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.

Selective and Bioavailable HDAC6 2-(Difluoromethyl)-1,3,4-oxadiazole Substrate Inhibitors and Modeling of Their Bioactivation Mechanism.

Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family mainly targeting cytosolic nonhistone substrates, such as α-tubulin, cortactin, and heat shock protein 90 to regulate cell proliferation, metastasis, invasion, and mitosis in tumors. We describe the identification and characterization of a series of 2-(difluoromethyl)-1,3,4-oxadiazoles (DFMOs) as selective nonhydroxamic acid HDAC6 inhibitors. By comparing structure-activity relationships and performing quantum mechanical calculations of the HDAC6 catalytic mechanism, we show that potent oxadiazoles are electrophilic substrates of HDAC6 and propose a mechanism for the bioactivation. We also observe that the inherent electrophilicity of the oxadiazoles makes them prone to degradation in water solution and the generation of potentially toxic products cannot be ruled out, limiting the developability for chronic diseases. However, the oxadiazoles demonstrate high oral bioavailability and low in vivo clearance and are excellent tools for studying the role of HDAC6 in vitro and in vivo in rats and mice.

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.

Fragment-Based Discovery of Novel Allosteric MEK1 Binders.

The MEK1 kinase plays a critical role in key cellular processes, and as such, its dysfunction is strongly linked to several human diseases, particularly cancer. MEK1 has consequently received considerable attention as a drug target, and a significant number of small-molecule inhibitors of this kinase have been reported. The majority of these inhibitors target an allosteric pocket proximal to the ATP binding site which has proven to be highly druggable, with four allosteric MEK1 inhibitors approved to date. Despite the significant attention that the MEK1 allosteric site has received, chemotypes which have been shown structurally to bind to this site are limited. With the aim of discovering novel allosteric MEK1 inhibitors using a fragment-based approach, we report here a screening method which resulted in the discovery of multiple allosteric MEK1 binders, one series of which was optimized to sub-µM affinity for MEK1 with promising physicochemical and ADMET properties.

Theoretical Studies of the Mechanism of Carbamoylation of Nucleobases by Isocyanates.

Isocyanates with the -N═C═O functional group are highly reactive compounds. They are used in various industrial applications and have been found as possible metabolites of hydroxamic acids. Isocyanates interact with biopolymers and are notorious mutagens. Mutagenic effects of isocyanates are caused by the formation of covalent adducts with nucleobases of DNA, primarily cytosines, through carbamoylation of NH2 groups to give the corresponding urea. The mechanism of carbamoylation of nucleobases by aryl isocyanates is studied by high-level density functional theory calculations. Three possible pathways are analyzed. It is demonstrated that the reaction follows the stepwise pathway, which starts with the formation of a π-complex followed by a rate-determining C-N covalent bond formation via the reactive tautomeric imine forms of the nucleobases. The reaction proceeds further through two consecutive proton transfers mediated by water molecules to give the final adduct. The predicted activation free energies of the rate-determining step in water agree with experimental data. In line with experiments, the reactivity of isocyanates toward nucleobases decreases in the order cytosine > adenine > guanine, and we rationalize this order of reactivity by the fall of their basicity and destabilization of the imine forms. Activation barriers of the alternative concerted pathways are higher than that of the preferred stepwise mechanism, and the match to experiment is poor. The kinetic effect of adding electron-withdrawing or electron-donating groups to the aryl group of aryl isocyanate is minute, which suggests that mutagenicity of isocyanates is determined exclusively by the reactivity of the -N═C═O group and as such cannot be removed by structural alterations of the adjacent aryl.

Identification and Optimization of Pyrrolidine Derivatives as Highly Potent Ghrelin Receptor Full Agonists.

Muscle atrophy and cachexia are common comorbidities among patients suffering from cancer, chronic obstructive pulmonary disease, and several other chronic diseases. The peptide hormone ghrelin exerts pleiotropic effects including the stimulation of growth hormone secretion and subsequent increase of insulin-like growth factor-1 levels, an important mediator of muscle growth and repair. Ghrelin also acts on inflammation, appetite, and adipogenesis and therefore has been considered a promising therapeutic target for catabolic conditions. We previously reported on the synthesis and properties of an indane based series of ghrelin receptor full agonists which led to a sustained increase of insulin-like growth factor-1 in a dog pharmacodynamic study. Herein we report on the identification of a series of pyrrolidine or piperidine based full agonists and attempted optimization to give compounds with profiles suitable for progression as clinical candidates.

