Pushing the Limits of Computational Structure-Based Drug Design with a Cryo-EM Structure: The Ca2+ Channel α2δ-1 Subunit as a Test Case.

Cryo-electron microscopy (cryo-EM) is emerging as a real alternative for structural elucidation. In spite of this, very few cryo-EM structures have been described so far as successful platforms for in silico drug design. Gabapentin and pregabalin are some of the most successful drugs in the treatment of epilepsy and neuropathic pain. Although both are in clinical use and are known to exert their effects by binding to the regulatory α2δ subunit of voltage gated calcium channels, their binding modes have never been characterized. We describe here the successful use of an exhaustive protein-ligand sampling algorithm on the α2δ-1 subunit of the recently published cryo-EM structure, with the goal of characterizing the ligand entry path and binding mode for gabapentin, pregabalin, and several other amino acidic α2δ-1 ligands. Our studies indicate that (i) all simulated drugs explore the same path for accessing the occluded binding site on the interior of the α2δ-1 subunit; (ii) they all roughly occupy the same pocket; (iii) the plasticity of the binding site allows the accommodation of a variety of amino acidic modulators, driven by the flexible "capping loop" delineated by residues Tyr426-Val435 and the floppy nature of Arg217; (iv) the predicted binding modes are in line with previously available mutagenesis data, confirming Arg217 as key for binding, with Asp428 and Asp467 highlighted as additional anchoring points for all amino acidic drugs. The study is one of the first proofs that latest-generation cryo-EM structures combined with exhaustive computational methods can be exploited in early drug discovery.

WLB-87848, a Selective σ1 Receptor Agonist, with an Unusually Positioned NH Group as Positive Ionizable Moiety and Showing Neuroprotective Activity.

The synthesis and pharmacological activity of a new series of thieno[2,3-d]pyrimidin-4(3H)-one derivatives as sigma-1 receptor (σ1R) ligands are reported. A hit from a high-throughput screening program was evolved into a highly potent and selective σ1R agonist (14qR) that contains a free NH group as positive ionizable moiety, not fulfilling the usual pharmacophoric features of the σ1R. The compound shows good physicochemical and ADMET characteristics, displays an agonist profile in the binding immunoglobulin protein/σ1R association assay, induces neuron viability in an in vitro model of ß-amyloid peptide intoxication, and presents positive results against recognition memory impairment induced by hippocampal injection of Aß peptide in rats after oral treatment, altogether making 14qR (WLB-87848) an interesting candidate for neuroprotection.

Towards Multitargeted Ligands as Pain Therapeutics: Dual Ligands of the Cavα2δ-1 Subunit of Voltage-Gated Calcium Channel and the µ-Opioid Receptor.

The synthesis and pharmacological activity of a new series of dual ligands combining activities towards the α2δ-1 subunit of voltage-gated calcium channels (Cavα2δ-1) and the µ-opioid receptor (MOR) as novel pain therapeutics are reported. A careful exploration of the pharmacophores related to both targets, which in principle had few common characteristics, led to the design of novel compounds exhibiting both activities. The construction of the dual ligands started from published Cavα2δ-1 ligands, onto which MOR ligand pharmacophoric elements were added. This exercise led to new amino-acidic substances with good affinities on both targets as well as good metabolic and physicochemical profiles and low potential for drug-drug interactions. A representative compound, (2S,4S)-4-(4-chloro-3-(((cis)-4-(dimethylamino)-4-phenylcyclohexyl)methyl)-5-fluorophenoxy)pyrrolidine-2-carboxylic acid, displayed promising analgesic activities in several in vivo pain models as well as a reduced side-effect profile in relation to morphine.

Discovery of WLB-89462, a New Drug-like and Highly Selective σ2 Receptor Ligand with Neuroprotective Properties.

