Discovery and Characterization of a Bicyclic Peptide (Bicycle) Binder to Thymic Stromal Lymphopoietin.

Thymic stromal lymphopoietin (TSLP) is an epithelial-derived pro-inflammatory cytokine involved in the development of asthma and other atopic diseases. We used Bicycle Therapeutics' proprietary phage display platform to identify bicyclic peptides (Bicycles) with high affinity for TSLP, a target that is difficult to drug with conventional small molecules due to the extended protein-protein interactions it forms with both receptors. The hit series was shown to bind to TSLP in a hotspot, that is also used by IL-7Rα. Guided by the first X-ray crystal structure of a small peptide binding to TSLP and the identification of key metabolites, we were able to improve the proteolytic stability of this series in lung S9 fractions without sacrificing binding affinity. This resulted in the potent Bicycle 46 with nanomolar affinity to TSLP (KD = 13 nM), low plasma clearance of 6.4 mL/min/kg, and an effective half-life of 46 min after intravenous dosing to rats.

A cell-active cyclic peptide targeting the Nrf2/Keap1 protein-protein interaction.

The disruption of the protein-protein interaction (PPI) between Nrf2 and Keap1 is an attractive strategy to counteract the oxidative stress that characterises a variety of severe diseases. Peptides represent a complementary approach to small molecules for the inhibition of this therapeutically important PPI. However, due to their polar nature and the negative net charge required for binding to Keap1, the peptides reported to date exhibit either mid-micromolar activity or are inactive in cells. Herein, we present a two-component peptide stapling strategy to rapidly access a variety of constrained and functionalised peptides that target the Nrf2/Keap1 PPI. The most promising peptide, P8-H containing a fatty acid tag, binds to Keap1 with nanomolar affinity and is effective at inducing transcription of ARE genes in a human lung epithelial cell line at sub-micromolar concentration. Furthermore, crystallography of the peptide in complex with Keap1 yielded a high resolution X-ray structure, adding to the toolbox of structures available to develop cell-permeable peptidomimetic inhibitors.

Kinetic Modeling of PROTAC-Induced Protein Degradation.

Kinetics of the PROTAC-induced protein degradation were modelled using the equilibrium approximation, accounting for the protein recovery rate with a time lag. The simulated kinetic curves resemble what is experimentally observed, and the physical formulas of the half-maximal degradation concentration (DC50 ) were derived from them. The equations reveal that DC50 is proportional to the dissociation constant of the ternary complex (Kd ) and inversely proportional to the expression level of the E3 ligase and the effective ubiquitylation rate (kub ). The predicted relationships were rigorously confirmed by experimental evidences from a matched molecular pair analysis using a set of published PROTACs.

Analyzing proteolytic stability and metabolic hotspots of therapeutic peptides in two rodent pulmonary fluids.

Peptides and peptide drug conjugates are emerging modalities to treat pulmonary diseases. Peptides are susceptible to proteolytic cleavage. Expression levels of specific proteases in the lung can be significantly increased in disease state and may lead to exaggerated peptide proteolysis. To support optimization of peptides for inhaled administration, we have recently reported a streamlined high-throughput LC-HRMS protocol to determine enzymatic protease stability of peptides. This method has now been complemented with profiling of peptide metabolic stability in two respiratory fluids, a lung supernatant (lung S9) and a bronchioalveolar lavage fluid (BALF) taken from rats. We have tested a set of 28 peptides with high structural diversity, analyzed the whole data set for formed metabolites, and identified the differences of cleavage pattern in the two test fluids. Comparison of our experimental results and literature-derived cleavage site estimates based on e.g. MEROPS show significant differences for a number of peptides. This indicates the need for an experimental workflow using both protease panels and testing of metabolic stability in lung fluid (BALF) to guide peptide optimization and selection of peptides for inhaled in vivo PK/PD studies in our drug discovery projects.

Automated high-throughput in vitro assays to identify metabolic hotspots and protease stability of structurally diverse, pharmacologically active peptides for inhalation.

