Discovery of macrocyclic HDACs 1, 2, and 3 selective inhibitors for HIV latency reactivation.

A series of unique macrocyclic HDACs 1, 2, and 3 selective inhibitors were identified with good enzymatic activity and high selectivity over HDACs 6 and 8. These macrocyclic HDAC inhibitors used an ethyl ketone as the zinc-binding group. Compounds 25 and 26 stood out as leads due to their low double-digit nM EC50s in the 2C4 cell-based HIV latency reactivation assay. The PK profiles of these macrocyclic HDAC inhibitors still needed improvement.

Identification of free fatty acid receptor 2 agonists using virtual screening.

Structure- and ligand-based virtual-screening methods (docking, 2D- and 3D-similarity searching) were analyzed for their effectiveness in virtual screening against FFAR2. To evaluate the performance of these methods, retrospective virtual screening was performed. Statistical quality of the methods was evaluated by BEDROC and RIE. The results revealed that electrostatic similarity search protocol using EON (ET combo) outperformed all other protocols with outstanding enrichment of >95% in top 1% and 2% of the dataset with an AUC of 0.958. Interestingly, the hit lists that are obtained from different virtual-screening methods are generally highly complementary to hits found from electrostatic similarity searching. These results suggest that considering electrostatic similarity searching first increases the chance of identifying more (and more diverse) active compounds from a virtual-screening campaign. Accordingly, prospective virtual screening using electrostatic similarity searching was used to identify novel FFAR2 ligands. The discovered compounds provide new chemical matter starting points for the initiation of a medicinal chemistry campaign.

Discovery of ethyl ketone-based HDACs 1, 2, and 3 selective inhibitors for HIV latency reactivation.

A novel series of ethyl ketone based HDACs 1, 2, and 3 selective inhibitors have been identified with good enzymatic and cellular activity and high selectivity over HDACs 6 and 8. These inhibitors contain a spirobicyclic group in the amide region. Compound 13 stands out as a lead due to its good potency, high selectivity, and reasonable rat and dog PK. Compounds 33 and 34 show good potency and rat PK profiles as well.

Development of a selective HDAC inhibitor aimed at reactivating the HIV latent reservoir.

The synthesis and SAR development of a trisubstituted imidazole HDAC inhibitor is described. The compounds were synthesized with high diastereocontrol by leveraging Ellman sulfinyl imine chemistry. Structural elucidation provided insight into binding mode and supported design rational. Pharmacokinetic properties of lead compounds were determined.

Workflows and performances in the ranking prediction of 2016 D3R Grand Challenge 2: lessons learned from a collaborative effort.

The 2016 D3R Grand Challenge 2 includes both pose and affinity or ranking predictions. This article is focused exclusively on affinity predictions submitted to the D3R challenge from a collaborative effort of the modeling and informatics group. Our submissions include ranking of 102 ligands covering 4 different chemotypes against the FXR ligand binding domain structure, and the relative binding affinity predictions of the two designated free energy subsets of 15 and 18 compounds. Using all the complex structures prepared in the same way allowed us to cover many types of workflows and compare their performances effectively. We evaluated typical workflows used in our daily structure-based design modeling support, which include docking scores, force field-based scores, QM/MM, MMGBSA, MD-MMGBSA, and MacroModel interaction energy estimations. The best performing methods for the two free energy subsets are discussed. Our results suggest that affinity ranking still remains very challenging; that the knowledge of more structural information does not necessarily yield more accurate predictions; and that visual inspection and human intervention are considerably important for ranking. Knowledge of the mode of action and protein flexibility along with visualization tools that depict polar and hydrophobic maps are very useful for visual inspection. QM/MM-based workflows were found to be powerful in affinity ranking and are encouraged to be applied more often. The standardized input and output enable systematic analysis and support methodology development and improvement for high level blinded predictions.

Performance of multiple docking and refinement methods in the pose prediction D3R prospective Grand Challenge 2016.

