The discovery of potent small molecule cyclic urea activators of STING.

STING mediates innate immune responses that are triggered by the presence of cytosolic DNA. Activation of STING to boost antigen recognition is a therapeutic modality that is currently being tested in cancer patients using nucleic-acid based macrocyclic STING ligands. We describe here the discovery of 3,4-dihydroquinazolin-2(1H)-one based 6,6-bicyclic heterocyclic agonists of human STING that activate all known human variants of STING with high potency.

The discovery of potent small molecule activators of human STING.

The adaptor protein STING plays a major role in innate immune sensing of cytosolic nucleic acids, by triggering a robust interferon response. Despite the importance of this protein as a potential therapeutic target for serious unmet medical conditions including cancer and infectious disease there remains a paucity of STING ligands. Starting with a benzothiazinone series of weak STING activators (human EC50 ∼10 µM) we identified several chemotypes with sub-micromolar STING activity across all the major protein polymorphs. An example compound 53 based on an oxindole core structure demonstrated robust on-target functional activation of STING (human EC50 185 nM) in immortalised and primary cells and a cytokine induction fingerprint consistent with STING activation. Our study has identified several related series of potent small molecule human STING activators with potential to be developed as immunomodulatory therapeutics.

G10 is a direct activator of human STING.

The cGAS/STING pathway initiates an innate immune response when DNA is detected in the cytosol. DNA bound cGAS synthesizes cyclic dinucleotides which bind and activate the adaptor STING, leading to downstream secretion of Type I interferons and other pro-inflammatory NFκB pathway cytokines. In the mouse, the STING driven innate immune response is central to immune based clearance of various tumors and this has triggered a significant effort focused on the discovery of human STING agonists for the treatment of cancer. This report uses an in vitro kinase assay to show that G10, a previously identified STING pathway activator is actually a weak but direct STING agonist and identifies other more potent leads.

Valproic acid interactions with the NavMs voltage-gated sodium channel.

Valproic acid (VPA) is an anticonvulsant drug that is also used to treat migraines and bipolar disorder. Its proposed biological targets include human voltage-gated sodium channels, among other membrane proteins. We used the prokaryotic NavMs sodium channel, which has been shown to be a good exemplar for drug binding to human sodium channels, to examine the structural and functional interactions of VPA. Thermal melt synchrotron radiation circular dichroism spectroscopic binding studies of the full-length NavMs channel (which includes both pore and voltage sensor domains), and a pore-only construct, undertaken in the presence and absence of VPA, indicated that the drug binds to and destabilizes the channel, but not the pore-only construct. This is in contrast to other antiepileptic compounds that have previously been shown to bind in the central hydrophobic core of the pore region of the channel, and that tend to increase the thermal stability of both pore-only constructs and full-length channels. Molecular docking studies also indicated that the VPA binding site is associated with the voltage sensor, rather than the hydrophobic cavity of the pore domain. Electrophysiological studies show that VPA influences the block and inactivation rates of the NavMs channel, although with lower efficacy than classical channel-blocking compounds. It thus appears that, while VPA is capable of binding to these voltage-gated sodium channels, it has a very different mode and site of action than other anticonvulsant compounds.

Design and Identification of a Novel, Functionally Subtype Selective GABAA Positive Allosteric Modulator (PF-06372865).

The design, optimization, and evaluation of a series of novel imidazopyridazine-based subtype-selective positive allosteric modulators (PAMs) for the GABAA ligand-gated ion channel are described. From a set of initial hits multiple subseries were designed and evaluated based on binding affinity and functional activity. As designing in the desired level of functional selectivity proved difficult, a probability-based assessment was performed to focus the project's efforts on a single subseries that had the greatest odds of delivering the target profile. These efforts ultimately led to the identification of two precandidates from this subseries, which were advanced to preclinical safety studies and subsequently to the identification of the clinical candidate PF-06372865.

A pharmacological master key mechanism that unlocks the selectivity filter gate in K+ channels.