The synthesis and pharmacological activity of a new series of isoxazolylpyrimidines as sigma-2 receptor (σ2R) ligands are reported. Modification of a new hit retrieved in an HTS campaign allowed the identification of the compound WLB-89462 (20c) with good σ2R affinity (Ki = 13 nM) and high selectivity vs both the σ1R (Ki = 1777 nM) and a general panel of 180 targets. It represents one of the first σ2R ligands with drug-like properties, linked to a good physicochemical and ADMET profile (good solubility, no CYP inhibition, good metabolic stability, high permeability, brain penetration, and high oral exposure in rodents). Compound 20c shows neuroprotective activity in vitro and improves short-term memory impairment induced by hippocampal injection of amyloid ß peptide in rats. Together with the promising effects in the chronic models where 20c is currently being evaluated, these results pave the way toward its clinical development as a neuroprotective agent.

Bispecific sigma-1 receptor antagonism and mu-opioid receptor partial agonism: WLB-73502, an analgesic with improved efficacy and safety profile compared to strong opioids.

Opioids are the most effective painkillers, but their benefit-risk balance often hinder their therapeutic use. WLB-73502 is a dual, bispecific compound that binds sigma-1 (S1R) and mu-opioid (MOR) receptors. WLB-73502 is an antagonist at the S1R. It behaved as a partial MOR agonist at the G-protein pathway and produced no/unsignificant ß-arrestin-2 recruitment, thus demonstrating low intrinsic efficacy on MOR at both signalling pathways. Despite its partial MOR agonism, WLB-73502 exerted full antinociceptive efficacy, with potency superior to morphine and similar to oxycodone against nociceptive, inflammatory and osteoarthritis pain, and superior to both morphine and oxycodone against neuropathic pain. WLB-73502 crosses the blood-brain barrier and binds brain S1R and MOR to an extent consistent with its antinociceptive effect. Contrary to morphine and oxycodone, tolerance to its antinociceptive effect did not develop after repeated 4-week administration. Also, contrary to opioid comparators, WLB-73502 did not inhibit gastrointestinal transit or respiratory function in rats at doses inducing full efficacy, and it was devoid of proemetic effect (retching and vomiting) in ferrets at potentially effective doses. WLB-73502 benefits from its bivalent S1R antagonist and partial MOR agonist nature to provide an improved antinociceptive and safety profile respect to strong opioid therapy.

Chemoenzymatic synthesis of 2,6-disubstituted tetrahydropyrans with high σ1 receptor affinity, antitumor and analgesic activity.

1,3-Dioxanes 1 and cyclohexanes 2 bearing a phenyl ring and an aminoethyl moiety in 1,3-relationship to each other represent highly potent σ1 receptor antagonists. In order to increase the chemical stability of the acetalic 1,3-dioxanes 1 and the polarity of the cyclohexanes 2, tetrahydropyran derivatives 3 equipped with the same substituents were designed, synthesized and pharmacologically evaluated. The key step of the synthesis was a lipase-catalyzed enantioselective acetylation of the alcohol (R)-5 leading finally to enantiomerically pure test compounds 3a-g. With respect to σ1 receptor affinity and selectivity over a broad range of related (σ2, PCP binding site) and further targets, the enantiomeric benzylamines 3a and cyclohexylmethylamines 3b represent the most promising drug candidates of this series. However, the eudismic ratio for σ1 binding is only in the range of 2.5-3.3. Classical molecular dynamics (MD) simulations confirmed the same binding pose for both the tetrahydropyran 3 and cyclohexane derivatives 2 at the σ1 receptor, according to which: i) the protonated amino moiety of (2S,6R)-3a engages the same key polar interactions with Glu172 (ionic) and Phe107 (π-cation), ii) the lipophilic parts of (2S,6R)-3a are hosted in three hydrophobic regions of the σ1 receptor, and iii) the O-atom of the tetrahydropyran derivatives 3 does not show a relevant interaction with the σ1 receptor. Further in silico evidences obtained by the application of free energy perturbation and steered MD techniques fully supported the experimentally observed difference in receptor/ligand affinities. Tetrahydropyrans 3 require a lower dissociative force peak than cyclohexane analogs 2. Enantiomeric benzylamines 3a and cyclohexylmethylamines 3b were able to inhibit the growth of the androgen negative human prostate cancer cell line DU145. The cyclohexylmethylamine (2S,6R)-3b showed the highest σ1 affinity (Ki(σ1) = 0.95 nM) and the highest analgesic activity in vivo (67%).

