Delineating the Ligand-Receptor Interactions That Lead to Biased Signaling at the µ-Opioid Receptor.

Biased agonists, which selectively stimulate certain signaling pathways controlled by a G protein-coupled receptor (GPCR), hold great promise as drugs that maximize efficacy while minimizing dangerous side effects. Biased agonists of the µ-opioid receptor (µOR) are of particular interest as a means to achieve analgesia through G protein signaling without dose-limiting side effects such as respiratory depression and constipation. Rational structure-based design of biased agonists remains highly challenging, however, because the ligand-mediated interactions that are key to activation of each signaling pathway remain unclear. We identify several compounds for which the R- and S-enantiomers have distinct bias profiles at the µOR. These compounds serve as excellent comparative tools to study bias because the identical physicochemical properties of enantiomer pairs ensure that differences in bias profiles are due to differences in interactions with the µOR binding pocket. Atomic-level simulations of compounds at µOR indicate that R- and S-enantiomers adopt different poses that form distinct interactions with the binding pocket. A handful of specific interactions with highly conserved binding pocket residues appear to be responsible for substantial differences in arrestin recruitment between enantiomers. Our results offer guidance for rational design of biased agonists at µOR and possibly at related GPCRs.

The role of organic anion-transporting polypeptides and formulation in the clearance and distribution of a novel Nav 1.7 channel blocker.

PF-06456384 is an extremely potent and selective blocker of the Nav 1.7 sodium channel designed as a potential intravenous (i.v.) analgesic targeting high potency and rapid clearance to minimize the potential for residual effects following the end of infusion. In our previous experience targeting oral molecules, the requirement to obtain potent, Nav 1.7 selective molecules led to a focus on acidic, amphipilic compounds cleared primarily by organic anion-transporting polypeptide mediated hepatic uptake and subsequent biliary excretion. However, the physicochemical properties of the i.v. lead matter were substantially different, moving from acidic, amphiphilic chemical space to zwitterions as well as substantially increasing molecular weight. This report describes the continued relevance of organic anion-transporting polypeptide driven hepatic uptake in this physicochemical space and highlights an apparent impact of the formulation excipient Solutol on the clearance and distribution of PF-06456384.

Highly potent and selective NaV1.7 inhibitors for use as intravenous agents and chemical probes.

The discovery and selection of a highly potent and selective NaV1.7 inhibitor PF-06456384, designed specifically for intravenous infusion, is disclosed. Extensive in vitro pharmacology and ADME profiling followed by in vivo preclinical PK and efficacy model data are discussed. A proposed protein-ligand binding mode for this compound is also provided to rationalise the high levels of potency and selectivity over inhibition of related sodium channels. To further support the proposed binding mode, potent conjugates are described which illustrate the potential for development of chemical probes to enable further target evaluation.

Selective 5-HT2C receptor agonists: Design and synthesis of pyridazine-fused azepines.

Heterocycle-fused azepines are discussed as potent 5-HT2C receptor agonists with excellent selectivity over 5-HT2B agonism. Synthesis and structure activity relationships are outlined for a series of bicyclic pyridazino[3,4-d]azepines. By comparison with earlier published work, in vitro assays predict a high probability for achieving CNS penetration for a potent and selective compound 15a, a pre-requisite to achieve in vivo efficacy.

Recent progress in sodium channel modulators for pain.

Voltage-gated sodium channels (Navs) are an important family of transmembrane ion channel proteins and Nav drug discovery is an exciting field. Pharmaceutical investment in Navs for pain therapeutics has expanded exponentially due to genetic data such as SCN10A mutations and an improved ability to establish an effective screen sequence for example IonWorks Barracuda®, Synchropatch® and Qube®. Moreover, emerging clinical data (AZD-3161, XEN402, CNV1014802, PF-05089771, PF-04531083) combined with recent breakthroughs in Nav structural biology pave the way for a future of fruitful prospective Nav drug discovery.

