Logo: to the web site of Uppsala University

Macrocycles represent a promising class of beyond-rule-of-5 (bRo5) therapeutics, capable of targeting proteins traditionally considered undruggable by conventional small molecules. Macrocycles exhibit intrinsic flexibility and often display a "chameleon-like" ability to adapt to their environment, thereby enhancing their oral bioavailability. Describing their structures and conformational changes is essential for advancing their development in the bRo5 space. In this study, we present the novel solid- and solution-state structures of the macrocycle grazoprevir, determined using microcrystal electron diffraction (MicroED) and nuclear magnetic resonance (NMR) spectroscopy. A low-energy core conformation was consistently identified throughout the conformational journey, from solid formulation to solvated states and upon complexation with the biological target, while the vinyl cyclopropyl sulfonamide side chain reoriented. The presence of a common core conformation (RMSD up to 0.273 Å) suggests that grazoprevir adopts a partial pre-organized state, optimizing its suitability for target binding. In apolar environment, mimicking the cell membrane, intramolecular hydrogen bonding promoted the formation of compact conformations with reduced radii of gyration and solvent accessible 3D polar surface area, likely facilitating cell permeability. This work presents a comprehensive conformational trajectory of a macrocyclic chameleon and provides potential insights into understanding and characterizing cell permeability and chameleonicity of similar bRo5 therapeutics.

Peptide-activated G protein-coupled receptors (GPCRs) play crucial roles in numerous diseases, but remain difficult therapeutic targets due to the challenges in developing small-molecule drugs. Here, we explore structure-based strategies to identify small-molecule agonists of neurotensin (NTS) receptors, which hold promise for developing non-opioid analgesics. Chemical libraries of drug-like molecules are first designed based on a receptor-peptide complex, and then 14.5 million compounds are computationally docked to the orthosteric binding site of the NTS₁ receptor. A set of 39 top-ranked compounds is synthesized, and seven of these are experimentally confirmed to activate the NTS₁ receptor. Structure-guided optimization yields NTS₁ ligands with signaling signatures distinct from the endogenous peptide, and these compounds also exhibit high affinity for the NTS₂ receptor. High-resolution crystal structures of two agonists bound to the NTS₁ receptor confirm predicted binding modes and reveal key determinants of activation. In vivo, the compounds produce robust antinociception in rodents without inducing hypotension, consistent with a contribution of NTS₂ receptor activity. To facilitate broader application of our virtual screening approach to peptide-binding GPCRs, we provide access to tailored chemical libraries containing billions of readily synthesizable compounds.

Achieving satisfactory permeability for beyond the rule of 5 (bRo5) molecules, such as bioactive macrocycles, remains a challenge for drug discovery. Gaining a mechanistic understanding of the correlation between their structure and permeability is expected to improve the foundation for rational prediction of the pharmacokinetic behavior of novel macrocyclic drug candidates. Based on a solution-phase NMR investigation, we suggest that griselimycin, a macrocyclic peptide that has remarkable antibiotic activity, permeates membranes while exposing its polar moieties in an apolar membrane-like environment and hiding them in a polar environment. This noncanonical behavior is driven by the simultaneous exposure of lipophilic moieties in an apolar environment and by their hydrophobic collapse in aqueous solution. The unexpected behavior of griselimycin opposes the concept of classical chameleonicity and may be described as "inverse chameleonicity".

The process of developing new drugs is arduous and costly, particularly for targets classified as "difficult-to-drug." Macrocycles show a particular ability to modulate difficult-to-drug targets, including protein-protein interactions, while still allowing oral administration. However, the determination of membrane permeability, critical for reaching intracellular targets and for oral bioavailability, is laborious and expensive. In silico methods are a cost-effective alternative, enabling predictions prior to compound synthesis. Here, we present a comprehensive online database (https://swemacrocycledb.com/), housing 5638 membrane permeability datapoints for 4216 nonpeptidic macrocycles, curated from the literature, patents, and bioactivity repositories. In addition, we present a new descriptor, the "amide ratio" (AR), that quantifies the peptidic nature of macrocyclic compounds, enabling the classification of peptidic, semipeptidic, and nonpeptidic macrocycles. Overall, this resource fills a gap among existing databases, offering valuable insights into the membrane permeability of nonpeptidic and semipeptidic macrocycles, and facilitating predictions for drug discovery projects.

Objective

For better management of RA, new biomarkers are needed to predict the development of different disease courses. This study aims to identify autoantibodies against epitopes on proteins in the joints and to predict disease outcome in patients with new onset RA.

Methods

Sera from new-onset RA patients from the Swedish BARFOT (Better Anti Rheumatic PharmacOTherapy) and TIRA-2 (Swedish acronym for 'tidiga insatser vid reumatoid artrit') cohorts (n = 1986) were screened for autoantibodies to selected peptides (JointIDs) in a bead-based multiplex flow immunoassay. Disease outcomes included Boolean remission 1.0, swollen joint count and radiographic destruction. Multivariate logistic regression and zero-inflated negative binomial models that accounted for clinical factors were used to identify JointIDs with the strongest potential to predict prognosis.

Results

Boolean remission was predicted with 42% sensitivity and 75% specificity in male patients positive for antibodies to a non-modified collagen type II (COL2) peptide at 12 months. When antibodies to a specific citrullinated cartilage oligomeric protein (COMP) peptide were absent and the patient was in Boolean remission at 6 months, the sensitivity was 13% and the specificity 99%. Positivity for the non-modified COL2 peptide also reduced the frequency of swollen joints by 41% and 33% at 6 and 12 months, respectively. Antibodies to CCP predicted joint destruction with low specificity (58%). Positivity for a COL2 and a glucose-6-phosphate dehydrogenase peptide in citrullinated forms increased specificity (86%) at the expense of sensitivity (39%).

