Therapy targeting antigen-specific T cells by a peptide-based tolerizing vaccine against autoimmune arthritis.

A longstanding goal has been to find an antigen-specific preventive therapy, i.e., a vaccine, for autoimmune diseases. It has been difficult to find safe ways to steer the targeting of natural regulatory antigen. Here, we show that the administration of exogenous mouse major histocompatibility complex class II protein bounding a unique galactosylated collagen type II (COL2) peptide (Aq-galCOL2) directly interacts with the antigen-specific TCR through a positively charged tag. This leads to expanding a VISTA-positive nonconventional regulatory T cells, resulting in a potent dominant suppressive effect and protection against arthritis in mice. The therapeutic effect is dominant and tissue specific as the suppression can be transferred with regulatory T cells, which downregulate various autoimmune arthritis models including antibody-induced arthritis. Thus, the tolerogenic approach described here may be a promising dominant antigen-specific therapy for rheumatoid arthritis, and in principle, for autoimmune diseases in general.

Going Viral: An Investigation into the Chameleonic Behaviour of Antiviral Compounds.

The ability to adjust conformations in response to the polarity of the environment, i.e. molecular chameleonicity, is considered to be important for conferring both high aqueous solubility and high cell permeability to drugs in chemical space beyond Lipinski's rule of 5. We determined the conformational ensembles populated by the antiviral drugs asunaprevir, simeprevir, atazanavir and daclatasvir in polar (DMSO-d6 ) and non-polar (chloroform) environments with NMR spectroscopy. Daclatasvir was fairly rigid, whereas the first three showed large flexibility in both environments, that translated into major differences in solvent accessible 3D polar surface area within each conformational ensemble. No significant differences in size and polar surface area were observed between the DMSO-d6 and chloroform ensembles of these three drugs. We propose that such flexible compounds are characterized as "partial molecular chameleons" and hypothesize that their ability to adopt conformations with low polar surface area contributes to their membrane permeability and oral absorption.

Simulation Reveals the Chameleonic Behavior of Macrocycles.

Conformational analysis is central to the design of bioactive molecules. It is particularly challenging for macrocycles due to noncovalent transannular interactions, steric interactions, and ring strain that are often coupled. Herein, we simulated the conformations of five macrocycles designed to express a progression of increasing complexity in environment-dependent intramolecular interactions and verified the results against NMR measurements in chloroform and dimethyl sulfoxide. Molecular dynamics using an explicit solvent model, but not the Monte Carlo method with implicit solvation, handled both solvents correctly. Refinement of conformations at the ab initio level was fundamental to reproducing the experimental observations─standard state-of-the-art molecular mechanics force fields were insufficient. Our simulations correctly predicted the intramolecular interactions between side chains and the macrocycle and revealed an unprecedented solvent-induced conformational switch of the macrocyclic ring. Our results provide a platform for the rational, prospective design of molecular chameleons that adapt to the properties of the environment.

Design of Drug Efficacy Guided by Free Energy Simulations of the ß2 -Adrenoceptor.

G-protein-coupled receptors (GPCRs) play important roles in physiological processes and are modulated by drugs that either activate or block signaling. Rational design of the pharmacological efficacy profiles of GPCR ligands could enable the development of more efficient drugs, but is challenging even if high-resolution receptor structures are available. We performed molecular dynamics simulations of the ß2 adrenergic receptor in active and inactive conformations to assess if binding free energy calculations can predict differences in ligand efficacy for closely related compounds. Previously identified ligands were successfully classified into groups with comparable efficacy profiles based on the calculated shift in ligand affinity upon activation. A series of ligands were then predicted and synthesized, leading to the discovery of partial agonists with nanomolar potencies and novel scaffolds. Our results demonstrate that free energy simulations enable design of ligand efficacy and the same approach can be applied to other GPCR drug targets.

Ultralarge Virtual Screening Identifies SARS-CoV-2 Main Protease Inhibitors with Broad-Spectrum Activity against Coronaviruses.