Discovery and Early Clinical Development of an Inhibitor of 5-Lipoxygenase Activating Protein (AZD5718) for Treatment of Coronary Artery Disease.

5-Lipoxygenase activating protein (FLAP) inhibitors attenuate 5-lipoxygenase pathway activity and reduce the production of proinflammatory and vasoactive leukotrienes. As such, they are hypothesized to have therapeutic benefit for the treatment of diseases that involve chronic inflammation including coronary artery disease. Herein, we disclose the medicinal chemistry discovery and the early clinical development of the FLAP inhibitor AZD5718 (12). Multiparameter optimization included securing adequate potency in human whole blood, navigation away from Ames mutagenic amine fragments while balancing metabolic stability and PK properties allowing for clinically relevant exposures after oral dosing. The superior safety profile of AZD5718 compared to earlier frontrunner compounds allowed us to perform a phase 1 clinical study in which AZD5718 demonstrated a dose dependent and greater than 90% suppression of leukotriene production over 24 h. Currently, AZD5718 is evaluated in a phase 2a study for treatment of coronary artery disease.

Identification and Pharmacological Profile of an Indane Based Series of Ghrelin Receptor Full Agonists.

Cachexia and muscle wasting are very common among patients suffering from cancer, chronic obstructive pulmonary disease, and other chronic diseases. Ghrelin stimulates growth hormone secretion via the ghrelin receptor, which subsequently leads to increase of IGF-1 plasma levels. The activation of the GH/IGF-1 axis leads to an increase of muscle mass and functional capacity. Ghrelin further acts on inflammation, appetite, and adipogenesis and for this reason was considered an important target to address catabolic conditions. We report the synthesis and properties of an indane based series of ghrelin receptor full agonists; they have been shown to generate a sustained increase of IGF-1 levels in dog and have been thoroughly investigated with respect to their functional activity.

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.

The discovery of a selective and potent A2a agonist with extended lung retention.

Although the anti-inflammatory role of the A2a receptor is well established, controversy remains with regard to the therapeutic value for A2a agonists in treatment of inflammatory lung diseases, also as a result of unwanted A2a-mediated cardiovascular effects. In this paper, we describe the discovery and characterization of a new, potent and selective A2a agonist (compound 2) with prolonged lung retention and limited systemic exposure following local administration. To support the lead optimization chemistry program with compound selection and profiling, multiple in vitro and in vivo assays were used, characterizing compound properties, pharmacodynamics (PD), and drug concentrations. Particularly, pharmacokinetic-PD modeling was applied to quantify the effects on the cardiovascular system, and an investigative toxicology study in rats was performed to explore potential myocardial toxicities. Compound 2, in comparison to a reference A2a agonist, UK-432,097, demonstrated higher solubility, lower lipophilicity, lower plasma protein binding, high rat lung retention (28% remaining after 24 h), and was efficacious in a lung inflammatory rat model following intratracheal dosing. Despite these properties, compound 2 did not provide a sufficient therapeutic index, that is, separation of local anti-inflammatory efficacy in the lung from systemic side effects in the cardiovascular system. The plasma concentration that resulted in induction of hypotension (half maximal effective concentration; EC50 0.5 nmol/L) correlated to the in vitro A2a potency (rIC50 0.6 nmol/L). Histopathological lesions in the heart were observed at a dose level which is threefold above the efficacious dose level in the inflammatory rat lung model. In conclusion, compound 2 is a highly potent and selective A2a agonist with significant lung retention after intratracheal administration. Despite its local anti-inflammatory efficacy in rat lung, small margins to the cardiovascular effects suggested limited therapeutic value of this compound for treatment of inflammatory lung disease by the inhaled route.

Theoretical studies of the mechanism of N-hydroxylation of primary aromatic amines by cytochrome P450 1A2: radicaloid or anionic?

