Pandemic drugs at pandemic speed: infrastructure for accelerating COVID-19 drug discovery with hybrid machine learning- and physics-based simulations on high-performance computers.

The race to meet the challenges of the global pandemic has served as a reminder that the existing drug discovery process is expensive, inefficient and slow. There is a major bottleneck screening the vast number of potential small molecules to shortlist lead compounds for antiviral drug development. New opportunities to accelerate drug discovery lie at the interface between machine learning methods, in this case, developed for linear accelerators, and physics-based methods. The two in silico methods, each have their own advantages and limitations which, interestingly, complement each other. Here, we present an innovative infrastructural development that combines both approaches to accelerate drug discovery. The scale of the potential resulting workflow is such that it is dependent on supercomputing to achieve extremely high throughput. We have demonstrated the viability of this workflow for the study of inhibitors for four COVID-19 target proteins and our ability to perform the required large-scale calculations to identify lead antiviral compounds through repurposing on a variety of supercomputers.

N-Terminus to Arginine Side-Chain Cyclization of Linear Peptidic Neuropeptide Y Y4 Receptor Ligands Results in Picomolar Binding Constants.

The family of neuropeptide Y (NPY) receptors comprises four subtypes (Y1R, Y2R, Y4R, Y5R), which are addressed by at least three endogenous peptides, i.e., NPY, peptide YY, and pancreatic polypeptide (PP), the latter showing a preference for Y4R. A series of cyclic oligopeptidic Y4R ligands were prepared by applying a novel approach, i.e., N-terminus to arginine side-chain cyclization. Most peptides acted as Y4R partial agonists, showing up to 60-fold higher Y4R affinity compared to the linear precursor peptides. Two cyclic hexapeptides (18, 24) showed higher Y4R potency (Ca2+ aequorin assay) and, with pKi values >10, also higher Y4R affinity compared to human pancreatic polypeptide (hPP). Compounds such as 18 and 24, exhibiting considerably lower molecular weight and considerably more pronounced Y4R selectivity than PP and previously described dimeric peptidic ligands with high Y4R affinity, represent promising leads for the preparation of labeled tool compounds and might support the development of drug-like Y4R ligands.

Dibenzodiazepinone-type muscarinic receptor antagonists conjugated to basic peptides: Impact of the linker moiety and unnatural amino acids on M2R selectivity.

The family of human muscarinic acetylcholine receptors (MRs) is characterized by a high sequence homology among the five subtypes (M1R-M5R), being the reason for a lack of subtype selective MR ligands. In continuation of our work on dualsteric dibenzodiazepinone-type M2R antagonists, a series of M2R ligands containing a dibenzodiazepinone pharmacophore linked to small basic peptides was synthesized (64 compounds). The linker moiety was varied with respect to length, number of basic nitrogens (0-2) and flexibility. Besides proteinogenic basic amino acids (Lys, Arg), shorter homologues of Lys and Arg, containing three and two methylene groups, respectively, as well as D-configured amino acids were incorporated. The type of linker had a marked impact on M2R affinity and also effected M2R selectivity. In contrast, the structure of the basic peptide rather determined M2R selectivity than M2R affinity. For example, the most M2R selective compound (UR-CG188, 89) with picomolar M2R affinity (pKi 9.60), exhibited a higher M2R selectivity (ratio of Ki M1R/M2R/M3R/M4R/M5R: 110:1:5200:55:2300) compared to the vast majority of reported M2R preferring MR ligands. For selected ligands, M2R antagonism was confirmed in a M2R miniG protein recruitment assay.

Water-soluble inhibitors of ABCG2 (BCRP) - A fragment-based and computational approach.