The inhalation of peptides comes with the advantage of directly targeting the lung as tissue of interest. However, peptides are often rapidly metabolized in lung tissue through proteolytic cleavage. We have developed an assay workflow to obtain half-life and metabolite ID data for peptides incubated with four proteases abundant in lungs of asthma and COPD patients. The assay system has been validated using 28 structurally diverse linear and cyclic peptides with a molecular weight between 708 and 5808 Da. Experimental conditions for incubation, sample preparation, chromatography, data acquisition and analysis are compatible with the required throughput in early stage peptide projects. Together with co-crystal structures and Ala scans, we are using the described assay workflow to guide the first chemical modifications of peptide hits in early respiratory drug discovery projects.

AZD0284, a Potent, Selective, and Orally Bioavailable Inverse Agonist of Retinoic Acid Receptor-Related Orphan Receptor C2.

Inverse agonists of the nuclear receptor RORC2 have been widely pursued as a potential treatment for a variety of autoimmune diseases. We have discovered a novel series of isoindoline-based inverse agonists of the nuclear receptor RORC2, derived from our recently disclosed RORC2 inverse agonist 2. Extensive structure-activity relationship (SAR) studies resulted in AZD0284 (20), which combined potent inhibition of IL-17A secretion from primary human TH17 cells with excellent metabolic stability and good PK in preclinical species. In two preclinical in vivo studies, compound 20 reduced thymocyte numbers in mice and showed dose-dependent reduction of IL-17A containing γδ-T cells and of IL-17A and IL-22 RNA in the imiquimod induced inflammation model. Based on these data and a favorable safety profile, 20 was progressed to phase 1 clinical studies.

Mechanism-Based Insights into Removing the Mutagenicity of Aromatic Amines by Small Structural Alterations.

Aromatic and heteroaromatic amines (ArNH2) are activated by cytochrome P450 monooxygenases, primarily CYP1A2, into reactive N-arylhydroxylamines that can lead to covalent adducts with DNA nucleobases. Hereby, we give hands-on mechanism-based guidelines to design mutagenicity-free ArNH2. The mechanism of N-hydroxylation of ArNH2 by CYP1A2 is investigated by density functional theory (DFT) calculations. Two putative pathways are considered, the radicaloid route that goes via the classical ferryl-oxo oxidant and an alternative anionic pathway through Fenton-like oxidation by ferriheme-bound H2O2. Results suggest that bioactivation of ArNH2 follows the anionic pathway. We demonstrate that H-bonding and/or geometric fit of ArNH2 to CYP1A2 as well as feasibility of both proton abstraction by the ferriheme-peroxo base and heterolytic cleavage of arylhydroxylamines render molecules mutagenic. Mutagenicity of ArNH2 can be removed by structural alterations that disrupt geometric and/or electrostatic fit to CYP1A2, decrease the acidity of the NH2 group, destabilize arylnitrenium ions, or disrupt their pre-covalent transition states with guanine.

Emerging modes-of-action in drug discovery.

An increasing focus on complex biology to cure diseases rather than merely treat symptoms has transformed how drug discovery can be approached. Instead of activating or blocking protein function, a growing repertoire of drug modalities can be leveraged or engineered to hijack cellular processes, such as translational regulation or degradation mechanisms. Drug hunters can therefore access a wider arsenal of modes-of-action to modulate biological processes and this review summarises these emerging strategies by highlighting the most representative examples of these approaches.

Discovery of Potent and Orally Bioavailable Inverse Agonists of the Retinoic Acid Receptor-Related Orphan Receptor C2.

The further optimization of a recently disclosed series of inverse agonists of the nuclear receptor RORC2 is described. Investigations into the left-hand side of compound 1, guided by X-ray crystal structures, led to the substitution of the 4-aryl-thiophenyl residue with the hexafluoro-2-phenyl-propan-2-ol moiety. This change resulted in to compound 28, which combined improved drug-like properties with good cell potency and a significantly lower dose, using an early dose to man prediction. Target engagement in vivo was demonstrated in the thymus of mice by a reduction in the number of double positive T cells after oral dosing.