Piperazinyl Bicyclic Derivatives as Selective Ligands of the α2δ-1 Subunit of Voltage-Gated Calcium Channels.

The synthesis and pharmacological activities of a new series of piperazinyl quinazolin-4-(3H)-one derivatives acting toward the α2δ-1 subunit of voltage-gated calcium channels (Cavα2δ-1) are reported. Different positions of a micromolar HTS hit were explored, and best activities were obtained for compounds containing a small alkyl group in position 3 of the quinazolin-4-(3H)-one scaffold and a 3-methyl-piperazin-1-yl- or 3,5-dimethyl-piperazin-1-yl-butyl group in position 2. The activity was shown to reside in the R enantiomer of the chain in position 2, and several eutomers reached single digit nanomolar affinities. Final modification of the central scaffold to reduce lipophilicity provided the pyrido[4,3-d]pyrimidin-4(3H)-one 16RR, which showed high selectivity for Cavα2δ-1 versus Cavα2δ-2, probably linked to its improved analgesic efficacy-safety ratio in mice over pregabalin.

Propionamide Derivatives as Dual µ-Opioid Receptor Agonists and σ1 Receptor Antagonists for the Treatment of Pain.

A new series of propionamide derivatives was developed as dual µ-opioid receptor agonists and σ1 receptor antagonists. Modification of a high-throughput screening hit originated a series of piperazinylcycloalkylmethyl propionamides, which were explored to overcome the challenge of achieving balanced dual activity and convenient drug-like properties. The lead compound identified, 18g, showed good analgesic effects in several animal models of both acute (paw pressure) and chronic (partial sciatic nerve ligation) pain, with reduced gastrointestinal effects in comparison with oxycodone.

Bicyclic Diazepinones as Dual Ligands of the α2δ-1 Subunit of Voltage-Gated Calcium Channels and the Norepinephrine Transporter.

The synthesis and pharmacological activity of a new series of bicyclic diazepinones with dual activity toward the α2δ-1 subunit of voltage-gated calcium channels (Cavα2δ-1) and the norepinephrine transporter (NET) are reported. Exploration of the positions amenable for substitution on a nonaminoacidic Cavα2δ-1 scaffold allowed the identification of favorable positions for the attachment of NET pharmacophores. Among the patterns explored, attachment of the 2-ethylamino-9-methyl-6-phenyl-6,7,8,9-tetrahydro-5H-pyrimido[4,5-e][1,4]diazepin-5-one framework to the meta-position of the phenyl ring of the 3-methylamino-1-phenylpropoxy and 3-methylamino-1-thiophenylpropoxy moieties provided dual compounds with excellent NET functionality. Alternative bicyclic frameworks were also explored, and some lead molecules were identified, which showed a balanced dual profile and exhibited good ADMET properties.

Tricyclic Triazoles as σ1 Receptor Antagonists for Treating Pain.

The synthesis and pharmacological activity of a new series of 5a,7,8,8a-tetrahydro-4H,6H-pyrrolo[3,4-b][1,2,3]triazolo[1,5-d][1,4]oxazine derivatives as potent sigma-1 receptor (σ1R) ligands are reported. A lead optimization program aimed at improving the aqueous solubility of parent racemic nonpolar derivatives led to the identification of several σ1R antagonists with a good absorption, distribution, metabolism, and excretion in vitro profile, no off-target affinities, and characterized by a low basic pKa (around 5) that correlates with high exposure levels in rodents. Two compounds displaying a differential brain-to-plasma ratio distribution profile, 12lR and 12qS, exhibited a good analgesic profile and were selected as preclinical candidates for the treatment of pain.

Pharmacological characterization of high-affinity σ1 receptor ligands with spirocyclic thienopyran and thienofuran scaffold.

