary uses of existing, Food and Drug Administration- or European Medicines Agency-approved therapies have been predicted in silico and successfully validated in vitro and/or in vivo (i.e., animal models and human clinical trials) for certain oncology indications. Finally, we describe the strategies that the pharmaceutical industry has previously employed to navigate regulatory considerations and successfully commercialize their drug products. These factors must be carefully considered when repurposing existing drugs for cancer to best benefit patients and drug developers alike

Small molecule in situ resin capture provides a compound first approach to natural product discovery

Culture-based microbial natural product discovery strategies fail to realize the extraordinary biosynthetic potential detected across earth's microbiomes. Here we introduce Small Molecule In situ Resin Capture (SMIRC), a culture-independent method to obtain natural products directly from the environments in which they are produced. We use SMIRC to capture numerous compounds including two new carbon skeletons that were characterized using NMR and contain structural features that are, to the best of our knowledge, unprecedented among natural products. Applications across diverse marine habitats reveal biome-specific metabolomic signatures and levels of chemical diversity in concordance with sequence-based predictions. Expanded deployments, in situ cultivation, and metagenomics facilitate compound discovery, enhance yields, and link compounds to candidate producing organisms, although microbial community complexity creates challenges for the later. This compound-first approach to natural product discovery provides access to poorly explored chemical space and has implications for drug discovery and the detection of chemically mediated biotic interactions