Optimization of Potent Ligands for the E3 Ligase DCAF15 and Evaluation of Their Use in Heterobifunctional Degraders.

Unlocking novel E3 ligases for use in heterobifunctional PROTAC degraders is of high importance to the pharmaceutical industry. Over-reliance on the current suite of ligands used to recruit E3 ligases could limit the potential of their application. To address this, potent ligands for DCAF15 were optimized using cryo-EM supported, structure-based design to improve on micromolar starting points. A potent binder, compound 24, was identified and subsequently conjugated into PROTACs against multiple targets. Following attempts on degrading a number of proteins using DCAF15 recruiting PROTACs, only degradation of BRD4 was observed. Deconvolution of the mechanism of action showed that this degradation was not mediated by DCAF15, thereby highlighting both the challenges faced when trying to expand the toolbox of validated E3 ligase ligands for use in PROTAC degraders and the pitfalls of using BRD4 as a model substrate.

Design and Evaluation of a Low Hydrogen Bond Donor Count Fragment Screening Set to Aid Hit Generation of PROTACs Intended for Oral Delivery.

The development of orally bioavailable PROTACs presents a significant challenge due to the inflated physicochemical properties of such heterobifunctional molecules. Molecules occupying this "beyond rule of five" space often demonstrate limited oral bioavailability due to the compounding effects of elevated molecular weight and hydrogen bond donor count (among other properties), but it is possible to achieve sufficient oral bioavailability through physicochemical optimization. Herein, we disclose the design and evaluation of a low hydrogen bond donor count (≤1 HBD) fragment screening set to aid hit generation of PROTACs intended for an oral route of delivery. We demonstrate that application of this library can enhance fragment screens against PROTAC proteins of interest and ubiquitin ligases, yielding fragment hits containing ≤1 HBD suitable for optimizing toward orally bioavailable PROTACs.

Rapid PROTAC Discovery Platform: Nanomole-Scale Array Synthesis and Direct Screening of Reaction Mixtures.

Precise length, shape, and linker attachment points are all integral components to designing efficacious proteolysis targeting chimeras (PROTACs). Due to the synthetic complexity of these heterobifunctional degraders and the difficulty of computational modeling to aid PROTAC design, the exploration of structure-activity relationships remains mostly empirical, which requires a significant investment of time and resources. To facilitate rapid hit finding, we developed capabilities for PROTAC parallel synthesis and purification by harnessing an array of preformed E3-ligand-linker intermediates. In the next iteration of this approach, we developed a rapid, nanomole-scale PROTAC synthesis methodology using amide coupling that enables direct screening of nonpurified reaction mixtures in cell-based degradation assays, as well as logD and EPSA measurements. This approach greatly expands and accelerates PROTAC SAR exploration (5 days instead of several weeks) as well as avoids laborious and solvent-demanding purification of the reaction mixtures, thus making it an economical and more sustainable methodology for PROTAC hit finding.

Discovery and Optimization of the First ATP Competitive Type-III c-MET Inhibitor.

Recent clinical reports have highlighted the need for wild-type (WT) and mutant dual inhibitors of c-MET kinase for the treatment of cancer. We report herein a novel chemical series of ATP competitive type-III inhibitors of WT and D1228V mutant c-MET. Using a combination of structure-based drug design and computational analyses, ligand 2 was optimized to a highly selective chemical series with nanomolar activities in biochemical and cellular settings. Representatives of the series demonstrate excellent pharmacokinetic profiles in rat in vivo studies with promising free-brain exposures, paving the way for the design of brain permeable drugs for the treatment of c-MET driven cancers.

Squaramides: physical properties, synthesis and applications.

Squaramides are remarkable four-membered ring systems derived from squaric acid that are able to form up to four hydrogen bonds. A high affinity for hydrogen bonding is driven through a concomitant increase in aromaticity of the ring. This hydrogen bonding and aromatic switching, in combination with structural rigidity, have been exploited in many of the applications of squaramides. Substituted squaramides can be accessed via modular synthesis under relatively mild or aqueous conditions, making them ideal units for bioconjugation and supramolecular chemistry. In this tutorial review the fundamental electronic and structural properties of squaramides are explored to rationalise the geometry, conformation, reactivity and biological activity.