Potassium (K+) channels have been evolutionarily tuned for activation by diverse biological stimuli, and pharmacological activation is thought to target these specific gating mechanisms. Here we report a class of negatively charged activators (NCAs) that bypass the specific mechanisms but act as master keys to open K+ channels gated at their selectivity filter (SF), including many two-pore domain K+ (K2P) channels, voltage-gated hERG (human ether-à-go-go-related gene) channels and calcium (Ca2+)-activated big-conductance potassium (BK)-type channels. Functional analysis, x-ray crystallography, and molecular dynamics simulations revealed that the NCAs bind to similar sites below the SF, increase pore and SF K+ occupancy, and open the filter gate. These results uncover an unrecognized polypharmacology among K+ channel activators and highlight a filter gating machinery that is conserved across different families of K+ channels with implications for rational drug design.

TRPA1 ankyrin repeat six interacts with a small molecule inhibitor chemotype.

TRPA1, a member of the transient receptor potential channel (TRP) family, is genetically linked to pain in humans, and small molecule inhibitors are efficacious in preclinical animal models of inflammatory pain. These findings have driven significant interest in development of selective TRPA1 inhibitors as potential analgesics. The majority of TRPA1 inhibitors characterized to date have been reported to interact with the S5 transmembrane helices forming part of the pore region of the channel. However, the development of many of these inhibitors as clinical drug candidates has been prevented by high lipophilicity, low solubility, and poor pharmacokinetic profiles. Identification of alternate compound interacting sites on TRPA1 provides the opportunity to develop structurally distinct modulators with novel structure-activity relationships and more desirable physiochemical properties. In this paper, we have identified a previously undescribed potent and selective small molecule thiadiazole structural class of TRPA1 inhibitor. Using species ortholog chimeric and mutagenesis strategies, we narrowed down the site of interaction to ankyrinR #6 within the distal N-terminal region of TRPA1. To identify the individual amino acid residues involved, we generated a computational model of the ankyrinR domain. This model was used predictively to identify three critical amino acids in human TRPA1, G238, N249, and K270, which were confirmed by mutagenesis to account for compound activity. These findings establish a small molecule interaction region on TRPA1, expanding potential avenues for developing TRPA1 inhibitor analgesics and for probing the mechanism of channel gating.

An Intracellular Allosteric Modulator Binding Pocket in SK2 Ion Channels Is Shared by Multiple Chemotypes.

Small conductance potassium (SK) ion channels define neuronal firing rates by conducting the after-hyperpolarization current. They are key targets in developing therapies where neuronal firing rates are dysfunctional, such as in epilepsy, Parkinson's, and amyotrophic lateral sclerosis (ALS). Here, we characterize a binding pocket situated at the intracellular interface of SK2 and calmodulin, which we show to be shared by multiple small-molecule chemotypes. Crystallization of this complex revealed that riluzole (approved for ALS) and an analog of the anti-ataxic agent (4-chloro-phenyl)-[2-(3,5-dimethyl-pyrazol-1-yl)-pyrimidin-4-yl]-amine (CyPPA) bind to and allosterically modulate via this site. Solution-state nuclear magnetic resonance demonstrates that riluzole, NS309, and CyPPA analogs bind at this bipartite pocket. We demonstrate, by patch-clamp electrophysiology, that both classes of ligand interact with overlapping but distinct residues within this pocket. These data define a clinically important site, laying the foundations for further studies of the mechanism of action of riluzole and related molecules.

Discovery of a Series of Indazole TRPA1 Antagonists.

A series of TRPA1 antagonists is described which has as its core structure an indazole moiety. The physical properties and in vitro DMPK profiles are discussed. Good in vivo exposure was obtained with several analogs, allowing efficacy to be assessed in rodent models of inflammatory pain. Two compounds showed significant activity in these models when administered either systemically or topically. Protein chimeras were constructed to indicate compounds from the series bound in the S5 region of the channel, and a computational docking model was used to propose a binding mode for example compounds.

