Promoting canonical signaling in constrictive airway diseases
In drug discovery and development, patient welfare demands that we never allow the perfect to be the enemy of the good. We use the best available information about target and pathway function to design, discover, and develop therapeutic candidates that improve patient outcomes and quality of life. Increased understanding of target and pathway function creates opportunities to improve efficacy and ameliorate liabilities. Therapeutics that modulate the activity of adrenergic receptors are an instructive example. Agonists of the beta-2-adrenergic receptor (B2AR) are critical bronchodilators in management of constrictive airway diseases, but many commonly used bronchodilators were developed before mechanisms for receptor desensitization, non-canonical signaling, recycling and degradation were elucidated. Although these drugs have improved quality of life for millions of patients, chronic use of bronchodilators (especially long-acting agonists) can increase the risk of fatal asthmatic attacks, which is why these drugs carry a black box warning. Mounting evidence suggests that non-canonical signaling, which is downstream of receptor desensitization, may play a role in the etiology of adverse events. Through the use of a double filter high-throughput screening strategy whereby we evaluate both canonical signaling and desensitization (via arrestin recruitment), my laboratory has discovered allosteric effectors that promote canonical signaling and suppress receptor desensitization. Bronchodilators that strongly bias towards canonical signaling should display enhanced efficacy while reducing tolerance and adverse events. We are currently evaluating and improving biased agonist candidates and allosteric modulators as part of a multidisciplinary program aimed at developing improved modalities for intervention in constrictive airway diseases.
Promoting non-canonical signaling in heart failure
As hearts fail, adrenal glands increase production of catecholamines (epinephrine, norepinephrine) to compensate for reduced cardiac output. Unfortunately, the resulting mechanical and metabolic stress can accelerate heart damage, which is why drugs that counteract catecholamine stimulation (beta-blockers) are standard therapy for heart failure. While most of these drugs compete with catecholamines for receptor binding without promoting increased heart rate or contractile force (antagonists), biased agonists that promote GRK and arrestin-mediated receptor desensitization and internalization while suppressing canonical signaling offer an alternative mechanism for beta-blocking activity that has the dual advantages of sequestering the receptor from catecholamine stimulation and promoting non-canonical signaling, which is cardioprotective. Preliminary studies with biased agonists (pepducins) that promote non-canonical signaling support their utility not only for enhancing survival of heart tissue following ischemic insult (Tilley, unpublished), but, unexpectedly, also for increasing cardiac contractility (Carr, et al., PNAS 2016). Considering that suppression of cardiac contractility is one of the dose-limiting deficiencies of traditional beta-blockers, beta-agonists or allosteric modulators that bias towards non-canonical signaling may offer substantial improvement over standard of care.
Image courtesy of Richard Carr, Ph.D. (copyright 2016)
One of the virtues of the double-filter high-throughput screening strategy for canonical signaling and desensitization that we are pursuing is that it enables us to discover compounds that bias B2AR signaling in both directions. Biased candidates that promote canonical signaling are evaluated in models of constrictive airway diseases, while those that promote non-canonical signaling are evaluated in heart failure models. By pursuing a screening strategy informed by a deeper understanding of B2AR signaling, we are able to find new value in old libraries – in essence, fishing where the fish are, but with better bait.
Exploring and expanding the druggable proteome
When looking for novel or improved therapeutics that regulate pharmacologically validated targets (such as B2AR), exploring established collections of drug-like molecules in novel ways offers a practical approach to focus discovery efforts in functionally relevant regions of chemical space. However, whether established molecular diversity is well suited for the discovery and validation of novel targets is an open question. Existing collections of molecular diversity are biased towards existing targets, but these constitute only a small fraction of the proteome (~3%). Worse still, traditional small molecule discovery is both expensive and exclusive (rather than inclusive) because it relies on a skill set (synthetic/medicinal chemistry) that is relatively rare in the scientific community. Natural products offer an alternative source of molecular diversity that is evolutionarily enriched for bioactive molecules, as evidence by the preponderance of natural products in the pharmacopeia. Pharmaceutically active natural products have been isolated from every corner of the globe, but perhaps the most significant and optimal repository of human-specific natural products – the human proteome itself – has never been exhaustively interrogated for drug-like function.
My laboratory has developed a method (Expressed Peptide Libraries from Open Reading Frames (ExPLORF)) to mine the proteome for short peptides that can be used to identify novel, druggable targets. We use inverse PCR to diversify open reading frames into comprehensive libraries that express every constituent peptide (six amino acids or greater) derived from a candidate open frame. Libraries can be expressed as linear or constrained peptides in the cytoplasm or directed to intracellular compartments. Minimal sequence elements that promote a phenotype of interest are enriched from expressed peptide libraries, and enriched sequences are used as biochemical probes to isolate druggable targets. B2AR is an ideal model system to develop ExPLORF since peptides and pepducins derived from B2AR have been reported to promote signaling bias, and our canonical and non-canonical HTS platforms are equally amenable for FACS enrichment of single cells that express therapeutically interesting clones. Our long-term goal is to empower crowd-sourcing of probe discovery and target validation and catalyze ORF by ORF interrogation of the druggability of the human proteome by harnessing the evolutionarily optimal source of molecular diversity for pharmacologic validation of human targets through an affordable method built upon a molecular biology platform with which the majority of biomedical scientists are familiar and comfortable.