Fragment screening at AstraZeneca: developing the next generation biophysics fragment set.

Fragment based drug discovery is a critical part of the lead generation toolbox and relies heavily on a readily available, high quality fragment library. Over years of use, the AstraZeneca fragment set had become partially depleted and instances of compound deterioration had been found. It was recognised that a redevelopment was required. This provided an opportunity to evolve our screening sets strategy, whilst ensuring that the quality of the fragment set met the robust requirements of fragment screening campaigns. In this communication we share the strategy employed, in particular highlighting two aspects of our approach that we believe others in the community would benefit from, namely that; (i) fragments were selected with input from Medicinal Chemists at an early stage, and (ii) the library was arranged in a layered format to ensure maximum flexibility on a per target basis.

Identification of Histone Peptide Binding Specificity and Small-Molecule Ligands for the TRIM33α and TRIM33ß Bromodomains.

TRIM33 is a member of the tripartite motif (TRIM) family of proteins, some of which possess E3 ligase activity and are involved in the ubiquitin-dependent degradation of proteins. Four of the TRIM family proteins, TRIM24 (TIF1α), TRIM28 (TIF1ß), TRIM33 (TIF1γ) and TRIM66, contain C-terminal plant homeodomain (PHD) and bromodomain (BRD) modules, which bind to methylated lysine (KMen) and acetylated lysine (KAc), respectively. Here we investigate the differences between the two isoforms of TRIM33, TRIM33α and TRIM33ß, using structural and biophysical approaches. We show that the N1039 residue, which is equivalent to N140 in BRD4(1) and which is conserved in most BRDs, has a different orientation in each isoform. In TRIM33ß, this residue coordinates KAc, but this is not the case in TRIM33α. Despite these differences, both isoforms show similar affinities for H31-27K18Ac, and bind preferentially to H31-27K9Me3K18Ac. We used this information to develop an AlphaScreen assay, with which we have identified four new ligands for the TRIM33 PHD-BRD cassette. These findings provide fundamental new information regarding which histone marks are recognized by both isoforms of TRIM33 and suggest starting points for the development of chemical probes to investigate the cellular function of TRIM33.

Pyrimido[4,5-d]azepines as potent and selective 5-HT2C receptor agonists: design, synthesis, and evaluation of PF-3246799 as a treatment for urinary incontinence.

New pyrimido[4,5-d]azepines 7 are disclosed as potent 5-HT(2C) receptor agonists. A preferred example, 7b had minimal activation at either the 5-HT(2A) or 5-HT(2B) receptors combined with robust efficacy in a preclinical canine model of stress urinary incontinence (SUI) and attractive pharmacokinetic and safety properties. Based on this profile, 7b (PF-3246799) was identified as a candidate for clinical development for the treatment of SUI. In addition, it proved to be critical to build an understanding of the translation between recombinant cell-based systems, native tissue preparations and in vivo preclinical models. This was a significant undertaking and proved to be crucial in compound selection.

Put a ring on it: application of small aliphatic rings in medicinal chemistry.

Aliphatic three- and four-membered rings including cyclopropanes, cyclobutanes, oxetanes, azetidines and bicyclo[1.1.1]pentanes have been increasingly exploited in medicinal chemistry for their beneficial physicochemical properties and applications as functional group bioisosteres. This review provides a historical perspective and comparative up to date overview of commonly applied small rings, exemplifying key principles with recent literature examples. In addition to describing the merits and advantages of each ring system, potential hazards and liabilities are also illustrated and explained, including any significant chemical or metabolic stability and toxicity risks.

Structural Basis for Targeting the Folded P-Loop Conformation of c-MET.