Drugs targeting SARS-CoV-2 could have saved millions of lives during the COVID-19 pandemic, and it is now crucial to develop inhibitors of coronavirus replication in preparation for future outbreaks. We explored two virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. First, structure-based docking was used to screen a diverse library of 235 million virtual compounds against the active site. One hundred top-ranked compounds were tested in binding and enzymatic assays. Second, a fragment discovered by crystallographic screening was optimized guided by docking of millions of elaborated molecules and experimental testing of 93 compounds. Three inhibitors were identified in the first library screen, and five of the selected fragment elaborations showed inhibitory effects. Crystal structures of target-inhibitor complexes confirmed docking predictions and guided hit-to-lead optimization, resulting in a noncovalent main protease inhibitor with nanomolar affinity, a promising in vitro pharmacokinetic profile, and broad-spectrum antiviral effect in infected cells.

Key interactions in the trimolecular complex consisting of the rheumatoid arthritis-associated DRB1*04:01 molecule, the major glycosylated collagen II peptide and the T-cell receptor.

OBJECTIVES: Rheumatoid arthritis (RA) is an autoimmune disease strongly associated with the major histocompatibility complex (MHC) class II allele DRB1*04:01, which encodes a protein that binds self-peptides for presentation to T cells. This study characterises the autoantigen-presenting function of DRB1*04:01 (HLA-DRA*01:01/HLA-DRB1*04:01) at a molecular level for prototypic T-cell determinants, focusing on a post-translationally modified collagen type II (Col2)-derived peptide. METHODS: The crystal structures of DRB1*04:01 molecules in complex with the peptides HSP70289-306, citrullinated CILP982-996 and galactosylated Col2259-273 were determined on cocrystallisation. T cells specific for Col2259-273 were investigated in peripheral blood mononuclear cells from patients with DRB1*04:01-positive RA by cytofluorometric detection of the activation marker CD154 on peptide stimulation and binding of fluorescent DRB1*0401/Col2259-273 tetramer complexes. The cDNAs encoding the T-cell receptor (TCR) α-chains and ß-chains were cloned from single-cell sorted tetramer-positive T cells and transferred via a lentiviral vector into TCR-deficient Jurkat 76 cells. RESULTS: The crystal structures identified peptide binding to DRB1*04:01 and potential side chain exposure to T cells. The main TCR recognition sites in Col2259-273 were lysine residues that can be galactosylated. RA T-cell responses to DRB1*04:01-presented Col2259-273 were dependent on peptide galactosylation at lysine 264. Dynamic molecular modelling of a functionally characterised Col2259-273-specific TCR complexed with DRB1*04:01/Col2259-273 provided evidence for differential allosteric T-cell recognition of glycosylated lysine 264. CONCLUSIONS: The MHC-peptide-TCR interactions elucidated in our study provide new molecular insights into recognition of a post-translationally modified RA T-cell determinant with a known dominant role in arthritogenic and tolerogenic responses in murine Col2-induced arthritis.

Structure-Guided Design of G-Protein-Coupled Receptor Polypharmacology.

Many diseases are polygenic and can only be treated efficiently with drugs that modulate multiple targets. However, rational design of compounds with multi-target profiles is rarely pursued because it is considered too difficult, in particular if the drug must enter the central nervous system. Here, a structure-based strategy to identify dual-target ligands of G-protein-coupled receptors is presented. We use this approach to design compounds that both antagonize the A2A adenosine receptor and activate the D2 dopamine receptor, which have excellent potential as antiparkinson drugs. Atomic resolution models of the receptors guided generation of a chemical library with compounds designed to occupy orthosteric and secondary binding pockets in both targets. Structure-based virtual screens identified ten compounds, of which three had affinity for both targets. One of these scaffolds was optimized to nanomolar dual-target activity and showed the predicted pharmacodynamic effect in a rat model of Parkinsonism.

Drug Syntheses Beyond the Rule of 5.

Drugs in the chemical space beyond the rule of 5 (bRo5) can modulate targets with difficult binding sites while retaining cell permeability and oral absorption. Reviewing the syntheses of bRo5 drugs approved since 1990 highlights synthetic chemistry's contribution to drug discovery in this space. Initially, bRo5 drugs were mainly natural products and semi-synthetic derivatives. Later, peptidomimetics and de novo designed compounds, that include up to seven chiral centres and macrocyclic rings became dominant. These drugs are prepared by total synthesis, sometimes by routes of more than 25 steps with stereocentres originating from the chiral pool, or being installed by chiral induction or enzymatic resolution. Interestingly, ring-closing metathesis proved to be the method of choice for macrocyclisation in hepatitis C virus protease inhibitors. We conclude that structural simplification, planning of synthetic routes regarding incorporation of stereocentres and macrocyclisation, as well as incorporation of structural knowledge and consideration of chameleonic properties in design, should facilitate drug discovery in bRo5 space.