Potent and Orally Bioavailable Inverse Agonists of RORγt Resulting from Structure-Based Design.

Retinoic acid receptor related orphan receptor γt (RORγt), has been identified as the master regulator of TH17-cell function and development, making it an attractive target for the treatment of autoimmune diseases by a small-molecule approach. Herein, we describe our investigations on a series of 4-aryl-thienyl acetamides, which were guided by insights from X-ray cocrystal structures. Efforts in targeting the cofactor-recruitment site from the 4-aryl group on the thiophene led to a series of potent binders with nanomolar activity in a primary human-TH17-cell assay. The observation of a DMSO molecule binding in a subpocket outside the LBD inspired the introduction of an acetamide into the benzylic position of these compounds. Hereby, a hydrogen-bond interaction of the introduced acetamide oxygen with the backbone amide of Glu379 was established. This greatly enhanced the cellular activity of previously weakly cell-active compounds. The best compounds combined potent inhibition of IL-17 release with favorable PK in rodents, with compound 32 representing a promising starting point for future investigations.

Theoretical studies of the second step of the nitric oxide synthase reaction: Electron tunneling prevents uncoupling.

Nitric oxide (NO·) is a messenger molecule with diverse physiological roles including host defense, neurotransmission and vascular function. The synthesis of NO· from l-arginine is catalyzed by NO-synthases and occurs in two steps through the intermediary Nω-hydroxy-l-arginine (NHA). In both steps the P450-like reaction cycle is coupled with the redox cycle of the cofactor tetrahydrobiopterin (H4B). The mechanism of the second step is studied by Density Functional Theory calculations to ascertain the canonical sequence of proton and electron transfer (PT and ET) events. The proposed mechanism is controlled by the interplay of two electron donors, H4B and NHA. Consistent with experimental data, the catalytic cycle proceeds through the ferric-hydroperoxide complex (Cpd 0) and the following aqua-ferriheme resting state, and involves interim partial oxidation of H4B. The mechanism starts with formation of Cpd 0 from the ferrous-dioxy reactant complex by PT from the C-ring heme propionate coupled with hole transfer to H4B through the highest occupied π-orbital of NHA as a bridge. This enables PT from NHA+· to the proximal oxygen leading to the shallow ferriheme-H2O2 oxidant. Subsequent Fenton-like peroxide bond cleavage triggered by ET from the NHA-derived iminoxy-radical leads to the protonated Cpd II diradicaloid singlet stabilized by spin delocalization in H4B, and the closed-shell coordination complex of HO- with iminoxy-cation. The complex is converted to the transient C-adduct, which releases intended products upon PT to the ferriheme-HO- complex coupled with ET to the H4B+·. Deferred ET from the substrate or undue ET from/to the cofactor leads to side products.

Benzoxazepines Achieve Potent Suppression of IL-17 Release in Human T-Helper 17 (TH 17) Cells through an Induced-Fit Binding Mode to the Nuclear Receptor RORγ.

RORγt, an isoform of the retinoic acid-related orphan receptor gamma (RORc, RORγ), has been identified as the master regulator of T-helper 17 (TH 17) cell function and development, making it an attractive target for the treatment of autoimmune diseases. Validation for this target comes from antibodies targeting interleukin-17 (IL-17), the signature cytokine produced by TH 17 cells, which have shown impressive results in clinical trials. Through focused screening of our compound collection, we identified a series of N-sulfonylated benzoxazepines, which displayed micromolar affinity for the RORγ ligand-binding domain (LBD) in a radioligand binding assay. Optimization of these initial hits resulted in potent binders, which dose-dependently decreased the ability of the RORγ-LBD to interact with a peptide derived from steroid receptor coactivator 1, and inhibited the release of IL-17 secretion from isolated and cultured human TH 17 cells with nanomolar potency. A cocrystal structure of inverse agonist 15 (2-chloro-6-fluoro-N-(4-{[3-(trifluoromethyl)phenyl]sulfonyl}-2,3,4,5-tetrahydro-1,4-benzoxazepin-7-yl)benzamide) bound to the RORγ-LBD illustrated that both hydrophobic interactions, leading to an induced fit around the substituted benzamide moiety of 15, as well as a hydrogen bond from the amide NH to His479 seemed to be important for the mechanism of action. This structure is compared with the structure of agonist 25 (N-(2-fluorophenyl)-4-[(4-fluorophenyl)sulfonyl]-2,3,4,5-tetrahydro-1,4-benzoxazepin-6-amine ) and structures of other known RORγ modulators.

