From structure to clinic: Design of a muscarinic M1 receptor agonist with potential to treatment of Alzheimer's disease.

Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.

Impact of GPCR Structures on Drug Discovery.

Structures of 70 unique G protein-coupled receptors (GPCRs) have been determined, with over 370 structures in total bound to different ligands and the receptors in various conformational states. Structure-based drug design has been applied to an increasing number of GPCR targets over the past decade and now a few of these drug candidates have entered clinical trials. Given the length of time required for a drug to reach the market, there are no documented examples of licensed drugs being developed with the aid of a structure, but this is likely to change as current efforts come to fruition.

Accurate structure prediction of biomolecular interactions with AlphaFold 3.

The introduction of AlphaFold 21 has spurred a revolution in modelling the structure of proteins and their interactions, enabling a huge range of applications in protein modelling and design2-6. Here we describe our AlphaFold 3 model with a substantially updated diffusion-based architecture that is capable of predicting the joint structure of complexes including proteins, nucleic acids, small molecules, ions and modified residues. The new AlphaFold model demonstrates substantially improved accuracy over many previous specialized tools: far greater accuracy for protein-ligand interactions compared with state-of-the-art docking tools, much higher accuracy for protein-nucleic acid interactions compared with nucleic-acid-specific predictors and substantially higher antibody-antigen prediction accuracy compared with AlphaFold-Multimer v.2.37,8. Together, these results show that high-accuracy modelling across biomolecular space is possible within a single unified deep-learning framework.

Corrigendum: Crystal structure of the GLP-1 receptor bound to a peptide agonist.

This corrects the article DOI: 10.1038/nature22800.

Crystal structure of the GLP-1 receptor bound to a peptide agonist.

Glucagon-like peptide 1 (GLP-1) regulates glucose homeostasis through the control of insulin release from the pancreas. GLP-1 peptide agonists are efficacious drugs for the treatment of diabetes. To gain insight into the molecular mechanism of action of GLP-1 peptides, here we report the crystal structure of the full-length GLP-1 receptor bound to a truncated peptide agonist. The peptide agonist retains an α-helical conformation as it sits deep within the receptor-binding pocket. The arrangement of the transmembrane helices reveals hallmarks of an active conformation similar to that observed in class A receptors. Guided by this structural information, we design peptide agonists with potent in vivo activity in a mouse model of diabetes.

Intracellular allosteric antagonism of the CCR9 receptor.

Chemokines and their G-protein-coupled receptors play a diverse role in immune defence by controlling the migration, activation and survival of immune cells. They are also involved in viral entry, tumour growth and metastasis and hence are important drug targets in a wide range of diseases. Despite very significant efforts by the pharmaceutical industry to develop drugs, with over 50 small-molecule drugs directed at the family entering clinical development, only two compounds have reached the market: maraviroc (CCR5) for HIV infection and plerixafor (CXCR4) for stem-cell mobilization. The high failure rate may in part be due to limited understanding of the mechanism of action of chemokine antagonists and an inability to optimize compounds in the absence of structural information. CC chemokine receptor type 9 (CCR9) activation by CCL25 plays a key role in leukocyte recruitment to the gut and represents a therapeutic target in inflammatory bowel disease. The selective CCR9 antagonist vercirnon progressed to phase 3 clinical trials in Crohn's disease but efficacy was limited, with the need for very high doses to block receptor activation. Here we report the crystal structure of the CCR9 receptor in complex with vercirnon at 2.8 Å resolution. Remarkably, vercirnon binds to the intracellular side of the receptor, exerting allosteric antagonism and preventing G-protein coupling. This binding site explains the need for relatively lipophilic ligands and describes another example of an allosteric site on G-protein-coupled receptors that can be targeted for drug design, not only at CCR9, but potentially extending to other chemokine receptors.

First-in-man study to investigate safety, pharmacokinetics and exploratory pharmacodynamics of HTL0018318, a novel M1 -receptor partial agonist for the treatment of dementias.