Solution Conformations Explain the Chameleonic Behaviour of Macrocyclic Drugs.

It has been hypothesised that drugs in the chemical space "beyond the rule of 5" (bRo5) must behave as molecular chameleons to combine otherwise conflicting properties, including aqueous solubility, cell permeability and target binding. Evidence for this has, however, been limited to the cyclic peptide cyclosporine A. Herein, we show that the non-peptidic and macrocyclic drugs roxithromycin, telithromycin and spiramycin behave as molecular chameleons, with rifampicin showing a less pronounced behaviour. In particular roxithromycin, telithromycin and spiramycin display a marked, yet limited flexibility and populate significantly less polar and more compact conformational ensembles in an apolar than in a polar environment. In addition to balancing of membrane permeability and aqueous solubility, this flexibility also allows binding to targets that vary in structure between species. The drugs' passive cell permeability correlates to their 3D polar surface area and corroborate two theoretical models for permeability, developed for cyclic peptides. We conclude that molecular chameleonicity should be incorporated in the design of orally administered drugs in the bRo5 space.

Is GPR146 really the receptor for proinsulin C-peptide?

Proinsulin C-peptide has previously been proposed to interact with a G-protein coupled receptor (GPCR), specifically the orphan receptor GPR146. To investigate the potential of C-peptide in treating complications of diabetes, such as kidney damage, it is necessary to understand its mode of action. We used CHO-K1 cells expressing human GPR146 to study human and murine C-peptide in dynamic mass redistribution and GPCR ß-arrestin assays, as well as with fluorescence confocal microscopy. Neither assay revealed any significant intracellular response to C-peptide at concentrations of up to 33 µM. We observed no internalisation of C-peptide by fluorescence microscopy. Our results do not support GPR146 as the receptor for C-peptide, but suggest that further investigations of the mode of action of C-peptide should be undertaken.

Intramolecular hydrogen bonding: An opportunity for improved design in medicinal chemistry.

Recent literature shows that intramolecular hydrogen bond (IMHB) formation can positively impact upon the triad of permeability, solubility, and potency of drugs and candidates. IMHB modulation can be applied to compounds in any chemical space as a means for discovering drug candidates with both acceptable potency and absorption, distribution, metabolism, and excretion-Tox profiles. Integrating IMHB formation in design of drugs is, therefore, an exciting and timely challenge for modern medicinal chemistry. In this review, we first provide some background about IMHBs from the medicinal chemist's point of view and highlight some IMHB-associated misconceptions. Second, we propose a classification of IMHBs for drug discovery purposes, review the most common in silico tactics to include IMHBs in lead optimization and list some experimental physicochemical descriptors, which quantify the propensity of compounds to form IMHBs. By focusing on the compounds size and the number of IMHBs that can potentially be formed, we also outline the major difficulties encountered when designing compounds based on the inclusion of IMHBs. Finally, we discuss recent case studies illustrating the application of IMHB to optimize cell permeability and physicochemical properties of small molecules, cyclic peptides and macrocycles.

Chemical and Biochemical Approaches for the Synthesis of Substituted Dihydroxybutanones and Di- and Tri-Hydroxypentanones.

Polyhydroxylated compounds are building blocks for the synthesis of carbohydrates and other natural products. Their synthesis is mainly achieved by different synthetic versions of aldol-coupling reactions, catalyzed either by organocatalysts, enzymes, or metal-organic catalysts. We have investigated the formation of 1,4-substituted 2,3-dihydroxybutan-1-one derivatives from para- and meta-substituted phenylacetaldehydes by three distinctly different strategies. The first involved a direct aldol reaction with hydroxyacetone, dihydroxyacetone, or 2-hydroxyacetophenone, catalyzed by the cinchona derivative cinchonine. The second was reductive cross-coupling with methyl- or phenylglyoxal promoted by SmI2, resulting in either 5-substituted 3,4-dihydroxypentan-2-ones or 1,4 bis-phenyl-substituted butanones, respectively. Finally, in the third case, aldolase catalysis was employed for synthesis of the corresponding 1,3,4-trihydroxylated pentan-2-one derivatives. The organocatalytic route with cinchonine generated distereomerically enriched syn-products (de = 60-99%), with moderate enantiomeric excesses (ee = 43-56%) but did not produce aldols with either hydroxyacetone or dihydroxyacetone as donor ketones. The SmI2-promoted reductive cross-coupling generated product mixtures with diastereomeric and enantiomeric ratios close to unity. This route allowed for the production of both 1-methyl- and 1-phenyl-substituted 2,3-dihydroxybutanones at yields between 40-60%. Finally, the biocatalytic approach resulted in enantiopure syn-(3 R,4 S) 1,3,4-trihydroxypentan-2-ones.