OBJECTIVES: In this study, the pharmacological properties of six spirocyclic piperidines 1-6 showing very high σ1 receptor affinity (Ki  = 0.2-16 nm) were investigated. METHODS: In vitro receptor binding studies, retinal ganglion assay and in vivo capsaicin assay were used to determine the affinity, selectivity and activity. Influence on human tumour cell growth (cell lines A427, LCLC-103H, 5637 and DAN-G) was determined in different assays. The effect on the ergosterol and cholesterol biosynthesis was determined by GLC/MS analysis. KEY FINDINGS: Receptor binding studies demonstrated high selectivity for the σ1 receptor. The increased Ca2+ influx mediated by 2 and the analgesic activity of 1, 4, 5 and 6 confirm σ1 receptor antagonistic activity. Inhibition of human tumour cell growth further supports the σ1 antagonistic effects. Treatment of A427 tumour cells with 2 led to cell detachment and cell degradation. Whereas the ergosterol biosynthesis was not affected, the sterol C14-reductase, a key enzyme in the cholesterol biosynthesis, was weakly inhibited. CONCLUSIONS: Due to the high selectivity, off-target effects are not expected. The antiallodynic activity underlines the clinical potential of the spirocyclic piperidines for the treatment of neuropathic pain. Due to the antiproliferative activity, the spirocyclic σ1 antagonists represent promising antitumour agents.

4-Aryl-1-oxa-4,9-diazaspiro[5.5]undecane Derivatives as Dual µ-Opioid Receptor Agonists and σ1 Receptor Antagonists for the Treatment of Pain.

The synthesis and pharmacological activity of a new series of 1-oxa-4,9-diazaspiro[5.5]undecane derivatives as potent dual ligands for the sigma-1 receptor (σ1R) and the µ-opioid receptor (MOR) are reported. The different positions of the central scaffold, designed using a merging strategy of both target pharmacophores, were explored using a versatile synthetic approach. Phenethyl derivatives in position 9, substituted pyridyl moieties in position 4 and small alkyl groups in position 2 provided the best profiles. One of the best compounds, 15au, showed a balanced dual profile (i.e., MOR agonism and sigma antagonism) and a potent analgesic activity, comparable to the MOR agonist oxycodone in the paw pressure test in mice. Contrary to oxycodone, as expected from the addition of σ1R antagonism, 15au showed local, peripheral activity in this test, which was reversed by the σ1R agonist PRE-084. At equianalgesic doses, 15au showed less constipation than oxycodone, providing evidence that dual MOR agonism and σ1R antagonism may be a useful strategy for obtaining potent and safer analgesics.

EST64454: a Highly Soluble σ1 Receptor Antagonist Clinical Candidate for Pain Management.

The synthesis and pharmacological activity of a new series of pyrazoles that led to the identification of 1-(4-(2-((1-(3,4-difluorophenyl)-1H-pyrazol-3-yl)methoxy)ethyl)piperazin-1-yl)ethanone (9k, EST64454) as a σ1 receptor (σ1R) antagonist clinical candidate for the treatment of pain are reported. The compound 9k is easily obtained through a five-step synthesis suitable for the production scale and shows an outstanding aqueous solubility, which together with its high permeability in Caco-2 cells will allow its classification as a BCS class I compound. It also shows high metabolic stability in all species, linked to an adequate pharmacokinetic profile in rodents, and antinociceptive properties in the capsaicin and partial sciatic nerve ligation models in mice.

Discovery of EST73502, a Dual µ-Opioid Receptor Agonist and σ1 Receptor Antagonist Clinical Candidate for the Treatment of Pain.

The synthesis and pharmacological activity of a new series of 4-alkyl-1-oxa-4,9-diazaspiro[5.5]undecane derivatives as potent dual ligands for the σ1 receptor (σ1R) and the µ-opioid receptor (MOR) are reported. A lead optimization program over the initial 4-aryl analogues provided 4-alkyl derivatives with the desired functionality and good selectivity and ADME profiles. Compound 14u (EST73502) showed MOR agonism and σ1R antagonism and a potent analgesic activity, comparable to the MOR agonist oxycodone in animal models of acute and chronic pain after single and repeated administration. Contrary to oxycodone, 14u produces analgesic activity with reduced opioid-induced relevant adverse events, like intestinal transit inhibition and naloxone-precipitated behavioral signs of opiate withdrawal. These results provide evidence that dual MOR agonism and σ1R antagonism may be a useful strategy for obtaining potent and safer analgesics and were the basis for the selection of 14u as a clinical candidate for the treatment of pain.