Site-selective Suzuki-Miyaura coupling of heteroaryl halides - understanding the trends for pharmaceutically important classes.

Suzuki-Miyaura cross-coupling reactions of heteroaryl polyhalides with aryl boronates are surveyed. Drawing on data from literature sources as well as bespoke searches of Pfizer's global chemistry RKB and CAS Scifinder® databases, the factors that determine the site-selectivity of these reactions are discussed with a view to rationalising the trends found.

Analysis of Differential Efficacy and Affinity of GABAA (α1/α2) Selective Modulators.

Selective modulators of the γ-amino butyric acid (GABAA) family of receptors have the potential to treat a range of disease states related to cognition, pain, and anxiety. While the development of various α subunit-selective modulators is currently underway for the treatment of anxiety disorders, a mechanistic understanding of the correlation between their bioactivity and efficacy, based on ligand-target interactions, is currently still lacking. In order to alleviate this situation, in the current study we have analyzed, using ligand- and structure-based methods, a data set of 5440 GABAA modulators. The Spearman correlation (ρ) between binding activity and efficacy of compounds was calculated to be 0.008 and 0.31 against the α1 and α2 subunits of GABA receptor, respectively; in other words, the compounds had little diversity in structure and bioactivity, but they differed significantly in efficacy. Two compounds were selected as a case study for detailed interaction analysis due to the small difference in their structures and affinities (ΔpKi(comp1_α1 - comp2_α1) = 0.45 log units, ΔpKi(comp1_α2 - comp2_α2) = 0 log units) as compared to larger relative efficacies (ΔRE(comp1_α1 - comp2_α1) = 1.03, ΔRE(comp1_α2 - comp2_α2) = 0.21). Docking analysis suggested that His-101 is involved in a characteristic interaction of the α1 receptor with both compounds 1 and 2. Residues such as Phe-77, Thr-142, Asn-60, and Arg-144 of the γ chain of the α1γ2 complex also showed interactions with heterocyclic rings of both compounds 1 and 2, but these interactions were disturbed in the case of α2γ2 complex docking results. Binding pocket stability analysis based on molecular dynamics identified three substitutions in the loop C region of the α2 subunit, namely, G200E, I201T, and V202I, causing a reduction in the flexibility of α2 compared to α1. These amino acids in α2, as compared to α1, were also observed to decrease the vibrational and dihedral entropy and to increase the hydrogen bond content in α2 in the apo state. However, freezing of both α1 and α2 was observed in the ligand-bound state, with an increased number of internal hydrogen bonds and increased entropy. Therefore, we hypothesize that the amino acid differences in the loop C region of α2 are responsible for conformational changes in the protein structure compared to α1, as well as for the binding modes of compounds and hence their functional signaling.

Investigation of the structure activity relationship of flufenamic acid derivatives at the human TRESK channel K2P18.1.

TRESK (Twik RElated Spinal cord K+ channel) is a member of the Twin Pore Domain potassium channel (K2P) family responsible for regulating neuronal excitability in dorsal root ganglion (DRG) and trigeminal (TG) neurons, peripheral neurons involved in pain transmission. As channel opening causes an outward K+ current responsible for cell hyperpolarisation, TRESK represents a potentially interesting target for pain treatment. However, as no crystal structure exists for this protein, the mechanisms involved in the opening action of its ligands are still poorly understood, making the development of new potent and selective openers challenging. In this work we present a structure activity relationship (SAR) of the known TRESK opener flufenamic acid (FFA) and some derivatives, investigating the functional effects of chemical modifications to build a TRESK homology model to support the biological results. A plausible binding mode is proposed, providing the first predictive hypothesis of a human TRESK opener binding site.

Structural basis for inhibition of a voltage-gated Ca2+ channel by Ca2+ antagonist drugs.