A good balance between hydrophilicity and lipophilicity is a prerequisite for all bioactive compounds. If the hydrophilicity of a compound is low, its solubility in water will be meager. Many drug development failures have been attributed to poor aqueous solubility. ABCG2 inhibitors are especially prone to be insoluble since they have to address the extremely large and hydrophobic multidrug binding site in ABCG2. For instance, our previous, tariquidar-related ABCG2 inhibitor UR-MB108 (1) showed high potency (79 nM), but very low aqueous solubility (78 nM). To discover novel potent ABCG2 inhibitors with improved solubility we pursued a fragment-based approach. Substructures of 1 were optimized and the fragments 'enlarged' to obtain inhibitors, supported by molecular docking studies. Synthesis was achieved, i.a., via Sonogashira coupling, click chemistry and amide coupling. A kinetic solubility assay revealed that 1 and most novel inhibitors did not precipitate during the short time period of the applied biological assays. The solubility of the compounds in aqueous media at equilibrium was investigated in a thermodynamic solubility assay, where UR-Ant116 (40), UR-Ant121 (41), UR-Ant131 (48) and UR-Ant132 (49) excelled with solubilities between 1 µM and 1.5 µM - an up to 19-fold improvement compared to 1. Moreover, these novel N-phenyl-chromone-2-carboxamides inhibited ABCG2 in a Hoechst 33342 transport assay with potencies in the low three-digit nanomolar range, reversed MDR in cancer cells, were non-toxic and proved stable in blood plasma. All properties make them attractive candidates for in vitro assays requiring long-term incubation and in vivo studies, both needing sufficient solubility at equilibrium. 41 and 49 were highly ABCG2-selective, a precondition for developing PET tracers. The triple ABCB1/C1/G2 inhibitor 40 qualifies for potential therapeutic applications, given the concerted role of the three transporter subtypes at many tissue barriers, e.g. the BBB.

Oligopeptides as Neuropeptide Y Y4 Receptor Ligands: Identification of a High-Affinity Tetrapeptide Agonist and a Hexapeptide Antagonist.

Within the family of neuropeptide Y (NPY) receptors, the Y4 receptor (Y4R) is unique as it prefers pancreatic polypeptide over NPY and peptide YY. Today, low-molecular-weight Y4R ligands are lacking, in particular antagonists. We synthesized a series of peptidic NPY Y4R ligands, derived from the hexapeptide acetyl-Arg-Tyr-Arg-Leu-Arg-Tyr-NH2 (1), reported to be a Y4R partial agonist with high affinity (pKi Y4R: 8.43). Peptide 1 was N-terminally extended as well as truncated and subjected to a d-amino acid scan, and Leu was replaced by different amino acids. Compounds were characterized by radioligand competition binding and functional studies (Cai2+ mobilization and ß-arrestin 1/2 recruitment). N-terminal truncation of 1 resulted in a tetrapeptide (Arg-Leu-Arg-Tyr-NH2), being a Y4R partial agonist with unchanged Y4R affinity (pKi: 8.47). Remarkably, replacement of Leu in 1 and in derivatives of 1 by Trp turned Y4R agonism to antagonism, giving Y4R antagonists with pKi values ≤7.57.

UR-DEBa242: A Py-5-Labeled Fluorescent Multipurpose Probe for Investigations on the Histamine H3 and H4 Receptors.

Comprehensively characterized fluorescent probes for the histamine H3 receptor (H3R) and especially for the H4R orthologs [e.g., human (h) and mouse (m)] are highly needed as versatile complementary tools to radioligands. In view of fluorescent probes for BRET-based binding studies and for localizing the H4R in live cells, we synthesized and biologically characterized Py-5-labeled histamine derivatives. The most notable compound was UR-DEBa242 (26, 1-[4-(1H-Imidazol-4-yl)butyl]-4-{(1E,3E)-4-[4-(dimethylamino)phenyl]buta-1,3-dienyl}-2,6-dimethylpyridinium hydrotrifluoroacetate trifluoroacetate), acting as a partial agonist at the hH3R [pEC50 (reporter gene) 8.77] and as an inverse agonist/antagonist at the h/mH4Rs [pIC50 (reporter gene) 8.76/7.08; pIC50/pKb (ß-arrestin2) 7.81/7.30]. In confocal microscopy, 26 proved suitable for hH4R localization and trafficking studies in live cells. BRET-based binding at the NLuc-hH3,4Rs/mH4R [pKd 8.78/7.75/7.18, comparable to binding constants from radioligand binding/flow cytometry; fast association/dissociation (∼2 min)] revealed 26 as a useful molecular tool to determine hH3,4Rs/mH4R binding affinities of ligands binding to these receptors.

Red-Emitting Dibenzodiazepinone Derivatives as Fluorescent Dualsteric Probes for the Muscarinic Acetylcholine M2 Receptor.

