D3R grand challenge 4: blind prediction of protein-ligand poses, affinity rankings, and relative binding free energies.

The Drug Design Data Resource (D3R) aims to identify best practice methods for computer aided drug design through blinded ligand pose prediction and affinity challenges. Herein, we report on the results of Grand Challenge 4 (GC4). GC4 focused on proteins beta secretase 1 and Cathepsin S, and was run in an analogous manner to prior challenges. In Stage 1, participant ability to predict the pose and affinity of BACE1 ligands were assessed. Following the completion of Stage 1, all BACE1 co-crystal structures were released, and Stage 2 tested affinity rankings with co-crystal structures. We provide an analysis of the results and discuss insights into determined best practice methods.

D3R Grand Challenge 3: blind prediction of protein-ligand poses and affinity rankings.

The Drug Design Data Resource aims to test and advance the state of the art in protein-ligand modeling by holding community-wide blinded, prediction challenges. Here, we report on our third major round, Grand Challenge 3 (GC3). Held 2017-2018, GC3 centered on the protein Cathepsin S and the kinases VEGFR2, JAK2, p38-α, TIE2, and ABL1, and included both pose-prediction and affinity-ranking components. GC3 was structured much like the prior challenges GC2015 and GC2. First, Stage 1 tested pose prediction and affinity ranking methods; then all available crystal structures were released, and Stage 2 tested only affinity rankings, now in the context of the available structures. Unique to GC3 was the addition of a Stage 1b self-docking subchallenge, in which the protein coordinates from all of the cocrystal structures used in the cross-docking challenge were released, and participants were asked to predict the pose of CatS ligands using these newly released structures. We provide an overview of the outcomes and discuss insights into trends and best-practices.

A Single Amino Acid Difference between Mouse and Human 5-Lipoxygenase Activating Protein (FLAP) Explains the Speciation and Differential Pharmacology of Novel FLAP Inhibitors.

5-Lipoxygenase activating protein (FLAP) plays a critical role in the metabolism of arachidonic acid to leukotriene A4, the precursor to the potent pro-inflammatory mediators leukotriene B4 and leukotriene C4 Studies with small molecule inhibitors of FLAP have led to the discovery of a drug binding pocket on the protein surface, and several pharmaceutical companies have developed compounds and performed clinical trials. Crystallographic studies and mutational analyses have contributed to a general understanding of compound binding modes. During our own efforts, we identified two unique chemical series. One series demonstrated strong inhibition of human FLAP but differential pharmacology across species and was completely inactive in assays with mouse or rat FLAP. The other series was active across rodent FLAP, as well as human and dog FLAP. Comparison of rodent and human FLAP amino acid sequences together with an analysis of a published crystal structure led to the identification of amino acid residue 24 in the floor of the putative binding pocket as a likely candidate for the observed speciation. On that basis, we tested compounds for binding to human G24A and mouse A24G FLAP mutant variants and compared the data to that generated for wild type human and mouse FLAP. These studies confirmed that a single amino acid mutation was sufficient to reverse the speciation observed in wild type FLAP. In addition, a PK/PD method was established in canines to enable preclinical profiling of mouse-inactive compounds.

Computational chemistry at Janssen.

Computer-aided drug discovery activities at Janssen are carried out by scientists in the Computational Chemistry group of the Discovery Sciences organization. This perspective gives an overview of the organizational and operational structure, the science, internal and external collaborations, and the impact of the group on Drug Discovery at Janssen.

Analysis of the structural and molecular basis of voltage-sensitive sodium channel inhibition by the spider toxin huwentoxin-IV (µ-TRTX-Hh2a).