We report here a fragment screen directed toward the c-MET kinase from which we discovered a series of inhibitors able to bind to a rare conformation of the protein in which the P-loop adopts a collapsed, or folded, arrangement. Preliminary SAR exploration led to an inhibitor (7) with nanomolar biochemical activity against c-MET and promising cell activity and kinase selectivity. These findings increase our structural understanding of the folded P-loop conformation of c-MET and provide a sound structural and chemical basis for further investigation of this underexplored yet potentially therapeutically exploitable conformational state.

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.

Enantioselective Brønsted acid-catalyzed N-acyliminium cyclization cascades.

An enantioselective Brønsted acid-catalyzed N-acyliminium cyclization cascade of tryptamines with enol lactones to form architecturally complex heterocycles in high enantiomeric excess has been developed. The reaction is technically simple to perform as well as atom-efficient and may be coupled to a gold(I)-catalyzed cycloisomerization of alkynoic acids whereby the key enol lactone reaction partner is generated in situ. Employing up to 10 mol % bulky chiral phosphoric acid catalysts in boiling toluene allowed the product materials to be generated in good overall yields (63-99%) and high enantioselectivities (72-99% ee). With doubly substituted enol lactones, high diastereo- and enantioselectivities were obtained, thus providing a new example of a dynamic kinetic asymmetric cyclization reaction.

Physiology of Hyperuricemia and Urate-Lowering Treatments.

Gout is the most common form of inflammatory arthritis and is a multifactorial disease typically characterized by hyperuricemia and monosodium urate crystal deposition predominantly in, but not limited to, the joints and the urinary tract. The prevalence of gout and hyperuricemia has increased in developed countries over the past two decades and research into the area has become progressively more active. We review the current field of knowledge with emphasis on active areas of hyperuricemia research including the underlying physiology, genetics and epidemiology, with a focus on studies which suggest association of hyperuricemia with common comorbidities including cardiovascular disease, renal insufficiency, metabolic syndrome and diabetes. Finally, we discuss current therapies and emerging drug discovery efforts aimed at delivering an optimized clinical treatment strategy.

Discovery of Compounds that Positively Modulate the High Affinity Choline Transporter.

Cholinergic hypofunction is associated with decreased attention and cognitive deficits in the central nervous system in addition to compromised motor function. Consequently, stimulation of cholinergic neurotransmission is a rational therapeutic approach for the potential treatment of a variety of neurological conditions. High affinity choline uptake (HACU) into acetylcholine (ACh)-synthesizing neurons is critically mediated by the sodium- and pH-dependent high-affinity choline transporter (CHT, encoded by the SLC5A7 gene). This transporter is comparatively well-characterized but otherwise unexplored as a potential drug target. We therefore sought to identify small molecules that would enable testing of the hypothesis that positive modulation of CHT mediated transport would enhance activity-dependent cholinergic signaling. We utilized existing and novel screening techniques for their ability to reveal both positive and negative modulation of CHT using literature tools. A screening campaign was initiated with a bespoke compound library comprising both the Pfizer Chemogenomic Library (CGL) of 2,753 molecules designed specifically to help enable the elucidation of new mechanisms in phenotypic screens and 887 compounds from a virtual screening campaign to select molecules with field-based similarities to reported negative and positive allosteric modulators. We identified a number of previously unknown active and structurally distinct molecules that could be used as tools to further explore CHT biology or as a starting point for further medicinal chemistry.

Design and Synthesis of a Pan-Janus Kinase Inhibitor Clinical Candidate (PF-06263276) Suitable for Inhaled and Topical Delivery for the Treatment of Inflammatory Diseases of the Lungs and Skin.

By use of a structure-based computational method for identification of structurally novel Janus kinase (JAK) inhibitors predicted to bind beyond the ATP binding site, a potent series of indazoles was identified as selective pan-JAK inhibitors with a type 1.5 binding mode. Optimization of the series for potency and increased duration of action commensurate with inhaled or topical delivery resulted in potent pan-JAK inhibitor 2 (PF-06263276), which was advanced into clinical studies.