A New Pharmacophore Model for the Design of Sigma-1 Ligands Validated on a Large Experimental Dataset.

The recent publication of the σ1R crystal structure is an important cornerstone for the derivation of more accurate activity prediction models. We report here a comparative study involving a set of more than 25,000 structures from our internal database that had been screened for σ1R affinity. Using the recently published crystal structure, 5HK1, two new pharmacophore models were generated. The first one, 5HK1-Ph.A, was obtained by an algorithm that identifies the most important receptor-ligand interactions including volume restrictions enforced by the atomic structure of the recognition site. The second, 5HK1-Ph.B, resulted from a manual edition of the first one by the fusion of two hydrophobic (HYD) features. Finally, we also docked the database using a high throughput docking technique and scored the resulting poses with seven different scoring functions. Statistical performance measures were obtained for the two models, comparing them with previously published σ1R pharmacophores (Hit Rate, sensitivity, specificity, and Receiver Operator Characteristic) and 5HK1-Ph.B emerged as the best one in discriminating between active and inactive compounds, with a ROC-AUC value above 0.8 and enrichment values above 3 at different fractions of screened samples. 5HK1-Ph.B also showed better results than the direct docking, which may be due to the rigidity of the crystal structure in the docking process (i.e., feature tolerances in the pharmacophore model). Additionally, the impact of the HYD interactions and the penalty for desolvating ligands with polar atoms may be not adequately captured by scoring functions, whereas HYD groups filling up such regions of the binding site are entailed in the pharmacophore model. Altogether, using annotated data from a large and diverse compound collection together with crystal structure information provides a sound basis for the generation and validation of predictive models to design new molecules.

Co-crystals as a new approach to multimodal analgesia and the treatment of pain.

Pain is highly prevalent, but frequently untreated or under-treated, and health care professionals are faced with a range of treatment challenges. Multimodal therapy is recommended and can be achieved using open combinations (ie, concomitant administration) of individual agents, fixed-dose combinations (FDCs), or multimodal agents (ie, single agents with multiple mechanisms of action). Co-crystallization of active pharmaceutical ingredients (APIs) offers another approach, with the potential to provide drugs with unique properties and advantages for therapeutic applications compared to combinations. API-API co-crystals are single-entity forms that offer a unique possibility of improving the physicochemical properties of both constituent APIs, as well as permitting their synchronous release. Consequently, this may positively impact on their pharmacokinetic (PK) properties and profiles, providing a potential advantage over FDCs and translating into improved clinical efficacy and safety profiles. We report here a revision of the literature concerning API-API co-crystals for the treatment of pain. It becomes apparent that identifying APIs with complementary mechanisms of action that can be adequately co-crystallized in an appropriate molecular ratio applicable for therapeutic use is challenging. In addition, API-API co-crystals normally result in a mere increased exposure of an API without defined clinical benefits (since, to maintain the benefit-risk, the dose needs to be proportionally reduced to adjust for the increased exposure). An exception to this is the co-crystal of tramadol-celecoxib (CTC), that represents a unique concept in co-crystal technology. In CTC neither of its three active components that have complementary mechanisms of action (ie, the two enantiomers of tramadol and celecoxib) show increased exposure levels versus commercially available single-entity reference products, but rather show a change in their PK profile with potential clinical advantages. CTC is in Phase III clinical development for the treatment of pain.

Synthesis and Structure-Affinity Relationships of Spirocyclic Benzopyrans with Exocyclic Amino Moiety.