Voltage-gated sodium channels (VGSCs) are essential to the normal function of the vertebrate nervous system. Aberrant function of VGSCs underlies a variety of disorders, including epilepsy, arrhythmia, and pain. A large number of animal toxins target these ion channels and may have significant therapeutic potential. Most of these toxins, however, have not been characterized in detail. Here, by combining patch clamp electrophysiology and radioligand binding studies with peptide mutagenesis, NMR structure determination, and molecular modeling, we have revealed key molecular determinants of the interaction between the tarantula toxin huwentoxin-IV and two VGSC isoforms, Nav1.7 and Nav1.2. Nine huwentoxin-IV residues (F6A, P11A, D14A, L22A, S25A, W30A, K32A, Y33A, and I35A) were important for block of Nav1.7 and Nav1.2. Importantly, molecular dynamics simulations and NMR studies indicated that folding was normal for several key mutants, suggesting that these amino acids probably make specific interactions with sodium channel residues. Additionally, we identified several amino acids (F6A, K18A, R26A, and K27A) that are involved in isoform-specific VGSC interactions. Our structural and functional data were used to model the docking of huwentoxin-IV into the domain II voltage sensor of Nav1.7. The model predicts that a hydrophobic patch composed of Trp-30 and Phe-6, along with the basic Lys-32 residue, docks into a groove formed by the Nav1.7 S1-S2 and S3-S4 loops. These results provide new insight into the structural and molecular basis of sodium channel block by huwentoxin-IV and may provide a basis for the rational design of toxin-based peptides with improved VGSC potency and/or selectivity.

Pharmacological characterization of 1-(5-chloro-6-(trifluoromethoxy)-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid (JNJ-42041935), a potent and selective hypoxia-inducible factor prolyl hydroxylase inhibitor.

The hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) enzymes represent novel targets for the treatment of anemia, ulcerative colitis, and ischemic and metabolic disease inter alia. We have identified a novel small-molecule inhibitor of PHD, 1-(5-chloro-6-(trifluoromethoxy)-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid (JNJ-42041935), through structure-based drug design methods. The pharmacology of JNJ-42041935 was investigated in enzyme, cellular, and whole-animal systems and was compared with other compounds described in the literature as PHD inhibitors. JNJ-42041935, was a potent (pK(I) = 7.3-7.9), 2-oxoglutarate competitive, reversible, and selective inhibitor of PHD enzymes. In addition, JNJ-42041935 was used to compare the effect of selective inhibition of PHD to intermittent, high doses (50 µg/kg i.p.) of an exogenous erythropoietin receptor agonist in an inflammation-induced anemia model in rats. JNJ-42041935 (100 µmol/kg, once a day for 14 days) was effective in reversing inflammation-induced anemia, whereas erythropoietin had no effect. The results demonstrate that JNJ-42041935 is a new pharmacological tool, which can be used to investigate PHD inhibition and demonstrate that PHD inhibitors offer great promise for the treatment of inflammation-induced anemia.

Highly potent, non-basic 5-HT6 ligands. Site mutagenesis evidence for a second binding mode at 5-HT6 for antagonism.

A series of 5-HT(6) ligands derived from (R)-1-(amino)methyl-6-(phenyl)sulfonyltetralin was prepared that yielded several non-basic analogs having sub-nanomolar affinity. Ligand structure-activity relationships, receptor point mutation studies, and molecular modeling of these novel ligands all combined to reveal a new alternative binding mode to 5-HT(6) for antagonism.

Molecular interactions of CCR5 with major classes of small-molecule anti-HIV CCR5 antagonists.

In addition to being an important receptor in leukocyte activation and mobilization, CCR5 is the essential coreceptor for human immunodeficiency virus (HIV). A large number of small-molecule CCR5 antagonists have been reported that show potent activities in blocking chemokine function and HIV entry. To facilitate the design and development of next generation CCR5 antagonists, docking models for major classes of CCR5 antagonists were created by using site-directed mutagenesis and CCR5 homology modeling. Five clinical candidates: maraviroc, vicriviroc, aplaviroc, TAK-779, and TAK-220 were used to establish the nature of the binding pocket in CCR5. Although the five antagonists are very different in structure, shape, and electrostatic potential, they were able to fit in the same binding pocket formed by the transmembrane (TM) domains of CCR5. It is noteworthy that each antagonist displayed a unique interaction profile with amino acids lining the pocket. Except for TAK-779, all antagonists showed strong interaction with Glu283 in TM 7 via their central basic nitrogen. The fully mapped binding pocket of CCR5 is being used for structure-based design and lead optimization of novel anti-HIV CCR5 inhibitors with improved potency and better resistance profile.

Recent progress in the design of small molecule inhibitors of HIV RNase H.

