18 research outputs found
The Structural Basis of Peptide Binding at Class A G Protein-Coupled Receptors
G protein-coupled receptors (GPCRs) represent the largest membrane protein family and a significant target class for therapeutics. Receptors from GPCRs’ largest class, class A, influence virtually every aspect of human physiology. About 45% of the members of this family endogenously bind flexible peptides or peptides segments within larger protein ligands. While many of these peptides have been structurally characterized in their solution state, the few studies of peptides in their receptor-bound state suggest that these peptides interact with a shared set of residues and undergo significant conformational changes. For the purpose of understanding binding dynamics and the development of peptidomimetic drug compounds, further studies should investigate the peptide ligands that are complexed to their cognate receptor
Computational assessment of pharmacological similarity between Class A GPCRs
Though G protein-coupled receptors (GPCRs) are the target of 30% of the clinically-approved drug market, these drugs target just ~25% of the non-olfactory GPCR superfamily (108). Almost half of the remaining untargeted GPCRs are “orphans” with no known endogenous ligand(s), representing a rich and underutilised source of pharmacotherapeutic targets. To facilitate ligand identification, the Kufareva and Smith research groups previously developed GPCR-Contact-Informed Neighbouring Pocket (GPCR-CoINPocket). It is a metric of pharmacological similarity between Class A GPCRs, incorporating sequence similarity and structurally-observed ligand interaction patterns derived from liganded GPCRs in the Pocketome, an annotated encyclopaedia of liganded protein structures. In this thesis, I re-evaluate previous GPCR-CoINPocket predictions and develop an upgraded and improved version of GPCR-CoINPocket.
In Chapter 2, I re-investigated the pharmacology of the orphan receptor, GPR37L1. In the previous iteration of GPCR-CoINPocket, GPR37L1 was used as a prototypical orphan for the prospective validation of the metric. However, previous retractions from our lab meant that clear evidence was once again lacking for the signalling pathways and activity of GPCR-CoINPocket-predicted ligands at GPR37L1. In the present study, I found no evidence of ligand-dependent or constitutive Gαi- or GαS-directed agonism in HEK293 or CHO-K1 cells, leaving the pharmacological toolbox of GPR37L1 once again empty.
In Chapter 3, I developed a method for quantifying the similarity of ligand sets and generated a benchmark of pharmacologically similar and dissimilar Class A GPCRs for the assessment of updates and upgrades to GPCR-CoINPocket. My method formalises the intuitive concept of pharmacological similarity by deriving receptor similarity scores from high quality ChEMBL-annotated receptor:ligand binding data via an approximation of the number of shared unique chemotypes.
In Chapter 4, I used my benchmarking set to evaluate four areas of potential improvement for GPCR-CoINPocket: the orthosteric contact fingerprints defining receptor pockets, the inclusion (or exclusion) of ECL2 fingerprints, the logic of score aggregation, and the amino acid similarity matrix used to score sequence similarity. The evaluation of these upgrades led to the development of GPCR-CoINPocket v2, which outperformed transmembrane similarity and GPCRdb-defined orthosteric pocket similarity in the discrimination of pharmacologically similar from dissimilar Class A GPCRs. The primary upgrade responsible for this improvement was the replacement of the Gonnet amino acid similarity matrix with a chemically-informed matrix developed in this thesis that directly (and solely) reflected structurally-observed ligand binding pattern similarities between amino acids.
In Chapter 5, I prospectively validated GPCR-CoINPocket v2 using the human β2-adrenoceptor as the prototypical target. I identified 4 compounds typically binding to the OT, MCH1, and SST2 receptors with nanomolar affinity/potency that unexpectedly had affinity for the β2-adrenoceptor in competitive radioligand binding assays.