Fluorescently labeled dibenzodiazepinone-type muscarinic acetylcholine receptor (MR) antagonists, including dimeric ligands, were prepared using red-emitting cyanine dyes. Probes containing a fluorophore with negative charge showed high M2R affinities (pKi (radioligand competition binding): 9.10-9.59). Binding studies at M1 and M3-M5 receptors indicated a M2R preference. Flow cytometric and high-content imaging saturation and competition binding (M1R, M2R, and M4R) confirmed occupation of the orthosteric site. Confocal microscopy revealed that fluorescence was located mainly at the cell membrane (CHO-hM2R cells). Results from dissociation and saturation binding experiments (M2R) in the presence of allosteric M2R modulators (dissociation: W84, LY2119620, and alcuronium; saturation binding: W84) were consistent with a competitive mode of action between the fluorescent probes and the allosteric ligands. Taken together, these lines of evidence indicate that these ligands are useful fluorescent molecular tools to label the M2R in imaging and binding studies and suggest that they have a dualsteric mode of action.

Argininamide-type neuropeptide Y Y1 receptor antagonists: the nature of N ω-carbamoyl substituents determines Y1R binding mode and affinity.

The recently resolved crystal structure of the neuropeptide Y Y1 receptor (Y1R), co-crystallized with the high-affinity (pK i: 10.11), argininamide-type Y1R antagonist UR-MK299 (2), revealed that the N ω-carbamoyl substituent (van der Waals volume: 139 Å3) is deeply buried in the receptor, occupying a hydrophobic pocket. We synthesized and characterized a series of argininamides, structurally related to 2. Y1R affinity decreased with increasing size of the carbamoyl residue (minimal pK i: 5.67). Exceeding a critical size of the substituent (van der Waals volume: 212 Å3), the ligands bound in an inverted mode with the carbamoyl side chain located at the surface of the receptor, as suggested by induced-fit docking and MD simulations.

Basal Histamine H4 Receptor Activation: Agonist Mimicry by the Diphenylalanine Motif.

Histamine H4 receptor (H4 R) orthologues are G-protein-coupled receptors (GPCRs) that exhibit species-dependent basal activity. In contrast to the basally inactive mouse H4 R (mH4 R), human H4 R (hH4 R) shows a high degree of basal activity. We have performed long-timescale molecular dynamics simulations and rigidity analyses on wild-type hH4 R, the experimentally characterized hH4 R variants S179M, F169V, F169V+S179M, F168A, and on mH4 R to investigate the molecular nature of the differential basal activity. H4 R variant-dependent differences between essential motifs of GPCR activation and structural stabilities correlate with experimentally determined basal activities and provide a molecular explanation for the differences in basal activation. Strikingly, during the MD simulations, F16945.55 dips into the orthosteric binding pocket only in the case of hH4 R, thus adopting the role of an agonist and contributing to the stabilization of the active state. The results shed new light on the molecular mechanism of basal H4 R activation that are of importance for other GPCRs.

Universal Activation Index for Class A GPCRs.

An index of the activation of Class A G-protein-coupled receptors (GPCRs) has been trained using interhelix distances from a series of microsecond molecular-dynamics simulations and tested for 268 published X-ray structures. In a three-class model that includes intermediate structures, 63% of the active structures are classified in agreement with the experimental assignment, 81% of the intermediate structures, and 89% of the inactives. An alternative two-state model classifies 94% of the actives and 99% of the inactives correctly. The intermediate structures are distributed 2:1 between actives and inactives. X-ray structures with protein nanobodies give good agreement between the assigned activation state and the predictions of the model, whereby many active nanobody structures are predicted to be weakly active. The five interhelix Cα-Cα distances that occur in the model relate clearly to the established activation mechanism. The model is available as a Python script or via an interactive web page. It can thus be used to classify both experimental and computational GPCR structures.

Conjugation of Short Peptides to Dibenzodiazepinone-Type Muscarinic Acetylcholine Receptor Ligands Determines M2R Selectivity.