Cutting Edge: Processing of Oxidized Peptides in Macrophages Regulates T Cell Activation and Development of Autoimmune Arthritis.

APCs are known to produce NADPH oxidase (NOX) 2-derived reactive oxygen species; however, whether and how NOX2-mediated oxidation affects redox-sensitive immunogenic peptides remains elusive. In this study, we investigated a major immunogenic peptide in glucose-6-phosphate isomerase (G6PI), a potential autoantigen in rheumatoid arthritis, which can form internal disulfide bonds. Ag presentation assays showed that presentation of this G6PI peptide was more efficient in NOX2-deficient (Ncf1m1J/m1J mutant) mice, compared with wild-type controls. IFN-γ-inducible lysosomal thiol reductase (GILT), which facilitates disulfide bond-containing Ag processing, was found to be upregulated in macrophages from Ncf1 mutant mice. Ncf1 mutant mice exhibited more severe G6PI peptide-induced arthritis, which was accompanied by the increased GILT expression in macrophages and enhanced Ag-specific T cell responses. Our results show that NOX2-dependent processing of the redox-sensitive autoantigens by APCs modify T cell activity and development of autoimmune arthritis.

Metabolite aberrations in early diabetes detected in rat kidney using mass spectrometry imaging.

Diabetic kidney disease is a serious complication of diabetes that can ultimately lead to end-stage renal disease. The pathogenesis of diabetic kidney disease is complex, and fundamental research is still required to provide a better understanding of the driving forces behind it. We report regional metabolic aberrations from an untargeted mass spectrometry imaging study of kidney tissue using an insulinopenic rat model of diabetes. Diabetes was induced by intravenous injection of streptozotocin, and kidneys were harvested 2 weeks thereafter. Imaging was performed using nanospray desorption electrospray ionization connected to a high-mass-resolving mass spectrometer. No histopathological changes were observed in the kidney sections; however, mass spectrometry imaging revealed a significant increase in several 18-carbon unsaturated non-esterified fatty acid species and monoacylglycerols. Notably, these 18-carbon acyl chains were also constituents of several increased diacylglycerol species. In addition, a number of short- and long-chain acylcarnitines were found to be accumulated while several amino acids were depleted. This study presents unique regional metabolic data indicating a dysregulated energy metabolism in renal mitochondria as an early response to streptozotocin-induced type I diabetes. Graphical abstract.

Structural and conformational determinants of macrocycle cell permeability.

Macrocycles are of increasing interest as chemical probes and drugs for intractable targets like protein-protein interactions, but the determinants of their cell permeability and oral absorption are poorly understood. To enable rational design of cell-permeable macrocycles, we generated an extensive data set under consistent experimental conditions for more than 200 non-peptidic, de novo-designed macrocycles from the Broad Institute's diversity-oriented screening collection. This revealed how specific functional groups, substituents and molecular properties impact cell permeability. Analysis of energy-minimized structures for stereo- and regioisomeric sets provided fundamental insight into how dynamic, intramolecular interactions in the 3D conformations of macrocycles may be linked to physicochemical properties and permeability. Combined use of quantitative structure-permeability modeling and the procedure for conformational analysis now, for the first time, provides chemists with a rational approach to design cell-permeable non-peptidic macrocycles with potential for oral absorption.

Hydroxyethylene isosteres introduced in type II collagen fragments substantially alter the structure and dynamics of class II MHC A(q)/glycopeptide complexes.