We describe the performance of multiple pose prediction methods for the D3R 2016 Grand Challenge. The pose prediction challenge includes 36 ligands, which represent 4 chemotypes and some miscellaneous structures against the FXR ligand binding domain. In this study we use a mix of fully automated methods as well as human-guided methods with considerations of both the challenge data and publicly available data. The methods include ensemble docking, colony entropy pose prediction, target selection by molecular similarity, molecular dynamics guided pose refinement, and pose selection by visual inspection. We evaluated the success of our predictions by method, chemotype, and relevance of publicly available data. For the overall data set, ensemble docking, visual inspection, and molecular dynamics guided pose prediction performed the best with overall mean RMSDs of 2.4, 2.2, and 2.2 Å respectively. For several individual challenge molecules, the best performing method is evaluated in light of that particular ligand. We also describe the protein, ligand, and public information data preparations that are typical of our binding mode prediction workflow.

Alternative core development around the tetracyclic indole class of HCV NS5A inhibitors.

Herein, we describe our research efforts to develop unique cores in molecules which function as HCV nonstructural protein 5A (NS5A) inhibitors. In particular, various fused tetracyclic cores were identified which showed genotype and mutant activities comparable to the indole-based tetracyclic core.

Discovery of fused tricyclic core containing HCV NS5A inhibitors with pan-genotype activity.

HCV NS5A inhibitors have demonstrated impressive in vitro potency profiles in HCV replicon assays and robust HCV RNA titer reduction in the clinic making them attractive components for inclusion in an all oral fixed dose combination regimen for the treatment of HCV infection. Herein, we describe research efforts that led to the discovery of a series of fused tricyclic core containing HCV NS5A inhibitors such as 24, 39, 40, 43, and 44 which have pan-genotype activity and are orally bioavailable in the rat.

Discovery of potent macrocyclic HCV NS5A inhibitors.

HCV NS5A inhibitors have demonstrated impressive in vitro virologic profiles in HCV replicon assays and robust HCV RNA titer reduction in the clinic making them attractive components for inclusion in an all oral fixed-dose combination (FDC) regimen for the treatment of HCV infection. Merck's effort in this area identified MK-4882 and MK-8325 as early development leads. Herein, we describe the discovery of potent macrocyclic NS5A inhibitors bearing the MK-8325 or MK-4882 core structure.

Structural determinants of the transient receptor potential 1 (TRPV1) channel activation by phospholipid analogs.

The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal protein that responds to various stimuli, including capsaicin (the pungent compound found in chili peppers), extracellular acid, and basic intracellular pH, temperatures close to 42 °C, and several lipids. Lysophosphatidic acid (LPA), an endogenous lipid widely associated with neuropathic pain, is an agonist of the TRPV1 channel found in primary afferent nociceptors and is activated by other noxious stimuli. Agonists or antagonists of lipid and other chemical natures are known to possess specific structural requirements for producing functional effects on their targets. To better understand how LPA and other lipid analogs might interact and affect the function of TRPV1, we set out to determine the structural features of these lipids that result in the activation of TRPV1. By changing the acyl chain length, saturation, and headgroup of these LPA analogs, we established strict requirements for activation of TRPV1. Among the natural LPA analogs, we found that only LPA 18:1, alkylglycerophosphate 18:1, and cyclic phosphatidic acid 18:1, all with a monounsaturated C18 hydrocarbon chain activate TRPV1, whereas polyunsaturated and saturated analogs do not. Thus, TRPV1 shows a more restricted ligand specificity compared with LPA G-protein-coupled receptors. We synthesized fatty alcohol phosphates and thiophosphates and found that many of them with a single double bond in position Δ9, 10, or 11 and Δ9 cyclopropyl group can activate TRPV1 with efficacy similar to capsaicin. Finally, we developed a pharmacophore and proposed a mechanistic model for how these lipids could induce a conformational change that activates TRPV1.

Mitigation of radiation injury by selective stimulation of the LPA(2) receptor.