AIMS: HTL0018318 is a selective M1 receptor partial agonist currently under development for the symptomatic treatment of cognitive and behavioural symptoms in Alzheimer's disease and other dementias. We investigated safety, tolerability, pharmacokinetics and exploratory pharmacodynamics (PD) of HTL0018318 following single ascending doses. METHODS: This randomized, double-blind, placebo-controlled study in 40 healthy younger adult and 57 healthy elderly subjects, investigated oral doses of 1-35 mg HTL0018318. Pharmacodynamic assessments were performed using a battery of neurocognitive tasks and electrophysiological measurements. Cerebrospinal fluid concentrations of HTL0018318 and food effects on pharmacokinetics of HTL0018318 were investigated in an open label and partial cross-over design in 14 healthy subjects. RESULTS: Pharmacokinetics of HTL0018318 were well-characterized showing dose proportional increases in exposure from 1-35 mg. Single doses of HTL0018318 were associated with mild dose-related adverse events of low incidence in both younger adult and elderly subjects. The most frequently reported cholinergic AEs included hyperhidrosis and increases in blood pressure up to 10.3 mmHg in younger adults (95% CI [4.2-16.3], 35-mg dose) and up to 11.9 mmHg in elderly subjects (95% CI [4.9-18.9], 15-mg dose). There were no statistically significant effects on cognitive function but the study was not powered to detect small to moderate effect sizes of clinical relevance. CONCLUSION: HTL0018318 showed well-characterized pharmacokinetics and following single doses were generally well tolerated in the dose range studied. These provide encouraging data in support of the development for HTL0018318 for Alzheimer's disease and other dementias.

Safety, pharmacokinetics and pharmacodynamics of HTL0009936, a selective muscarinic M1 -acetylcholine receptor agonist: A randomized cross-over trial.

AIMS: HTL0009936 is a selective M1 muscarinic receptor agonist in development for cognitive dysfunction in Alzheimer's disease. Safety, tolerability and pharmacokinetics and exploratory pharmacodynamic effects of HTL0009936 administered by continuous IV infusion at steady state were investigated in elderly subjects with below average cognitive functioning (BACF). METHODS: Part A was a four-treatment open label sequential study in healthy elderly investigating 10-83 mg HTL0009936 (IV) and a 24 mg HTL0009936 single oral dose. Part B was a five-treatment randomized, double-blind, placebo and physostigmine controlled cross-over study with IV HTL0009936 in elderly subjects with BACF. Pharmacodynamic assessments were performed using neurocognitive and electrophysiological tests. RESULTS: Pharmacokinetics of HTL0009936 showed dose-proportional increases in exposure with a mean half-life of 2.4 hours. HTL0009936 was well-tolerated with transient dose-related adverse events (AEs). Small increases in mean systolic blood pressure of 7.12 mmHg (95% CI [3.99-10.24]) and in diastolic of 5.32 mmHg (95% CI [3.18-7.47]) were noted at the highest dose in part B. Overall, there was suggestive, but no definitive, positive or negative pharmacodynamic effects. Statistically significant effects were observed on P300 with HTL0009936 and adaptive tracking with physostigmine. CONCLUSIONS: HTL0009936 showed well-characterized pharmacokinetics and single doses were safe and generally well-tolerated in healthy elderly subjects. Due to physostigmine tolerability issues and subject burden, the study design was changed and some pharmacodynamic assessments (neurocognitive) were performed at suboptimal drug exposures. Therefore no clear conclusions can be made on pharmacodynamic effects of HTL0009936, although an effect on P300 is suggestive of central target engagement.

Accurate Prediction of GPCR Ligand Binding Affinity with Free Energy Perturbation.

The computational prediction of relative binding free energies is a crucial goal for drug discovery, and G protein-coupled receptors (GPCRs) are arguably the most important drug target class. However, they present increased complexity to model compared to soluble globular proteins. Despite breakthroughs, experimental X-ray crystal and cryo-EM structures are challenging to attain, meaning computational models of the receptor and ligand binding mode are sometimes necessary. This leads to uncertainty in understanding ligand-protein binding induced changes such as, water positioning and displacement, side chain positioning, hydrogen bond networks, and the overall structure of the hydration shell around the ligand and protein. In other words, the very elements that define structure activity relationships (SARs) and are crucial for accurate binding free energy calculations are typically more uncertain for GPCRs. In this work we use free energy perturbation (FEP) to predict the relative binding free energies for ligands of two different GPCRs. We pinpoint the key aspects for success such as the important role of key water molecules, amino acid ionization states, and the benefit of equilibration with specific ligands. Initial calculations following typical FEP setup and execution protocols delivered no correlation with experiment, but we show how results are improved in a logical and systematic way. This approach gave, in the best cases, a coefficient of determination (R2) compared with experiment in the range of 0.6-0.9 and mean unsigned errors compared to experiment of 0.6-0.7 kcal/mol. We anticipate that our findings will be applicable to other difficult-to-model protein ligand data sets and be of wide interest for the community to continue improving FE binding energy predictions.

X-Ray Crystallography and Free Energy Calculations Reveal the Binding Mechanism of A2A Adenosine Receptor Antagonists.