Identification of indole inhibitors of human hematopoietic prostaglandin D2 synthase (hH-PGDS).

Human H-PGDS has shown promise as a potential target for anti-allergic and anti-inflammatory drugs. Here we describe the discovery of a novel class of indole inhibitors, identified through focused screening of 42,000 compounds and evaluated using a series of hit validation assays that included fluorescence polarization binding, 1D NMR, ITC and chromogenic enzymatic assays. Compounds with low nanomolar potency, favorable physico-chemical properties and inhibitory activity in human mast cells have been identified. In addition, our studies suggest that the active site of hH-PGDS can accommodate larger structural diversity than previously thought, such as the introduction of polar groups in the inner part of the binding pocket.

HTS followed by NMR based counterscreening. Discovery and optimization of pyrimidones as reversible and competitive inhibitors of xanthine oxidase.

The identification of novel, non-purine based inhibitors of xanthine oxidase is described. After a high-throughput screening campaign, an NMR based counterscreen was used to distinguish actives, which interact with XO in a reversible manner, from assay artefacts. This approach identified pyrimidone 1 as a reversible and competitive inhibitor with good lead-like properties. A hit to lead campaign gave compound 41, a nanomolar inhibitor of hXO with efficacy in the hyperuricemic rat model after oral dosing.

Discovery of (7R)-14-cyclohexyl-7-{[2-(dimethylamino)ethyl](methyl) amino}-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylic acid (MK-3281), a potent and orally bioavailable finger-loop inhibitor of the hepatitis C virus NS5B polymerase.

Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. The polymerase of HCV is responsible for the replication of viral genome and has been a prime target for drug discovery efforts. Here, we report on the further development of tetracyclic indole inhibitors, binding to an allosteric site on the thumb domain. Structure-activity relationship (SAR) studies around an indolo-benzoxazocine scaffold led to the identification of compound 33 (MK-3281), an inhibitor with good potency in the HCV subgenomic replication assay and attractive molecular properties suitable for a clinical candidate. The compound caused a consistent decrease in viremia in vivo using the chimeric mouse model of HCV infection.

Enhancement of intestinal absorption of 2-methyl cytidine prodrugs.

The purpose of this study was to investigate the in vivo absorption enhancement of a nucleoside (phosphoramidate prodrug of 2'-methyl-cytidine) anti-viral agent of proven efficacy by means of intestinal permeation enhancers. Natural nucleosides are hydrophilic molecules that do not rapidly penetrate cell membranes by diffusion and their absorption relies on specialized transporters. Therefore, the oral absorption of nucleoside prodrugs and the target organ concentration of the biologically active nucleotide can be limited due to poor permeation across the intestinal epithelium. In the present study, the specificity, concentration dependence, and effect of four classes of absorption promoters, i.e. fatty acids, steroidal detergents, mucoadhesive polymers, and secretory transport inhibitors, were evaluated in a rat in vivo model. Sodium caprate and alpha-tocopheryl-polyethyleneglycol-1000-succinate (TPGS) showed a significant effect in increasing liver concentration of nucleotide (5-fold). These results suggested that both excipients might be suited in a controlled release matrix for the synchronous release of the drug and absorption promoter directly to the site of absorption and highlights that the effect is strictly dependent on the absorption promoter dose. The feasibility of such a formulation approach in humans was evaluated with the aim of developing a solid dosage form for the peroral delivery of nucleosides and showed that these excipients do provide a potential valuable tool in pre-clinical efficacy studies to drive discovery programs forward.

