bioRxiv Science⌕ Search

Biology subjects

Hubner, H.

Publications and source records attributed to Hubner, H..

5 recordsLinked to original sources

Toward a Random Background for Ligand Optimization

Ligand optimization is central to drug discovery as hundreds of analogs might be designed and synthesized between an initial hit and a therapeutic candidate. The efficiency of this process is unclear, at least partly because there is no random background for optimization against which to compare. Such a random background might emerge from synthetically accessible but otherwise systematic random small substitutions across starting ligands, measuring likelihood of achieving a substantial improvement in affinity/potency or other property by any single perturbation. Recent literature and ligand-affinity/potency databases suggest that perhaps 10% of analogs with minor modifications improve upon a parents potency substantially (by [≥]10-fold), but this number is clouded by reporting bias, intentional improvement, and inter-group reproducibility. To begin to establish a background expectation for ligand optimization, we comprehensively and systematically modified 18 lead molecules across six targets with single atom changes; 257 compounds were synthesized. Unexpectedly, 11.2% of these random small perturbation analogs improved potency by [≥]10-fold over their parents. Conversely, these more potent analogs typically had worse in vitro pharmacokinetics (e.g. reduced metabolic stability, lower plasma free fraction). While it was possible to find analogs where the potency increase compensated for inferior exposure and half-life, resulting in more potent compounds in vivo, overall a frustrated landscape for ligand optimization is revealed. This study begins to establish a background expectation for ligand potency optimization and offers a simple strategy to do so. It also begins to quantify the challenges confronting the field in moving beyond in vitro potency.

pharmacology and toxicology↗

The role of intrinsically disordered domains in regulating G protein coupled receptor signaling

The 2A adrenergic receptor (2AAR) is a clinically important target for various diseases including hypertension, diabetes and chronic pain. Here, using single-molecule fluorescence resonance energy transfer imaging, we show how agonist-specific activation dynamics in both structured transmembrane domain (TMD) and intrinsically disorders regions (IDRs) of 2AAR lead to diverse signaling profiles. Through seven pairs of strategically designed fluorophore labels, we systematically investigate the real-time conformational changes of 2AAR. Our study reveals unique TM6 dynamics in 2AAR, featured by a high energy barrier for agonist-induced outward movements essential for activation. In contrast, we identify agonist-specific conformational dynamics of a partially disordered extracellular loop (ECL2), highlighting its role as a dynamic regulatory module that controls receptor function. Moreover, we characterize the conformational landscapes of the long third intracellular loop (ICL3), revealing its compact structural features and membrane-proximal localization in the basal state, where it acts as a negative allosteric regulator in transducer coupling. Furthermore, we identify multiful functional sub-states of ICL3 that are dynamically modulated by both kinase phosphorylation and drug efficacy. These findings offer previously underappreciated structural and dynamic insights into 2AAR function governed by both TMD and IDRs, and may open up new avenues for the development of better therapeutics.

biophysics↗

Molecular insights into G protein specificity and biased agonism at the β2-adrenergic receptor

G protein coupled receptors (GPCRs) exhibit varying degrees of selectivity for different G protein isoforms. Despite the abundant structures of GPCR-G protein complexes, little is known about the mechanism of G protein coupling specificity. The {beta}2-adrenergic receptor is an example of GPCR with high selectivity for Gs, the stimulatory G protein for adenylyl cyclase, and much weaker for the Gi family of G proteins inhibiting adenylyl cyclase. By developing a Gi-biased agonist (LM189), we provide structural and biophysical evidence supporting that distinct conformations at ICL2 and TM6 are required for coupling of the different G protein subtypes Gs and Gi. These results deepen our understanding of G protein specificity and bias and can accelerate the design of ligands that select for preferred signaling pathways.

molecular biology↗

Discovery of a functionally selective serotonin 5-HT1A receptor agonist for the treatment of pain

The G protein-coupled serotonin receptor 5-HT1AR mediates antinociception and may serve as a valuable target for the treatment of pain. Starting from a chemical library, ST171, a bitopic chemotype activating 5-HT1AR was evolved. In vitro pharmacological investigations of ST171 revealed potent and selective Gi activation (EC50 = 0.3 nM), with marginal Gs and {beta}-arrestin recruitment. Preclinical studies in mice showed that ST171 was effective in acute and chronic (inflammatory and neuropathic) pain models, without causing sedation. Comparison of cryo-EM structures of a 5-HT1AR-Gi complex bound to the functionally biased agonist ST171, with a structure bound to the functionally balanced agonist befiradol, showed that both ligands bind to the same orthosteric site, but address different exo-sites. The individual poses are associated with ligand-specific helical dispositions and rearrangements of microdomains. Complementation of these studies with molecular dynamics simulations allowed us to derive structural features associated with ST171s functional selectivity, a phenomenon that may be crucial to the discovery of more effective and safe GPCR drugs.

pharmacology and toxicology↗

An allosteric ligand stabilizes distinct conformations in the M2 muscarinic acetylcholine receptor

Allosteric modulators provide therapeutic advantages over orthosteric drugs. A plethora of allosteric modulators have been identified for several GPCRs, particularly for muscarinic receptors (mAChRs)1,2. To study the molecular mechanisms governing allosteric modulation, we utilized a recently developed NMR system to investigate the conformational changes in the M2 muscarinic receptor (M2R) in response to the positive allosteric modulator (PAM) LY2119620. Our studies provide the first biophysical data showing that LY2119620 can substantially change the structure and dynamics of M2R in both the extracellular and G-protein coupling domains during the activation process. These NMR data suggest that LY2119620 may function by stabilizing distinct sets of conformations not observed in the presence of orthosteric agonists alone, which may account for the different signaling behaviors of the M2R when bound to LY2119620. Our studies provide new structural information for understanding the mechanism of GPCR allostery, and may facilitate the rational design of allosteric therapeutics targeting muscarinic receptors.

biophysics↗