We present a robust protocol based on iterations of free energy perturbation (FEP) calculations, chemical synthesis, biophysical mapping and X-ray crystallography to reveal the binding mode of an antagonist series to the A2A adenosine receptor (AR). Eight A2A AR binding site mutations from biophysical mapping experiments were initially analyzed with sidechain FEP simulations, performed on alternate binding modes. The results distinctively supported one binding mode, which was subsequently used to design new chromone derivatives. Their affinities for the A2A AR were experimentally determined and investigated through a cycle of ligand-FEP calculations, validating the binding orientation of the different chemical substituents proposed. Subsequent X-ray crystallography of the A2A AR with a low and a high affinity chromone derivative confirmed the predicted binding orientation. The new molecules and structures here reported were driven by free energy calculations, and provide new insights on antagonist binding to the A2A AR, an emerging target in immuno-oncology.

Identification of a novel allosteric GLP-1R antagonist HTL26119 using structure- based drug design.

A series of novel allosteric antagonists of the GLP-1 receptor (GLP-1R), exemplified by HTL26119, are described. SBDD approaches were employed to identify HTL26119, exploiting structural understanding of the allosteric binding site of the closely related Glucagon receptor (GCGR) (Jazayeri et al., 2016) and the homology relationships between GCGR and GLP-1R. The region around residue C3476.36b of the GLP-1R receptor represents a key difference from GCGR and was targeted for selectivity for GLP-1R.

Pharmacological Analysis and Structure Determination of 7-Methylcyanopindolol-Bound ß1-Adrenergic Receptor.

Comparisons between structures of the ß1-adrenergic receptor (AR) bound to either agonists, partial agonists, or weak partial agonists led to the proposal that rotamer changes of Ser(5.46), coupled to a contraction of the binding pocket, are sufficient to increase the probability of receptor activation. (RS)-4-[3-(tert-butylamino)-2-hydroxypropoxy]-1H-indole-2-carbonitrile (cyanopindolol) is a weak partial agonist of ß1AR and, based on the hypothesis above, we predicted that the addition of a methyl group to form 4-[(2S)-3-(tert-butylamino)-2-hydroxypropoxy]-7-methyl-1H-indole-2-carbonitrile (7-methylcyanopindolol) would dramatically reduce its efficacy. An eight-step synthesis of 7-methylcyanopindolol was developed and its pharmacology was analyzed. 7-Methylcyanopindolol bound with similar affinity to cyanopindolol to both ß1AR and ß2AR. As predicted, the efficacy of 7-methylcyanopindolol was reduced significantly compared with cyanopindolol, acting as a very weak partial agonist of turkey ß1AR and an inverse agonist of human ß2AR. The structure of 7-methylcyanopindolol-bound ß1AR was determined to 2.4-Å resolution and found to be virtually identical to the structure of cyanopindolol-bound ß1AR. The major differences in the orthosteric binding pocket are that it has expanded by 0.3 Å in 7-methylcyanopindolol-bound ß1AR and the hydroxyl group of Ser(5.46) is positioned 0.8 Å further from the ligand, with respect to the position of the Ser(5.46) side chain in cyanopindolol-bound ß1AR. Thus, the molecular basis for the reduction in efficacy of 7-methylcyanopindolol compared with cyanopindolol may be regarded as the opposite of the mechanism proposed for the increase in efficacy of agonists compared with antagonists.

Structure-based drug design for G protein-coupled receptors.

Our understanding of the structural biology of G protein-coupled receptors has undergone a transformation over the past 5 years. New protein-ligand complexes are described almost monthly in high profile journals. Appreciation of how small molecules and natural ligands bind to their receptors has the potential to impact enormously how medicinal chemists approach this major class of receptor targets. An outline of the key topics in this field and some recent examples of structure- and fragment-based drug design are described. A table is presented with example views of each G protein-coupled receptor for which there is a published X-ray structure, including interactions with small molecule antagonists, partial and full agonists. The possible implications of these new data for drug design are discussed.

A phase 1b/2a multicenter study of the safety and preliminary pharmacodynamic effects of selective muscarinic M1 receptor agonist HTL0018318 in patients with mild-to-moderate Alzheimer's disease.