Muscarinic acetylcholine receptors (MRs), comprising five subtypes (M1R-M5R) in humans, exhibit a high degree of structural similarity. Therefore, subtype-selective MR agonists and antagonists are lacking. We present an approach to highly M2R-selective MR antagonists based on the conjugation of di- or tripeptides to M2R-preferring dibenzodiazepinone-type MR antagonists. M2R selectivity was dependent on the peptide sequence and on the type of linker. The introduction of basic amino acids resulted in improved M2R selectivity (e.g., UR-AP148 (48): p Ki (hM2R) of 8.97, ratio of Ki M1R/M2R/M3R/M4R/M5R of 49:1:6500:60:400) compared to reported pyridobenzo- and dibenzodiazepinone-type MR ligands. A supposed dualsteric binding mode of the DIBA-peptide conjugates, such as 48, at MRs was supported by molecular dynamics simulations.

Structure-Activity Relationship of Hetarylpropylguanidines Aiming at the Development of Selective Histamine Receptor Ligands†.

New classes of alkylated hetarylpropylguanidines with different functionality and variation in spacer length were synthesized to determine their behavior at the four histamine receptor (H1R, H2R, H3R, H4R) subtypes. Alkylated guanidines with different terminal functional groups and varied basicity, like amine, guanidine and urea were developed, based on the lead structure SK&F 91486 (2). Furthermore, heteroatomic exchange at the guanidine structure of 2 led to simple analogues of the lead compound. Radioassays at all histamine receptor subtypes were accomplished, as well as organ bath studies at the guinea pig (gp) ileum (gpH1R) and right atrium (gpH2R). Ligands with terminal functionalization led to, partially, highly affine and potent structures (two digit nanomolar), which showed up a bad selectivity profile within the histamine receptor family. While the benzoylurea derivative 144 demonstrated a preference towards the human (h) H3R, S-methylisothiourea analogue 143 obtained high affinity at the hH4R (pKi=8.14) with moderate selectivity. The molecular basis of the latter finding was supported by computational studies.

Highly Potent, Stable, and Selective Dimeric Hetarylpropylguanidine-Type Histamine H2 Receptor Agonists.

On the basis of the long-known prototypic pharmacophore 3-(1H-imidazol-4-yl)propylguanidine (SK&F 91486, 2), monomeric, homodimeric, and heterodimeric bisalkylguanidine-type histamine H2 receptor (H2R) agonists with various alkyl spacers were synthesized. Aiming at increased H2R selectivity of the ligands, the imidazol-4-yl moiety was replaced by imidazol-1-yl, 2-aminothiazol-5-yl or 2-amino-4-methylthiazol-5-yl according to a bioisosteric approach. All compounds turned out to be partial or full agonists at the h/gp/rH2R. The most potent analogue, the thiazole-type heterodimeric ligand 63 (UR-Po461), was a partial agonist (Emax = 88%) and 250 times more potent than histamine (pEC50: 8.56 vs 6.16, gpH2R, atrium). The homodimeric structures 56 (UR-Po395) and 58 (UR-Po448) exhibited the highest hH2R affinities (pKi: 7.47, 7.33) in binding studies. Dimeric amino(methyl)thiazole derivatives, such as 58, generated an increased hH2R selectivity compared to the monomeric analogues, e.g., 139 (UR-Po444). Although monomeric ligands showed up lower affinities and potencies at the H2R, compounds with a short alkylic side chain like 129 (UR-Po194) proved to be highly affine hH4R ligands.

Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor.

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology 1,2 . The NPY-Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y1, Y2, Y4 and Y5 receptors, with different affinity and selectivity 3 . NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y1 receptor (Y1R) 4 . A number of peptides and small-molecule compounds have been characterized as Y1R antagonists and have shown clinical potential in the treatment of obesity 4 , tumour 1 and bone loss 5 . However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability 6 . Here we report crystal structures of the human Y1R bound to the two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal the binding modes of Y1R to several structurally diverse antagonists and the determinants of ligand selectivity. The Y1R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance, photo-crosslinking and functional studies, provide insights into the binding behaviour of the agonist and for the first time, to our knowledge, determine the interaction of its N terminus with the receptor. These insights into Y1R can enable structure-based drug discovery that targets NPY receptors.

Radiolabeled Dibenzodiazepinone-Type Antagonists Give Evidence of Dualsteric Binding at the M2 Muscarinic Acetylcholine Receptor.