DNA polymerase and RNase H (RH) activities of HIV reverse transcriptase (RT) have been recognized as potential targets for antiretroviral therapy for more than 15 years. The development of medicines targeting the DNA polymerase activity has been highly successful, with currently 12 drugs approved for the treatment of HIV infection and more candidates in preclinical and clinical development. In contrast, the discovery of potent and selective inhibitors of HIV RH has been slow, and inhibitors of this enzyme function have yet to reach the clinical development stage. Selective HIV RH inhibitors are likely to provide significant clinical benefit in combination therapies, considering the high prevalence of HIV strains resistant to currently available antiretroviral therapies. Recent progress in a number of key areas has provided new impetus to the discovery of HIV RH inhibitors. High throughput assay systems based on fluorescence detection have been developed, which facilitate screening of inhibitor candidates. Substantial progress has been made in expression, purification, crystallisation and solution studies of HIV RT and RH, in particular with regards to aspects of structural dynamics. Crystal structures of active site binding and allosteric HIV RH inhibitors bound to HIV RT and RH have been obtained. Finally, an improved understanding of similarities and differences in enzymatic mechanisms between related nuclease enzymes has provided new concepts for achieving inhibitor selectivity. Together, these developments provide promising new starting points for the rational design of selective HIV RH inhibitors.

Lactams as EP4 prostanoid receptor agonists. 3. Discovery of N-ethylbenzoic acid 2-pyrrolidinones as subtype selective agents.

Two distinct synthetic schemes were applied to access heteroatom-containing alpha-chain lactams or lactams terminated as aryl acids. The latter lactams were devised using a pharmacophore for EP(4) receptor activity. gamma-Lactams were characterized for their prostanoid EP receptor affinities and EP(4) activity and found to be selective for the EP(2) and EP(4) receptors or selective for the EP(4) subtype. Benzoic acid 17 displayed enhanced in vivo exposure relative to 3.

Benzimidazole-2-pyrazole HIF Prolyl 4-Hydroxylase Inhibitors as Oral Erythropoietin Secretagogues.

HIF prolyl 4-hydroxylases (PHD) are a family of enzymes that mediate key physiological responses to hypoxia by modulating the levels of hypoxia inducible factor 1-α (HIF1α). Certain benzimidazole-2-pyrazole carboxylates were discovered to be PHD2 inhibitors using ligand- and structure-based methods and found to be potent, orally efficacious stimulators of erythropoietin secretion in vivo.

Substrate-dependent inhibition or stimulation of HIV RNase H activity by non-nucleoside reverse transcriptase inhibitors (NNRTIs).

HIV reverse transcriptase (HIV-RT) contains two distinct protein domains catalyzing DNA polymerase and RNase H activities. Non-nucleoside reverse transcriptase inhibitor (NNRTI) binding to HIV-RT can affect RNase H activity. The structurally diverse NNRTIs capravirine, efavirenz, GW8248, TMC-125, and nevirapine all inhibited 5'-RNA directed HIV RNase H activity as partial inhibitors with maximal inhibition of 40-65%. Potencies of RNase H inhibition correlated with the respective potencies of DNA polymerase inhibition. Mutations in the NNRTI binding site (K103N, Y181C, Y188L, and K103N/Y181C) reduced the potency of RNase H inhibition, similar to their effects on DNA polymerase activity. The NNRTIs did not affect the activity of the isolated HIV RNase H domain. In contrast, 3'-DNA directed RNase H activity of HIV-RT was mechanistically distinct from 5'-RNA directed RNase H activity and was stimulated rather than inhibited by NNRTI binding to HIV-RT. Therefore, NNRTI binding to the polymerase domain of HIV-RT interferes with RNase H activity through a long-range effect, which is affected by the structure of the RNA:DNA hybrid substrate, but is independent of NNRTI compound structure and nucleic acid substrate sequence.

Rational design of 6-methylsulfonylindoles as selective cyclooxygenase-2 inhibitors.

The introduction of 3-arylmethyl, 3-aryloxy and 3-arylthio moieties into a 6-methylsulfonylindole framework using rational drug design led to potent, selective COX-2 inhibitors having efficacy in a rat carrageenan air pouch model. Incorporation of a conformationally more rigid 3-aroyloxy substituent onto the 6-methylsulfonylindole scaffold led to selective, but considerably less potent COX-2 inhibitors. Variation of the hydrophilicity and size of the indole 2-substituent of 3-arylthio-6-methylsulfonylindole inhibitors led to modulation of the COX-2 human whole blood (HWB) potency and selectivity.