Due to its antiapoptotic action, derivatives of the lipid mediator lysophosphatidic acid (LPA) provide potential therapeutic utility in diseases associated with programmed cell death. Apoptosis is one of the major pathophysiological processes elicited by radiation injury to the organism. Consequently, therapeutic explorations applying compounds that mimic the antiapoptotic action of LPA have begun. Here we present a brief account of our decade-long drug discovery effort aimed at developing LPA mimics with a special focus on specific agonists of the LPA(2) receptor subtype, which was found to be highly effective in protecting cells from apoptosis. We describe new evidence that 2-((3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)thio)benzoic acid (GRI977143), a prototypic nonlipid agonist specific to the LPA(2) receptor subtype, rescues apoptotically condemned cells in vitro and in vivo from injury caused by high-dose γ-irradiation. GRI977143 shows the features of a radiomitigator because it is effective in rescuing the lives of mice from deadly levels of radiation when administered 24h after radiation exposure. Our findings suggest that by specifically activating LPA(2) receptors GRI977143 activates the ERK1/2 prosurvival pathway, effectively reduces Bax translocation to the mitochondrion, attenuates the activation of initiator and effector caspases, reduces DNA fragmentation, and inhibits PARP-1 cleavage associated with γ-irradiation-induced apoptosis. GRI977143 also inhibits bystander apoptosis elicited by soluble proapoptotic mediators produced by irradiated cells. Thus, GRI977143 can serve as a prototype scaffold for lead optimization paving the way to more potent analogs amenable for therapeutic exploration. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Hits of a high-throughput screen identify the hydrophobic pocket of autotaxin/lysophospholipase D as an inhibitory surface.

Autotaxin (ATX), a lysophospholipase D, plays an important role in cancer invasion, metastasis, tumor progression, tumorigenesis, neuropathic pain, fibrotic diseases, cholestatic pruritus, lymphocyte homing, and thrombotic diseases by producing the lipid mediator lysophosphatidic acid (LPA). A high-throughput screen of ATX inhibition using the lysophosphatidylcholine-like substrate fluorogenic substrate 3 (FS-3) and ∼10,000 compounds from the University of Cincinnati Drug Discovery Center identified several small-molecule inhibitors with IC50 vales ranging from nanomolar to low micromolar. The pharmacology of the three most potent compounds: 918013 (1; 2,4-dichloro-N-(3-fluorophenyl)-5-(4-morpholinylsulfonyl) benzamide), 931126 (2; 4-oxo-4-{2-[(5-phenoxy-1H-indol-2-yl)carbonyl]hydrazino}-N-(4-phenylbutan-2-yl)butanamide), and 966791 (3; N-(2,6-dimethylphenyl)-2-[N-(2-furylmethyl)(4-(1,2,3,4-tetraazolyl)phenyl)carbonylamino]-2-(4-hydroxy-3-methoxyphenyl) acetamide), were further characterized in enzyme, cellular, and whole animal models. Compounds 1 and 2 were competitive inhibitors of ATX-mediated hydrolysis of the lysophospholipase substrate FS-3. In contrast, compound 3 was a competitive inhibitor of both FS-3 and the phosphodiesterase substrate p-nitrophenyl thymidine 5'-monophosphate. Computational docking and mutagenesis suggested that compounds 1 and 2 target the hydrophobic pocket, thereby blocking access to the active site of ATX. The potencies of compounds 1-3 were comparable to each other in each of the assays. All of these compounds significantly reduced invasion of A2058 human melanoma cells in vitro and the colonization of lung metastases by B16-F10 murine melanoma cells in C57BL/6 mice. The compounds had no agonist or antagonist effects on select LPA or sphingosine 1-phosphate receptors, nor did they inhibit nucleotide pyrophosphatase/phosphodiesterase (NPP) enzymes NPP6 and NPP7. These results identify the molecular surface of the hydrophobic pocket of ATX as a target-binding site for inhibitors of enzymatic activity.

Virtual screening for LPA2-specific agonists identifies a nonlipid compound with antiapoptotic actions.