Primary aromatic and heteroaromatic amines are notoriously known as potential mutagens and carcinogens. The major event of the mechanism of their mutagenicity is N-hydroxylation by P450 enzymes, primarily P450 1A2 (CYP1A2), which leads to the formation of nitrenium ions that covalently modify nucleobases of DNA. Energy profiles of the NH bond activation steps of two possible mechanisms of N-hydroxylation of a number of aromatic amines by CYP1A2, radicaloid and anionic, are studied by dispersion-corrected DFT calculations. The classical radicaloid mechanism is mediated by H-atom transfer to the electrophilic ferryl-oxo intermediate of the P450 catalytic cycle (called Compound I or Cpd I), whereas the alternative anionic mechanism involves proton transfer to the preceding nucleophilic ferrous-peroxo species. The key structural features of the catalytic site of human CYP1A2 revealed by X-ray crystallography are maintained in calculations. The obtained DFT reaction profiles and additional calculations that account for nondynamical electron correlation suggest that Cpd I has higher thermodynamic drive to activate aromatic amines than the ferrous-peroxo species. Nevertheless, the anionic mechanism is demonstrated to be consistent with a variety of experimental observations. Thus, energy of the proton transfer from aromatic amines to the ferrous-peroxo dianion splits aromatic amines into two classes with different mutagenicity mechanisms. Favorable or slightly unfavorable barrier-free proton transfer is inherent in compounds that undergo nitrenium ion mediated mutagenicity. Monocyclic electron-rich aromatic amines that do not follow this mutagenicity mechanism show significantly unfavorable proton transfer. Feasibility of the entire anionic mechanism is demonstrated by favorable Gibbs energy profiles of both chemical steps, NH bond activation, and NO bond formation. Taken together, results suggest that the N-hydroxylation of aromatic amines in CYP1A2 undergoes the anionic mechanism. Possible reasons for the apparent inability of Cpd I to activate aromatic amines in CYP1A2 are discussed.

Theoretical studies of chemical reactivity of metabolically activated forms of aromatic amines toward DNA.

The metabolism of aromatic and heteroaromatic amines (ArNH2) results in nitrenium ions (ArNH⁺) that modify nucleobases of DNA, primarily deoxyguanosine (dG), by forming dG-C8 adducts. The activated amine nitrogen in ArNH⁺ reacts with the C8 of dG, which gives rise to mutations in DNA. For the most mutagenic ArNH2, including the majority of known genotoxic carcinogens, the stability of ArNH⁺ is of intermediate magnitude. To understand the origin of this observation as well as the specificity of reactions of ArNH⁺ with guanines in DNA, we investigated the chemical reactivity of the metabolically activated forms of ArNH2, that is, ArNHOH and ArNHOAc, toward 9-methylguanine by DFT calculations. The chemical reactivity of these forms is determined by the rate constants of two consecutive reactions leading to cationic guanine intermediates. The formation of ArNH⁺ accelerates with resonance stabilization of ArNH⁺, whereas the formed ArNH⁺ reacts with guanine derivatives with the constant diffusion-limited rate until the reaction slows down when ArNH⁺ is about 20 kcal/mol more stable than PhNH⁺. At this point, ArNHOH and ArNHOAc show maximum reactivity. The lowest activation energy of the reaction of ArNH⁺ with 9-methylguanine corresponds to the charge-transfer π-stacked transition state (π-TS) that leads to the direct formation of the C8 intermediate. The predicted activation barriers of this reaction match the observed absolute rate constants for a number of ArNH⁺. We demonstrate that the mutagenic potency of ArNH2 correlates with the rate of formation and the chemical reactivity of the metabolically activated forms toward the C8 atom of dG. On the basis of geometric consideration of the π-TS complex made of genotoxic compounds with long aromatic systems, we propose that precovalent intercalation in DNA is not an essential step in the genotoxicity pathway of ArNH2. The mechanism-based reasoning suggests rational design strategies to avoid genotoxicity of ArNH2 primarily by preventing N-hydroxylation of ArNH2.

Explanation for main features of structure-genotoxicity relationships of aromatic amines by theoretical studies of their activation pathways in CYP1A2.

Aromatic and heteroaromatic amines (ArNH(2)) represent a class of potential mutagens that after being metabolically activated covalently modify DNA. Activation of ArNH(2) in many cases starts with N-hydroxylation by P450 enzymes, primarily CYP1A2. Poor understanding of structure-mutagenicity relationships of ArNH(2) limits their use in drug discovery programs. Key factors that facilitate activation of ArNH(2) are revealed by exploring their reaction intermediates in CYP1A2 using DFT calculations. On the basis of these calculations and extensive analysis of structure-mutagenicity data, we suggest that mutagenic metabolites are generated by ferric peroxo intermediate, (CYP1A2)Fe(III)-OO(-), in a three-step heterolytic mechanism. First, the distal oxygen of the oxidant abstracts proton from H-bonded ArNH(2). The subsequent proximal protonation of the resulting (CYP1A2)Fe(III)-OOH weakens both the O-O and the O-H bonds of the oxidant. Heterolytic cleavage of the O-O bond leads to N-hydroxylation of ArNH(-) via S(N)2 mechanism, whereas cleavage of the O-H bond results in release of hydroperoxy radical. Thus, our proposed reaction offers a mechanistic explanation for previous observations that metabolism of aromatic amines could cause oxidative stress. The primary drivers for mutagenic potency of ArNH(2) are (i) binding affinity of ArNH(2) in the productive binding mode within the CYP1A2 substrate cavity, (ii) resonance stabilization of the anionic forms of ArNH(2), and (iii) exothermicity of proton-assisted heterolytic cleavage of N-O bonds of hydroxylamines and their bioconjugates. This leads to a strategy for designing mutagenicity free ArNH(2): Structural alterations in ArNH(2), which disrupt geometric compatibility with CYP1A2, hinder proton abstraction, or strongly destabilize the nitrenium ion, in this order of priority, prevent genotoxicity.