Ca2+ antagonist drugs are widely used in therapy of cardiovascular disorders. Three chemical classes of drugs bind to three separate, but allosterically interacting, receptor sites on CaV1.2 channels, the most prominent voltage-gated Ca2+ (CaV) channel type in myocytes in cardiac and vascular smooth muscle. The 1,4-dihydropyridines are used primarily for treatment of hypertension and angina pectoris and are thought to act as allosteric modulators of voltage-dependent Ca2+ channel activation, whereas phenylalkylamines and benzothiazepines are used primarily for treatment of cardiac arrhythmias and are thought to physically block the pore. The structural basis for the different binding, action, and therapeutic uses of these drugs remains unknown. Here we present crystallographic and functional analyses of drug binding to the bacterial homotetrameric model CaV channel CaVAb, which is inhibited by dihydropyridines and phenylalkylamines with nanomolar affinity in a state-dependent manner. The binding site for amlodipine and other dihydropyridines is located on the external, lipid-facing surface of the pore module, positioned at the interface of two subunits. Dihydropyridine binding allosterically induces an asymmetric conformation of the selectivity filter, in which partially dehydrated Ca2+ interacts directly with one subunit and blocks the pore. In contrast, the phenylalkylamine Br-verapamil binds in the central cavity of the pore on the intracellular side of the selectivity filter, physically blocking the ion-conducting pathway. Structure-based mutations of key amino-acid residues confirm drug binding at both sites. Our results define the structural basis for binding of dihydropyridines and phenylalkylamines at their distinct receptor sites on CaV channels and offer key insights into their fundamental mechanisms of action and differential therapeutic uses in cardiovascular diseases.

Exploring the Dynamics of the TWIK-1 Channel.

Potassium channels in the two-pore domain family (K2P) have various structural attributes that differ from those of other K(+) channels, including a dimeric assembly constituted of nonidentical domains and an expansive extracellular cap. Crystallization of the prototypical K2P channel, TWIK-1, finally revealed the structure of these characteristics in atomic detail, allowing computational studies to be undertaken. In this study, we performed molecular-dynamics simulations for a cumulative time of ∼1 µs to discern the mechanism of ion transport throughout TWIK-1. We observed the free passage of ions beneath the extracellular cap and identified multiple high-occupancy sites in close proximity to charged residues on the protein surface. Despite the overall topological similarity of the x-ray structure of the selectivity filter to other K(+) channels, the structure diverges significantly in molecular-dynamics simulations as a consequence of nonconserved residues in both pore domains contributing to the selectivity filter (T118 and L228). The behavior of such residues has been linked to channel inactivation and the phenomenon of dynamic selectivity, where TWIK-1 displays robust Na(+) inward flux in response to subphysiological K(+) concentrations.

Identification of Positive Allosteric Modulators of Glycine Receptors from a High-Throughput Screen Using a Fluorescent Membrane Potential Assay.

Glycine receptor 3 (GlyRα3) is a ligand-gated ion channel of the cys-loop family that plays a key role in mediating inhibitory neurotransmission and regulation of pain signaling in the dorsal horn. Potentiation of GlyRα3 function is therefore of interest as a putative analgesic mechanism with which to target new therapeutics. However, to date, positive allosteric modulators (PAMs) of this receptor with sufficient selectivity to enable target validation studies have not been described. To address this lack of pharmacological tools, we developed a suite of in vitro assays comprising a high-throughput fluorescent membrane potential screen and a medium-throughput electrophysiology assay using IonFlux HT together with conventional manual patch clamp. Using these assays, we conducted a primary screening campaign and report the structures of hit compounds identified as GlyR PAMs. Our functional characterization data reveal a hit compound with high efficacy relative to current known potentiators and selectivity over GABAAR, another major class of inhibitory neurotransmission receptors of importance to pain. These small-molecule GlyR PAMs have high potential both as early tool compounds to enable pharmacological studies of GlyR inhibitory neurotransmission and as a starting point for the development of potent, selective GlyRα3 PAMs as novel analgesics.

Azogabazine; a photochromic antagonist of the GABAA receptor.