The key output of this thesis is GPCR-CoINPocket v2, a direct upgrade to the original orthosteric pocket-based metric of pharmacological similarity between Class A GPCRs. I have validated it both retrospectively and prospectively, illustrating one use of the method that allows it to complement virtual and physical high-throughput screening technologies. Further applications and limitations are discussed in Chapter 6, but it is hoped that the methods and tools I have developed here can be easily adopted and aid in elucidating orphan GPCR physiology and pharmacology
Exploration of Somatostatin Binding Mechanism to Somatostatin Receptor Subtype 4
Somatostatin (also named as growth hormone-inhibiting -hormone or somatotropin release inhibiting factor) is a regulatory peptide important for the proper functioning of the endocrine system, local inflammatory reactions, mood and motor coordination, and behavioral responses to
stress. Somatostatin exerts its effects via binding to G-protein-coupled somatostatin receptors of which the fourth subtype (SSTR4) is a particularly important receptor mediating analgesic, anti-inflammatory, and anti-depressant effects without endocrine actions. Thus, SSTR4 agonists are
promising drug candidates. Although the knowledge of the atomic resolution-binding modes of SST would be essential for drug development, experimental elucidation of the structures of SSTR4 and its complexes is still awaiting. In the present study, structures of the somatostatin–SSTR4 complex were produced using an unbiased, blind docking approach. Beyond the static structures, the binding
mechanism of SST was also elucidated in the explicit water molecular dynamics (MD) calculations, and key binding modes (external, intermediate, and internal) were distinguished. The most important residues on both receptor and SST sides were identified. An energetic comparison of SST binding
to SSTR4 and 2 offered a residue-level explanation of receptor subtype selectivity. The calculated
structures show good agreement with available experimental results and indicate that somatostatin binding is realized via prerequisite binding modes and an induced fit mechanism. The identified binding modes and the corresponding key residues provide useful information for future drug design
targeting SSTR4
Ion Channels of Nociception
The Special Issue “Ion Channels of Nociception” contains 13 articles united by a focus on the peripheral mechanisms of pain. The content covers the mechanisms of neuropathic, inflammatory, and dental pain as well as pain in migraine and diabetes; nociceptive roles of P2X3, ASIC, Piezo and TRP channels; pain control through GPCRs and pharmacological agents; and nonpharmacological treatment with electroacupuncture
Biological and Biochemical Basis of the Differential Efficacy of First and Second Generation Somatostatin Receptor Ligands in Neuroendocrine Neoplasms
Endogenous somatostatin shows anti-secretory effects in both physiological and pathological settings, as well as inhibitory activity on cell growth. Since somatostatin is not suitable for clinical practice, researchers developed synthetic somatostatin receptor ligands (SRLs) to overcome this limitation. Currently, SRLs represent pivotal tools in the treatment algorithm of neuroendocrine tumors (NETs). Octreotide and lanreotide are the first-generation SRLs developed and show a preferential binding affinity to somatostatin receptor (SST) subtype 2, while pasireotide, which is a second-generation SRL, has high affinity for multiple SSTs (SST5 > SST2 > SST3 > SST1). A number of studies demonstrated that first-generation and second-generation SRLs show distinct functional properties, besides the mere receptor affinity. Therefore, the aim of the present review is to critically review the current evidence on the biological effects of SRLs in pituitary adenomas and neuroendocrine tumors, by mainly focusing on the differences between first-generation and second-generation ligands
International Union of Basic and Clinical Pharmacology. CV. Somatostatin Receptors: Structure, Function, Ligands, and New Nomenclature.
Somatostatin, also known as somatotropin-release inhibitory factor, is a cyclopeptide that exerts potent inhibitory actions on hormone secretion and neuronal excitability. Its physiologic functions are mediated by five G protein-coupled receptors (GPCRs) called somatostatin receptor (SST)1-5. These five receptors share common structural features and signaling mechanisms but differ in their cellular and subcellular localization and mode of regulation. SST2 and SST5 receptors have evolved as primary targets for pharmacological treatment of pituitary adenomas and neuroendocrine tumors. In addition, SST2 is a prototypical GPCR for the development of peptide-based radiopharmaceuticals for diagnostic and therapeutic interventions. This review article summarizes findings published in the last 25 years on the physiology, pharmacology, and clinical applications related to SSTs. We also discuss potential future developments and propose a new nomenclature
An investigation of the molecular pharmacology of G protein-coupled receptor 35
G protein-coupled receptors (GPCRs) are seven-pass integral membrane proteins that act as transducers of extracellular signals across the lipid bilayer. Their location and
involvement in basic and pathological physiological processes has secured their role as key targets for pharmaceutical intervention. GPCRs are targeted by many of the best-selling
drugs on the market and there are a substantial number of GPCRs that are yet to be
characterised; these could offer interest for therapeutic targeting. GPR35 is one such
receptor that, as a result of gene knockout and genome wide association studies, has
attracted interest through its association with cardiovascular and gastrointestinal disease.
Elucidation of the basic physiological function of GPR35 has, however, been difficult due a
paucity of potent and selective ligands in addition to a lack of consensus on the endogenous ligand. Herein, a focussed drug discovery effort was carried out to identify agonists of GPR35. Various in vitro cellular assays were employed in conjunction with N- or C-terminally
manipulated forms of the receptor to investigate GPR35’s signalling profile and to provide an
assay format suitable for the characterisation of newly identified ligands. Although GPR35
associates with both Gαi/o and Gα13 families of small heterotrimeric G proteins, the G
protein-independent β-arrestin-2 recruitment format was found to be the most suited to
drug screening efforts. Small molecule compound screening, carried out in conjunction with
the Medical Research Council Technology, identified compound 1 as the most potent ligand
of human GPR35 reported at that time. However, the lower efficacy and potency of
compound 1 at the rodent species orthologues of GPR35 prevented its use in in vivo studies. A subsequent effort, carried out with Novartis, focused on mast cell stabilisers as putative
agonists of GPR35, revealed lodoxamide and bufrolin as highly potent agonists that activated
human and rat GPR35 with equal potency. This finding offered–for the first time–the
opportunity to employ the same GPR35 ligand between species at a similar concentration, an important factor to
consider when translating rodent in vivo functional studies to those in man. Additionally,
using molecular modelling and site directed mutagenesis studies, these newly identified
compounds were used to aid characterisation of the ligand binding pockets of human and
rat GPR35 to reveal the molecular basis of species selectivity at this receptor. In summary, this research effort presents GPR35 tool compounds that can now be used to dissect the
basic biology of GPR35 and investigate its contribution to disease