Class II major histocompatibility complex (MHC) proteins are involved in initiation of immune responses to foreign antigens via presentation of peptides to receptors of CD4(+) T-cells. An analogous presentation of self-peptides may lead to autoimmune diseases, such as rheumatoid arthritis (RA). The glycopeptide fragment CII259-273, derived from type II collagen, is presented by A(q) MHCII molecules in the mouse and has a key role in development of collagen induced arthritis (CIA), a validated model for RA. We have introduced hydroxyethylene amide bond isosteres at the Ala(261)-Gly(262) position of CII259-273. Biological evaluation showed that A(q) binding and T cell recognition were dramatically reduced for the modified glycopeptides, although static models predicted similar binding modes as the native type II collagen fragment. Molecular dynamics (MD) simulations demonstrated that introduction of the hydroxyethylene isosteres disturbed the entire hydrogen bond network between the glycopeptides and A(q). As a consequence the hydroxyethylene isosteric glycopeptides were prone to dissociation from A(q) and unfolding of the ß1-helix. Thus, the isostere induced adjustment of the hydrogen bond network altered the structure and dynamics of A(q)/glycopeptide complexes leading to the loss of A(q) affinity and subsequent T cell response.

Molecular chameleons in drug discovery.

Molecular chameleons possess a flexibility that allows them to dynamically shield or expose polar functionalities in response to the properties of the environment. Although the concept of molecular chameleons was introduced already in 1970, interest in them has grown considerably since the 2010s, when drug discovery has focused to an increased extent on new chemical modalities. Such modalities include cyclic peptides, macrocycles and proteolysis-targeting chimeras, all of which reside in a chemical space far from that of traditional small-molecule drugs. Both cell permeability and aqueous solubility are required for the oral absorption of drugs. Engineering these properties, and potent target binding, into the larger new modalities is a more daunting task than for traditional small-molecule drugs. The ability of chameleons to adapt to different environments may be essential for success. In this Review, we provide both general and theoretical insights into the realm of molecular chameleons. We discuss why chameleons have come into fashion and provide a do-it-yourself toolbox for their design; we then provide a glimpse of how advanced in silico methods can support molecular chameleon design.

Exploring the chemical space of orally bioavailable PROTACs.

A principal challenge in the discovery of proteolysis targeting chimeras (PROTACs) as oral medications is their bioavailability. To facilitate drug design, it is therefore essential to identify the chemical space where orally bioavailable PROTACs are more likely to be situated. To this aim, we extracted structure-bioavailability insights from published data using traditional 2D descriptors, thereby shedding light on their potential and limitations as drug design tools. Subsequently, we describe cutting-edge experimental, computational and hybrid design strategies based on 3D descriptors, which show promise for enhancing the probability of discovering PROTACs with high oral bioavailability.

Multifunctional T cell reactivity with native and glycosylated type II collagen in rheumatoid arthritis.

OBJECTIVE: Type II collagen (CII) is a cartilage-specific protein to which a loss of immune tolerance may trigger autoimmune reactions and cause arthritis. The major T cell epitope on CII, amino acids 259-273, can be presented by several HLA-DRB1 04 alleles in its native or posttranslational glycosylated form. The present study was undertaken to functionally explore and compare CII-autoreactive T cells from blood and synovial fluid of patients with rheumatoid arthritis (RA). METHODS: Peripheral blood was obtained from HLA-DRB1 04-positive RA patients (n = 10) and control subjects (n = 10) and stimulated in vitro with several variants of the CII(259-273) epitope, i.e., unmodified, glycosylated on Lys-264, glycosylated on Lys-270, or glycosylated on both Lys-264 and Lys-270. Up-regulation of CD154 was used to identify responding T cells. These cells were further characterized by intracellular staining for interleukin-17 (IL-17), interferon-γ (IFNγ), and IL-2 by flow cytometry. Synovial T cells from RA patients were investigated in parallel. RESULTS: Multifunctional T cell responses toward all examined variants of the CII(259-273) peptide could be detected in RA patients and, to a lesser extent, also in healthy HLA-matched controls (P < 0.001). In RA patients, a comparison between blood- and joint-derived T cell function revealed a significant increase in levels of the proinflammatory cytokine IFNγ in synovial T cells (P = 0.027). Studies of longitudinally obtained samples showed that T cell responses were sustained over the course of disease, and even included epitope spreading. CONCLUSION: The identification of inflammatory T cell responses to both glycosylated and nonglycosylated variants of the major CII epitope in RA patients suggests that CII autoreactivity in RA may be more common than previously recognized.