INTRODUCTION: This study examined the safety and pharmacodynamic effects of selective muscarinic M1 receptor orthosteric agonist HTL0018318 in 60 patients with mild-to-moderate Alzheimer's disease (AD) on background donepezil 10 mg/day. METHODS: A randomized, double-blind, placebo-controlled 4-week safety study of HTL0018318 with up-titration and maintenance phases, observing exploratory effects on electrophysiological biomarkers and cognition. RESULTS: Treatment-emergent adverse events (TEAEs) were mild and less frequently reported during maintenance versus titration. Headache was most commonly reported (7-21%); 0 to 13% reported cholinergic TEAEs (abdominal pain, diarrhea, fatigue, nausea) and two patients discontinued due to TEAEs. At 1 to 2 hours post-dose, HTL0018318-related mean maximum elevations in systolic and diastolic blood pressure of 5 to 10 mmHg above placebo were observed during up-titration but not maintenance. Postive effects of HTL0018318 were found on specific attention and memory endpoints. DISCUSSION: HTL0018318 was well tolerated in mild-to-moderate AD patients and showed positive effects on attention and episodic memory on top of therapeutic doses of donepezil.

Novel Macrocyclic Antagonists of the CGRP Receptor Part 2: Stereochemical Inversion Induces an Unprecedented Binding Mode.

The diastereomeric macrocyclic calcitonin gene-related peptide (CGRP) antagonists HTL0029881 (3) and HTL0029882 (4), in which the stereochemistry of a spiro center is reversed, surprisingly demonstrate comparable potency. X-ray crystallographic characterization demonstrates that 3 binds to the CGRP receptor in a precedented manner but that 4 binds in an unprecedented, unexpected, and radically different manner. The observation of this phenomenon is noteworthy and may open novel avenues for CGRP receptor antagonist design.

Novel Macrocyclic Antagonists of the Calcitonin Gene-Related Peptide Receptor: Design, Realization, and Structural Characterization of Protein-Ligand Complexes.

A series of macrocyclic calcitonin gene-related peptide (CGRP) receptor antagonists identified using structure-based design principles, exemplified by HTL0028016 (1) and HTL0028125 (2), is described. Structural characterization by X-ray crystallography of the interaction of two of the macrocycle antagonists with the CGRP receptor ectodomain is described, along with structure-activity relationships associated with point changes to the macrocyclic antagonists. The identification of non-peptidic/natural product-derived, macrocyclic ligands for a G protein coupled receptor (GPCR) is noteworthy.

Acetylcholine prioritises direct synaptic inputs from entorhinal cortex to CA1 by differential modulation of feedforward inhibitory circuits.

Acetylcholine release in the hippocampus plays a central role in the formation of new memory representations. An influential but largely untested theory proposes that memory formation requires acetylcholine to enhance responses in CA1 to new sensory information from entorhinal cortex whilst depressing inputs from previously encoded representations in CA3. Here, we show that excitatory inputs from entorhinal cortex and CA3 are depressed equally by synaptic release of acetylcholine in CA1. However, feedforward inhibition from entorhinal cortex exhibits greater depression than CA3 resulting in a selective enhancement of excitatory-inhibitory balance and CA1 activation by entorhinal inputs. Entorhinal and CA3 pathways engage different feedforward interneuron subpopulations and cholinergic modulation of presynaptic function is mediated differentially by muscarinic M3 and M4 receptors, respectively. Thus, our data support a role and mechanisms for acetylcholine to prioritise novel information inputs to CA1 during memory formation.

Safety, pharmacokinetics and exploratory pro-cognitive effects of HTL0018318, a selective M1 receptor agonist, in healthy younger adult and elderly subjects: a multiple ascending dose study.

BACKGROUND: The cholinergic system and M1 receptor remain an important target for symptomatic treatment of cognitive dysfunction. The selective M1 receptor partial agonist HTL0018318 is under development for the symptomatic treatment of Dementia's including Alzheimer's disease (AD) and dementia with Lewy bodies (DLB). We investigated the safety, tolerability, pharmacokinetics and exploratory pharmacodynamics of multiple doses of HTL0018318 in healthy younger adults and elderly subjects. METHODS: This randomised, double blind, placebo-controlled study was performed, investigating oral doses of 15-35 mg/day HTL0018318 or placebo in 7 cohorts of healthy younger adult (n = 36; 3 cohorts) and elderly (n = 50; 4 cohorts) subjects. Safety, tolerability and pharmacokinetic measurements were performed. Pharmacodynamics were assessed using a battery of neurocognitive tasks and electrophysiological biomarkers of synaptic and cognitive functions. RESULTS: HTL0018318 was generally well-tolerated in multiple doses up to 35 mg/day and were associated with mild or moderate cholinergic adverse events. There were modest increases in blood pressure and pulse rate when compared to placebo-treated subjects, with tendency for the blood pressure increase to attenuate with repeated dosing. There were no clinically significant observations or changes in blood and urine laboratory measures of safety or abnormalities in the ECGs and 24-h Holter assessments. HTL0018318 plasma exposure was dose-proportional over the range 15-35 mg. Maximum plasma concentrations were achieved after 1-2 h. The apparent terminal half-life of HTL0018318 was 16.1 h (± 4.61) in younger adult subjects and 14.3 h (± 2.78) in elderly subjects at steady state. HTL0018318 over the 10 days of treatment had significant effects on tests of short-term (working) memory (n-back) and learning (Milner maze) with moderate to large effect sizes. CONCLUSION: Multiple doses of HTL0018138 showed well-characterised pharmacokinetics and were safe and generally well-tolerated in the dose range studied. Pro-cognitive effects on short-term memory and learning were demonstrated across the dose range. These data provide encouraging data in support of the development of HTL0018138 for cognitive dysfunction in AD and DLB. TRIAL REGISTRATION: Netherlands Trial Register identifier NTR5781 . Registered on 22 March 2016.