Lysophosphatidic acid (LPA) is a highly potent endogenous lipid mediator that protects and rescues cells from programmed cell death. Earlier work identified the LPA2 G protein-coupled receptor subtype as an important molecular target of LPA mediating antiapoptotic signaling. Here we describe the results of a virtual screen using single-reference similarity searching that yielded compounds 2-((9-oxo-9H-fluoren-2-yl)carbamoyl)benzoic acid (NSC12404), 2-((3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)thio)benzoic acid (GRI977143), 4,5-dichloro-2-((9-oxo-9H-fluoren-2-yl)carbamoyl)benzoic acid (H2L5547924), and 2-((9,10-dioxo-9,10-dihydroanthracen-2-yl)carbamoyl) benzoic acid (H2L5828102), novel nonlipid and drug-like compounds that are specific for the LPA2 receptor subtype. We characterized the antiapoptotic action of one of these compounds, GRI977143, which was effective in reducing activation of caspases 3, 7, 8, and 9 and inhibited poly(ADP-ribose)polymerase 1 cleavage and DNA fragmentation in different extrinsic and intrinsic models of apoptosis in vitro. Furthermore, GRI977143 promoted carcinoma cell invasion of human umbilical vein endothelial cell monolayers and fibroblast proliferation. The antiapoptotic cellular signaling responses were present selectively in mouse embryonic fibroblast cells derived from LPA(1&2) double-knockout mice reconstituted with the LPA2 receptor and were absent in vector-transduced control cells. GRI977143 was an effective stimulator of extracellular signal-regulated kinase 1/2 activation and promoted the assembly of a macromolecular signaling complex consisting of LPA2, Na⁺ - H⁺ exchange regulatory factor 2, and thyroid receptor interacting protein 6, which has been shown previously to be a required step in LPA-induced antiapoptotic signaling. The present findings indicate that nonlipid LPA2-specific agonists represent an excellent starting point for development of lead compounds with potential therapeutic utility for preventing the programmed cell death involved in many types of degenerative and inflammatory diseases.

Controlling cancer through the autotaxin-lysophosphatidic acid receptor axis.

LPA (lysophosphatidic acid, 1-acyl-2-hydroxy-sn-glycero-3-phosphate), is a growth factor-like lipid mediator that regulates many cellular functions, many of which are unique to malignantly transformed cells. The simple chemical structure of LPA and its profound effects in cancer cells has attracted the attention of the cancer therapeutics field and drives the development of therapeutics based on the LPA scaffold. In biological fluids, LPA is generated by ATX (autotaxin), a lysophospholipase D that cleaves the choline/serine headgroup from lysophosphatidylcholine and lysophosphatidylserine to generate LPA. In the present article, we review some of the key findings that make the ATX-LPA signalling axis an emerging target for cancer therapy.

Discovery of Ethyl Ketone-Based Highly Selective HDACs 1, 2, 3 Inhibitors for HIV Latency Reactivation with Minimum Cellular Potency Serum Shift and Reduced hERG Activity.

We describe the discovery of histone deacetylase (HDACs) 1, 2, and 3 inhibitors with ethyl ketone as the zinc-binding group. These HDACs 1, 2, and 3 inhibitors have good enzymatic and cellular activity. Their serum shift in cellular potency has been minimized, and selectivity against hERG has been improved. They are also highly selective over HDACs 6 and 8. These inhibitors contain a variety of substituted heterocycles on the imidazole or oxazole scaffold. Compounds 31 and 48 stand out due to their good potency, high selectivity over HDACs 6 and 8, reduced hERG activity, optimized serum shift in cellular potency, and good rat and dog PK profiles.

Unique ligand selectivity of the GPR92/LPA5 lysophosphatidate receptor indicates role in human platelet activation.

Lysophosphatidic acid (LPA) is a ligand for LPA(1-3) of the endothelial differentiation gene family G-protein-coupled receptors, and LPA(4-8) is related to the purinergic family G-protein-coupled receptor. Because the structure-activity relationship (SAR) of GPR92/LPA(5) is limited and whether LPA is its preferred endogenous ligand has been questioned in the literature, in this study we applied a combination of computational and experimental site-directed mutagenesis of LPA(5) residues predicted to interact with the headgroup of LPA. Four residues involved in ligand recognition in LPA(5) were identified as follows: R2.60N mutant abolished receptor activation, whereas H4.64E, R6.62A, and R7.32A greatly reduced receptor activation. We also investigated the SAR of LPA(5) using LPA analogs and other non-lysophospholipid ligands. SAR revealed that the rank order of agonists is alkyl glycerol phosphate > LPA > farnesyl phosphates >> N-arachidonoylglycine. These results confirm LPA(5) to be a bona fide lysophospholipid receptor. We also evaluated several compounds with previously established selectivity for the endothelial differentiation gene receptors and found several that are LPA(5) agonists. A pharmacophore model of LPA(5) binding requirements was developed for in silico screening, which identified two non-lipid LPA(5) antagonists. Because LPA(5) transcripts are abundant in human platelets, we tested its antagonists on platelet activation and found that these non-lipid LPA(5) antagonists inhibit platelet activation. The present results suggest that selective inhibition of LPA(5) may provide a basis for future anti-thrombotic therapies.