Increasing selectivity of CC chemokine receptor 8 antagonists by engineering nondesolvation related interactions with the intended and off-target binding sites.

The metabolic stability and selectivity of a series of CCR8 antagonists against binding to the hERG ion channel and cytochrome Cyp2D6 are studied by principal component analysis. It is demonstrated that an efficient way of increasing metabolic stability and selectivity of this series is to decrease compound lipophilicity by engineering nondesolvation related attractive interactions with CCR8, as rationalized by three-dimensional receptor models. Although such polar interactions led to increased compound selectivity, such a strategy could also jeopardize the DMPK profile of compounds. However, once increased potency is found, the lipophilicity can be readjusted by engineering hydrophobic substituents that fit to CCR8 but do not fit to hERG. Several such lipophilic fragments are identified by two-dimensional fragment-based QSAR analysis. Electrophysiological measurements and site-directed mutagenesis studies indicated that the repulsive interactions of these fragments with hERG are caused by steric hindrances with residue F656.

Overcoming undesirable HERG potency of chemokine receptor antagonists using baseline lipophilicity relationships.

The inhibition of the hERG channel by noncardiovascular drugs is a side effect that severely impedes the development of new medications. To increase hERG selectivity of preclinical compounds, we recommend the study of nondesolvation related interactions with the intended target and hERG using a baseline lipophilicity relationship approach. While this approach is conventionally used in studies of potency, we demonstrate here that it can help in selectivity issues. Studies of hERG selectivity in four in-house classes of chemokine receptor (CCR) antagonists suggest that the selectivity is improved most effectively by structural alterations that increase the lipophilicity-adjusted primary potency, pIC 50 (CCR) - Log D. Fragment-based QSAR analysis is performed using the lipophilicity-adjusted hERG potency, pIC 50 (hERG) - Log D, to identify moieties that form nonhydrophobic interactions with the hERG channel. These moieties, which erode hERG selectivity, can then be avoided. A novel two-dimensional fragment-based QSAR analysis helps visualizing the lipophilicity-adjusted hERG and CCR potencies within chemical series.

Differential activity of the nerve growth factor (NGF) antagonist PD90780 [7-(benzolylamino)-4,9-dihydro-4-methyl-9-oxo-pyrazolo[5,1-b]quinazoline-2-carboxylic acid] suggests altered NGF-p75NTR interactions in the presence of TrkA.

The neurotrophin nerve growth factor (NGF) binds to two receptor types: the tyrosine kinase receptor TrkA and the common neurotrophin receptor p75(NTR). Although many of the biological effects of NGF (such as neuronal growth and survival) are associated with TrkA activation, p75(NTR) also contributes to these activities by enhancing the action of TrkA when receptors are coexpressed. The NGF antagonist PD90780 [7-(benzolylamino)-4,9-dihydro-4-methyl-9-oxo-pyrazolo[5,1-b]quinazoline-2-carboxylic acid] interacts with NGF, preventing its binding to p75(NTR). In this study, the actions of this compound are further explored, and it is found that PD90780 is not able to inhibit the binding of either brain-derived neurotrophic factor or neurotrophin-3 to p75(NTR), consistent with the direct interactions of the antagonist with NGF. In addition, we demonstrate that the ability of PD90780 to inhibit NGF-p75(NTR) interactions is lower when receptors are coexpressed, compared with when p75(NTR) is the only neurotrophin receptor expressed. These results suggest that the interaction between NGF and the p75(NTR) receptor is altered when TrkA is coexpressed. This alteration can be exploited in the development of antagonists that will selectively inhibit the pro-apoptotic actions of p75(NTR) when expressed in the absence of TrkA, although having less effect on the pro-survival effects of p75(NTR) mediated by enhanced TrkA activation.