σ1 and/or σ2 receptors play a crucial role in pathological conditions such as pain, neurodegenerative disorders, and cancer. A set of spirocyclic cyclohexanes with diverse O-heterocycles and amino moieties (general structure III) was prepared and pharmacologically evaluated. In structure-activity relationships studies, the σ1 receptor affinity and σ1:σ2 selectivity were correlated with the stereochemistry, the kind and substitution pattern of the O-heterocycle, and the substituents at the exocyclic amino moiety. cis-configured 2-benzopyran cis-11b bearing a methoxy group and a tertiary cyclohexylmethylamino moiety showed the highest σ1 affinity ( Ki = 1.9 nM) of this series of compounds. In a Ca2+ influx assay, cis-11b behaved as a σ1 antagonist. cis-11b reveals high selectivity over σ2 and opioid receptors. The interactions of the novel σ1 ligands were analyzed on the molecular level using the recently reported X-ray crystal structure of the σ1 receptor protein. The protonated amino moiety forms a persistent salt bridge with E172. The spiro[benzopyran-1,1'-cyclohexane] scaffold and the cyclohexylmethyl moiety occupy two hydrophobic pockets. Exchange of the N-cyclohexylmethyl moiety by a benzyl group led unexpectedly to potent and selective µ-opioid receptor ligands.

Co-crystal of tramadol-celecoxib: preclinical and clinical evaluation of a novel analgesic.

INTRODUCTION: Pain management is a major unmet need due to the suboptimal efficacy and undesirable side effects of current analgesics. Multimodal therapies recruiting complementary mechanisms of action may help address this. Co-crystals incorporating two active pharmaceutical ingredients (APIs) constitute an innovative approach to multimodal therapy, particularly if modification of the physicochemical properties of constituent APIs can be translated into clinical benefits. AREAS COVERED: The preclinical and clinical profiles of Co-Crystal of Tramadol-Celecoxib (CTC), a novel API-API co-crystal (1:1 molecular ratio of rac-tramadol.hydrochloride and celecoxib) are described. EXPERT OPINION: CTC may provide a relevant addition to pain therapy due to its: i) unique co-crystal structure conferring differentiated intrinsic dissolution profiles on constituent APIs, ii) modified clinical pharmacokinetics (slower absorption of tramadol and faster absorption of celecoxib) compared with commercially available single-entity reference products (in agreement with modified dissolution rates), iii) superior benefit-risk ratio compared with reference products (suggested by preclinical synergistic antinociceptive effects, without potentiation of adverse effects), and iv) efficacy in a phase 2 trial of moderate to severe pain following extraction of ≥2 impacted third molars requiring bone removal, where CTC doses containing low doses of APIs exerted a significant effect. Phase 3 studies are currently ongoing.

Dissection of the recognition properties of p38 MAP kinase. Determination of the binding mode of a new pyridinyl-heterocycle inhibitor family.

The main recognition characteristics of the ATP binding site of p38 mitogen activated protein kinase alpha (p38alpha MAPK) have been explored by a combination of modeling and bioinformatics techniques, making special emphasis in the characteristics of the site that justifies binding specificity with respect to other MAP kinases. Particularly, we have analyzed the binding mode of a new family of p38 MAPK inhibitors based on the pyridinyl-heterocycle core. This family of compounds has a marked pseudosymmetry and can exist in different tautomeric forms, which makes the determination of the binding mode especially challenging. A combination of homology modeling, quantum mechanics, classical docking, and molecular dynamics calculations allowed us to determine the main characteristics defining the binding mode of this new scaffold in the ATP binding site of p38alpha. A set of free energy calculations allowed us to verify the binding mode proposed, giving an overall excellent agreement with the experimental values. Finally, the binding mode of this new family of compounds was compared to that of other members of the pyridinyl and pyrimidinyl heterocycle class.

Pyrazolo[3,4-d]pyrimidines as sigma-1 receptor ligands for the treatment of pain. Part 1: 4-acylamino derivatives.

The synthesis of a new series of 4-acylaminopyrazolo[3,4-d]pyrimidines active on the sigma-1 receptor (σ1R) is reported. Compounds were efficiently prepared using a two to three step process starting from commercially available 1H-pyrazolo[3,4-d]pyrimidin-4-amine. A SAR study shows that the σ1R requires the presence of relatively highly lipophilic substituents at opposite sides of the central scaffold, while selectivity versus the σ2R can be improved by shortening the distance of the basic nitrogen to it. Compound 9a was among the most active and selective in vitro derivatives and exhibited potent antinociceptive properties in several pain models in mice, indicating its antagonistic behaviour.