Selective Class I HDAC Inhibitors Based on Aryl Ketone Zinc Binding Induce HIV-1 Protein for Clearance.

HIV persistence in latently infected, resting CD4+ T cells is broadly considered a barrier to eradicate HIV. Activation of the provirus using latency-reversing agents (LRAs) followed by immune-mediated clearance to purge reservoirs has been touted as a promising therapeutic approach. Histone deacetylases (HDACs) and histone acetyltransferases (HATs) control the acetylation level of lysine residues in histones to regulate the gene transcription. Several clinical HDAC inhibitors had been examined as LRAs, which induced HIV activation in vitro and in vivo. Here we report the discovery of a series of selective and potent class I HDAC inhibitors based on aryl ketones as a zinc binding group, which reversed HIV latency using a Jurkat model of HIV latency in 2C4 cells. The SAR led to the discovery of a highly selective class I HDAC inhibitor 10 with excellent potency. HDACi 10 induces the HIV gag P24 protein in patient latent CD4+ T cells.

Structure-based drug design identifies novel LPA3 antagonists.

Compound 5 ([5-(3-nitrophenoxy)-1,3-dioxo-1,3-dihydro-2-isoindol-2-yl]acetic acid) was identified as a weak selective LPA(3) antagonist (IC(50)=4504 nM) in a virtual screening effort to optimize a dual LPA(2 and 3) antagonist. Structure-based drug design techniques were used to prioritize similarity search matches of compound 5. This strategy rapidly identified 10 novel antagonists. The two most efficacious compounds identified inhibit activation of the LPA(3) receptor by 200 nM LPA with IC(50) values of 752 nM and 2992 nM. These compounds additionally define changes to our previously reported pharmacophore that will improve its ability to identify more potent and selective LPA(3) receptor antagonists. The results of the combined computational and experimental screening are reported.

Molecular recognition in the sphingosine 1-phosphate receptor family.

Computational modeling and its application in ligand screening and ligand receptor interaction studies play important roles in structure-based drug design. A series of sphingosine 1-phosphate (S1P) receptor ligands with varying potencies and receptor selectivities were docked into homology models of the S1P(1-5) receptors. These studies provided molecular insights into pharmacological trends both across the receptor family as well as at single receptors. This study identifies ligand recognition features that generalize across the S1P receptor family, features unique to the S1P(4) and S1P(5) receptors, and suggests significant structural differences of the S1P(2) receptor. Docking results reveal a previously unknown sulfur-aromatic interaction between the S1P(4) C5.44 sulfur atom and the phenyl ring of benzimidazole as well as pi-pi interaction between F3.33 of S1P(1,4,5) and aromatic ligands. The findings not only confirm the importance of a cation-pi interaction between W4.64 and the ammonium of S1P at S1P(4) but also predict the same interaction at S1P(5). S1P receptor models are validated for pharmacophore development including database mining and new ligand discovery and serve as tools for ligand optimization to improve potency and selectivity.

Identification of non-lipid LPA3 antagonists by virtual screening.

In the present study, we utilized virtual screening to identify LPA(3) antagonists. We have developed a three-point structure-based pharmacophore model based on known LPA(3) antagonists. This model was used to mine the NCI database. Docking, pharmacophore development, and database mining produced new, non-lipid leads. Experimental testing of seven computationally selected pharmacophore hits produced one potentiator and three antagonists, one of which displays both LPA(3) selectivity and nanomolar potency. Similarity searching in the ChemBridge database using the most promising lead as the search target produced four additional LPA(3) antagonists and a potent dual LPA(1&2) antagonist.