Mechanism of hepatitis C virus RNA polymerase inhibition with dihydroxypyrimidines.

We studied the biochemical mechanisms associated with inhibition and resistance to a 4,5-dihydroxypyrimidine carboxylate that inhibits the hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B. On the basis of the structure of the pharmacophore, it has been suggested that these compounds may act as pyrophosphate (PP(i)) mimics. We monitored nucleotide incorporation events during the elongation phase and showed that the polymerase activity of wild-type NS5B was inhibited by the dihydroxypyrimidine at a 50% inhibitory concentration (IC(50)) of 0.73 muM. Enzymes with the G152E or P156L mutation, either of which confers resistance to this compound, showed four- to fivefold increases in IC(50)s. The inhibitor was competitive with respect to nucleotide incorporation. It was likewise effective at preventing the PP(i)-mediated excision of an incorporated chain terminator in a competitive fashion. In the absence of the dihydroxypyrimidine, the reaction was not significantly affected by the G152E or P156L mutation. These data suggest that the resistance associated with these two mutations is unlikely due to an altered interaction with the pyrophosphate-mimicking domain of the compound but, rather, is due to altered interactions with its specificity domain at a region distant from the active site. Together, our findings provide strong experimental evidence that supports the notion that the members of this class of compounds can act as PP(i) mimics that have the potential to mechanistically complement established nucleoside and nonnucleoside analogue inhibitors.

Identification and biological evaluation of a series of 1H-benzo[de]isoquinoline-1,3(2H)-diones as hepatitis C virus NS5B polymerase inhibitors.

The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in virus replication. NS5B has no functional equivalent in mammalian cells and, as a consequence, is an attractive target for inhibition. Herein, we present 1H-benzo[de]isoquinoline-1,3(2H)-diones as a new series of selective inhibitors of HCV NS5B polymerase. The HTS hit 1 shows submicromolar potency in two different HCV replicons (1b and 2b) and displays no activity on other polymerases (HIV-RT, Polio-pol, GBV-b-pol). These inhibitors act during the pre-elongation phase by binding to NS5B non-nucleoside binding site Thumb Site II as demonstrated by crystal structure of compound 1 with the DeltaC55-1b and DeltaC21-2b enzymes and by mutagenesis studies. SAR in this new series reveals inhibitors, such as 20, with low micromolar activity in the HCV replicon and with good activity/toxicity window in cells.

2-(3-Thienyl)-5,6-dihydroxypyrimidine-4-carboxylic acids as inhibitors of HCV NS5B RdRp.

A series of 2-(3-thienyl)-5,6-dihydroxypyrimidine-4-carboxylic acid inhibitors of the hepatitis C virus (HCV) NS5B polymerase enzyme are reported. Sulfonyl urea substituted analogs in this series proved to be the most potent active site non-nucleoside inhibitors of NS5B reported to date. These compounds had low nanomolar enzyme inhibition across HCV genotypes 1-3 and showed single digit micromolar inhibition in the HCV replicon assay. This improved cell-based activity allowed the binding mode of these compounds to be probed by selection of resistant mutations against compound 21. The results generated are in broad agreement with the previously proposed binding model for this compound class.

Phosphoramidate prodrugs of 2'-C-methylcytidine for therapy of hepatitis C virus infection.

The application of a phosphoramidate prodrug approach to 2'-C-methylcytidine (NM107), the first nucleoside inhibitor of the hepatitis C virus (HCV) NS5B polymerase, is reported. 2'-C-Methylcytidine, as its valyl ester prodrug (NM283), was efficacious in reducing the viral load in patients infected with HCV. Several of the phosphoramidates prepared demonstrated a 10- to 200-fold superior potency with respect to the parent nucleoside in the cell-based replicon assay. This is due to higher levels of 2'-C-methylcytidine triphosphate in the cells. These prodrugs are efficiently activated and converted to the triphosphate in hepatocytes of several species. Our SAR studies ultimately led to compounds that gave high levels of NTP in hamster and rat liver after subcutaneous dosing and that were devoid of the toxic phenol moiety usually found in ProTides.