Conclusion

Autoantibodies against joint-related proteins at RA diagnosis predict remission with high specificity and, in combination with clinical factors, may guide future treatment decisions.

The ability to adopt folded conformations that have a low solvent-accessible 3D polar surface area has been found to be important for PROTACs to display a high passive cell permeability. We have studied two VHL PROTACs that differ only by the replacement of two methylene groups in the linker by oxygen atoms but that displayed vast differences in their cell permeability. MD simulations and NMR spectroscopy revealed an unexpected, environment-dependent conformational behavior for the low-permeability PROTAC that has an alkyl linker. Hydrophobic interactions enforced extended and polar conformations for this PROTAC in nonpolar media, explaining its low cell permeability. In water, hydrophobic collapse around the linker led to folded and less polar conformations. In contrast, the highly permeable PROTAC having a PEG linker adopted conformations of similar shapes and polarities in polar and nonpolar environments.

Allosteric modulation of G protein–coupled receptors (GPCRs) is an exciting strategy for developing new therapeutic agents, and it has several advantages over more commonly used orthosteric drugs. Recently determined GPCR structures have revealed allosteric pockets facing the lipid bilayer, enabling rational drug design. Here, we develop a virtual screening strategy to discover ligands of extrahelical binding pockets and apply this approach to the adenosine A₁ receptor (A₁R). The A₁R is a high-value therapeutic target for ischemia–reperfusion injury and chronic neuropathic pain. Developing effective A₁R therapeutics remains challenging due to high structural conservation across orthosteric binding sites and on-target unwanted effects stimulated by prototypical A₁R agonists, such as bradycardia and atrioventricular block. However, A₁R positive allosteric modulators (PAMs) acting through spatially distinct allosteric sites can fine-tune A₁R activity with high subtype selectivity and spatiotemporal specificity, thereby overcoming current limitations. A chemical library of 160 million compounds was computationally docked to the allosteric pocket identified in a cryo-EM structure of the A₁R, and a set of 26 top-ranked compounds were selected for experimental evaluation. Pharmacological evaluation of these, and structure-guided hit optimization, led to the discovery of subtype-selective A₁R PAMs. These compounds demonstrated minimal allosteric agonism and negligible impact on A₁R-mediated beat rate of an orthosteric agonist. The discovered PAMs pave the way for potential treatments for neuropathic pain and ischemia–reperfusion injury without accompanying side effects. Our results demonstrate the utility of a synergistic computational and experimental approach in GPCR drug discovery.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint inflammation, cartilage damage, and bone erosion. Despite improvements with the introduction of biological disease-modifying anti-rheumatic drugs (DMARDs), RA remains an incurable life-long disease. Advancements in peptide-based vaccination may open new avenues for treating autoimmune diseases, including RA, by inducing immune tolerance while maintaining normal immune function. We have already demonstrated the efficacy of a potent vaccine against RA, consisting of the mouse major histocompatibility complex class II (A^q) protein bound to the immunodominant type II collagen peptide COL2_259-273, which needed to be galactosylated at position 264. To translate the vaccine to humans and to further enhance vaccine efficacy, we modified the glycine residue at position 265 and conjugated it with the human DRB1∗04:01 molecule. Remarkably, this modified vaccine (named DR4-AL179) provided robust effectiveness in suppressing arthritis in DRB1∗04:01-expressing mice without the need for galactosylation at position 264. DR4-AL179 vaccination induces tolerance involving multiple immunoregulatory pathways, including the activation of V-type immunoglobulin domain-containing suppressor of T cell activation (VISTA)-positive nonconventional regulatory T cells, which contribute to a potent suppressive response preventing arthritis development in mice. This modified RA vaccine offers a novel therapeutic potential for human autoimmune diseases.

The orphan G protein-coupled receptor (GPCR) GPR139 attracts interest as a target for neuropsychiatric disorders. Whereas the physiological functions of GPR139 remain elusive, a high-resolution receptor structure is now available. To assess whether structural information enables ligand discovery, we computationally dock 235 million compounds to the GPR139 binding site. Of 68 top-ranked compounds evaluated experimentally, five are full agonists with potencies ranging from 160 nM to 3.6 µM. Structure-guided optimization identifies one of the most potent GPR139 agonists, and a cryo-EM structure of the receptor-ligand complex confirms the predicted binding mode. Functional characterization provides insights into GPR139 signalling, and one agonist elicits behavioural effects in mice. We also explore the potential to replace experimental structure determination with the deep-learning method AlphaFold3, revealing a limited capability of artificial intelligence to model receptor-ligand interactions for understudied GPCRs. The results demonstrate how high-resolution GPCR structures combined with large-library docking can accelerate drug discovery.

Fragment-based screening can catalyze drug discovery by identifying novel scaffolds, but this approach is limited by the small chemical libraries studied by biophysical experiments and the challenging hit optimization step. In efforts to identify DNA repair inhibitors, we explored the use of structure-based virtual screening to access ultralarge fragment libraries that cover four orders of magnitude larger fractions of chemical space than traditional techniques. A set of 14 million fragments were docked to 8-oxoguanine DNA glycosylase (OGG1), a challenging drug target involved in cancer and inflammation. Of the 29 top-ranked fragments that were experimentally evaluated, four compounds were shown to bind to OGG1 and X-ray crystallography confirmed the predicted binding modes. Docking of readily synthesizable elaborations guided fragment optimization, leading to the discovery of submicromolar OGG1 inhibitors with anti-inflammatory and anti-cancer effects in cell models. Our results demonstrate that fragment-based virtual screening enables efficient exploration of vast chemical libraries.