The dualsteric ligand approach, aiming at ligands with improved subtype selectivity, has been increasingly applied to muscarinic receptors (MRs). In this article, we present the synthesis and characterization of a M2R subtype-preferring radiolabeled dibenzodiazepinone-type antagonist ([3H]UNSW-MK259, [3H]19) and its homodimeric analogue [3H]UR-AP060 ([3H]33). Saturation binding studies at the M2R, using the orthosteric antagonist atropine to determine unspecific binding, proved that the monomeric and the dimeric compound bind to the orthosteric binding site (apparent Kd: 0.87 and 0.31 nM, respectively). Various binding studies with [3H]19 and [3H]33 at the M2R, for instance, saturation binding experiments in the presence of the allosteric MR modulators W84 (8) or LY2119620 (9) (Schild-like analysis) suggested a competitive mechanism between the allosteric modulator and the dibenzodiazepinone derivatives, and thus a dualsteric binding mode of both 19 and 33. This was consistent with the results of M2R MD simulations (≥2 µs) performed with 19 and 33.

Mimicking of Arginine by Functionalized N(ω)-Carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples.

Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

The extracellular loop 2 (ECL2) of the human histamine H4 receptor substantially contributes to ligand binding and constitutive activity.

In contrast to the corresponding mouse and rat orthologs, the human histamine H4 receptor (hH4R) shows extraordinarily high constitutive activity. In the extracellular loop (ECL), replacement of F169 by V as in the mouse H4R significantly reduced constitutive activity. Stabilization of the inactive state was even more pronounced for a double mutant, in which, in addition to F169V, S179 in the ligand binding site was replaced by M. To study the role of the FF motif in ECL2, we generated the hH4R-F168A mutant. The receptor was co-expressed in Sf9 insect cells with the G-protein subunits Gαi2 and Gß1γ2, and the membranes were studied in [3H]histamine binding and functional [35S]GTPγS assays. The potency of various ligands at the hH4R-F168A mutant decreased compared to the wild-type hH4R, for example by 30- and more than 100-fold in case of the H4R agonist UR-PI376 and histamine, respectively. The high constitutive activity of the hH4R was completely lost in the hH4R-F168A mutant, as reflected by neutral antagonism of thioperamide, a full inverse agonist at the wild-type hH4R. By analogy, JNJ7777120 was a partial inverse agonist at the hH4R, but a partial agonist at the hH4R-F168A mutant, again demonstrating the decrease in constitutive activity due to F168A mutation. Thus, F168 was proven to play a key role not only in ligand binding and potency, but also in the high constitutive activity of the hH4R.

Luciferase reporter gene assay on human, murine and rat histamine H4 receptor orthologs: correlations and discrepancies between distal and proximal readouts.

The investigation of the (patho)physiological role of the histamine H4 receptor (H4R) and its validation as a possible drug target in translational animal models are compromised by distinct species-dependent discrepancies regarding potencies and receptor subtype selectivities of the pharmacological tools. Such differences were extremely pronounced in case of proximal readouts, e. g. [(32)P]GTPase or [(35)S]GTPγS binding assays. To improve the predictability of in vitro investigations, the aim of this study was to establish a reporter gene assay for human, murine and rat H4Rs, using bioluminescence as a more distal readout. For this purpose a cAMP responsive element (CRE) controlled luciferase reporter gene assay was established in HEK293T cells, stably expressing the human (h), the mouse (m) or the rat (r) H4R. The potencies and efficacies of 23 selected ligands (agonists, inverse agonists and antagonists) were determined and compared with the results obtained from proximal readouts. The potencies of the examined ligands at the human H4R were consistent with reported data from [(32)P]GTPase or [(35)S]GTPγS binding assays, despite a tendency toward increased intrinsic efficacies of partial agonists. The differences in potencies of individual agonists at the three H4R orthologs were generally less pronounced compared to more proximal readouts. In conclusion, the established reporter gene assay is highly sensitive and reliable. Regarding discrepancies compared to data from functional assays such as [(32)P]GTPase and [(35)S]GTPγS binding, the readout may reflect multifactorial causes downstream from G-protein activation, e.g. activation/amplification of or cross-talk between different signaling pathways.