The design and synthesis of azogabazine is described, which represents a highly potent (IC50 = 23 nM) photoswitchable antagonist of the GABAA receptor. An azologization strategy is adopted, in which a benzyl phenyl ether in a high affinity gabazine analogue is replaced by an azobenzene, with resultant retention of antagonist potency. We show that cycling from blue to UV light, switching between trans and cis isomeric forms, leads to photochemically controlled antagonism of the GABA ion channel.

Lateral Fenestrations in K(+)-Channels Explored Using Molecular Dynamics Simulations.

Potassium channels are of paramount physiological and pathological importance and therefore constitute significant drug targets. One of the keys to rationalize the way drugs modulate ion channels is to understand the ability of such small molecules to access their respective binding sites, from which they can exert an activating or inhibitory effect. Many computational studies have probed the energetics of ion permeation, and the mechanisms of voltage gating, but little is known about the role of fenestrations as possible mediators of drug entry in potassium channels. To explore the existence, structure, and conformational dynamics of transmembrane fenestrations accessible by drugs in potassium channels, molecular dynamics simulation trajectories were analyzed from three potassium channels: the open state voltage-gated channel Kv1.2, the G protein-gated inward rectifying channel GIRK2 (Kir3.2), and the human two-pore domain TWIK-1 (K2P1.1). The main results of this work were the identification of the sequence identity of four main lateral fenestrations of similar length and with bottleneck radius in the range of 0.9-2.4 Å for this set of potassium channels. It was found that the fenestrations in Kv1.2 and Kir3.2 remain closed to the passage of molecules larger than water. In contrast, in the TWIK-1 channel, both open and closed fenestrations are sampled throughout the simulation, with bottleneck radius shown to correlate with the random entry of lipid membrane molecules into the aperture of the fenestrations. Druggability scoring function analysis of the fenestration regions suggests that Kv and Kir channels studied are not druggable in practice due to steric constraining of the fenestration bottleneck. A high (>50%) fenestration sequence identity was found in each potassium channel subfamily studied, Kv1, Kir3, and K2P1. Finally, the reported fenestration sequence of TWIK-1 compared favorably with another channel, K2P channel TREK-2, reported to possess open fenestrations, suggesting that K2P channels could be druggable via fenestrations, for which we reported atomistic detail of the fenestration region, including the flexible residues M260 and L264 that interact with POPC membrane in a concerted fashion with the aperture and closure of the fenestrations.

Discovery of a Selective TRPM8 Antagonist with Clinical Efficacy in Cold-Related Pain.

The transient receptor potential (TRP) family of ion channels comprises nonselective cation channels that respond to a wide range of chemical and thermal stimuli. TRPM8, a member of the melastatin subfamily, is activated by cold temperatures (<28 °C), and antagonists of this channel have the potential to treat cold induced allodynia and hyperalgesia. However, TRPM8 has also been implicated in mammalian thermoregulation and antagonists have the potential to induce hypothermia in patients. We report herein the identification and optimization of a series of TRPM8 antagonists that ultimately led to the discovery of PF-05105679. The clinical finding with this compound will be discussed, including both efficacy and its ability to affect thermoregulation processes in humans.

In silico identification of PAP-1 binding sites in the Kv1.2 potassium channel.

Voltage-gated potassium channels of the Kv1 family play a crucial role in the generation and transmission of electrical signals in excitable cells affecting neuronal and cardiac activities. Small-molecule blockage of these channels has been proposed to occur via a cooperative mechanism involving two main blocking sites: the inner-pore site located below the selectivity filter, and a side-pocket cavity located between the pore and the voltage sensor. Using 0.5 µs molecular dynamics simulation trajectories complemented by docking calculations, the potential binding sites of the PAP-1 (5-(4-phenoxybutoxy)psoralen) blocker to the crystal structure of Kv1.2 channel have been studied. The presence of both mentioned blocking sites at Kv1.2 is confirmed, adding evidence in favor of a cooperative channel blockage mechanism. These observations provide insight into drug modulation that will guide further developments of Kv inhibitors.