Recent Developments in Therapeutic Peptides for the Glucagon-like Peptide 1 and 2 Receptors.

Glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 2 (GLP-2) are proglucagon derived peptides that are released from gut endocrine cells in response to nutrient intake. These molecules are rapidly inactivated by the action of dipeptidyl peptidase IV (DPP-4) which limits their use as therapeutic agents. The recent emergence of three-dimensional structures of GPCRs such as GLP-1R and glucagon receptor has helped to drive the rational design of innovative peptide molecules that hold promise as novel peptide therapeutics. One emerging area is the discovery of multifunctional molecules that act at two or more pharmacological systems to enhance therapeutic efficacy. In addition, drug discovery efforts are also focusing on strategies to improve patient convenience through alternative routes of peptide delivery. These novel strategies highlight the broad utility of peptide-based therapeutics in human disease settings where unmet needs still exist.

Small molecule AZD4635 inhibitor of A2AR signaling rescues immune cell function including CD103+ dendritic cells enhancing anti-tumor immunity.

Accumulation of extracellular adenosine within the microenvironment is a strategy exploited by tumors to escape detection by the immune system. Adenosine signaling through the adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects which promote tumor growth and limit the efficacy of immune checkpoint inhibitors. Preclinical data with A2AR inhibitors have demonstrated tumor regressions in mouse models by rescuing T cell function; however, the mechanism and role on other immune cells has not been fully elucidated. METHODS: We report here the development of a small molecule A2AR inhibitor including characterization of binding and inhibition of A2AR function with varying amounts of a stable version of adenosine. Functional activity was tested in both mouse and human T cells and dendritic cells (DCs) in in vitro assays to understand the intrinsic role on each cell type. The role of adenosine and A2AR inhibition was tested in DC differentiation assays as well as co-culture assays to access the cross-priming function of DCs. Syngeneic models were used to assess tumor growth alone and in combination with alphaprogrammed death-ligand 1 (αPD-L1). Immunophenotyping by flow cytometry was performed to examine global immune cell changes upon A2AR inhibition. RESULTS: We provide the first report of AZD4635, a novel small molecule A2AR antagonist which inhibits downstream signaling and increases T cell function as well as a novel mechanism of enhancing antigen presentation by CD103+ DCs. The role of antigen presentation by DCs, particularly CD103+ DCs, is critical to drive antitumor immunity providing rational to combine a priming agent AZD4635 with check point blockade. We find adenosine impairs the maturation and antigen presentation function of CD103+ DCs. We show in multiple syngeneic mouse tumor models that treatment of AZD4635 alone and in combination with αPD-L1 led to decreased tumor volume correlating with enhanced CD103+ function and T cell response. We extend these studies into human DCs to show that adenosine promotes a tolerogenic phenotype that can be reversed with AZD4635 restoring antigen-specific T cell activation. Our results support the novel role of adenosine signaling as an intrinsic negative regulator of CD103+ DCs maturation and priming. We show that potent inhibition of A2AR with AZD4635 reduces tumor burden and enhances antitumor immunity. This unique mechanism of action in CD103+ DCs may contribute to clinical responses as AZD4635 is being evaluated in clinical trials with IMFINZI (durvalumab, αPD-L1) in patients with solid malignancies. CONCLUSION: We provide evidence implicating suppression of adaptive and innate immunity by adenosine as a mechanism for immune evasion by tumors. Inhibition of adenosine signaling through selective small molecule inhibition of A2AR using AZD4635 restores T cell function via an internal mechanism as well as tumor antigen cross-presentation by CD103+ DCs resulting in antitumor immunity.