Crystallographic and Computational Insights into Isoform-Selective Dynamics in Nitric Oxide Synthase.

In our efforts to develop inhibitors selective for neuronal nitric oxide synthase (nNOS) over endothelial nitric oxide synthase (eNOS), we found that nNOS can undergo conformational changes in response to inhibitor binding that does not readily occur in eNOS. One change involves movement of a conserved tyrosine, which hydrogen bonds to one of the heme propionates, but in the presence of an inhibitor, changes conformation, enabling part of the inhibitor to hydrogen bond with the heme propionate. This movement does not occur as readily in eNOS and may account for the reason why these inhibitors bind more tightly to nNOS. A second structural change occurs upon the binding of a second inhibitor molecule to nNOS, displacing the pterin cofactor. Binding of this second site inhibitor requires structural changes at the dimer interface, which also occurs more readily in nNOS than in eNOS. Here, we used a combination of crystallography, mutagenesis, and computational methods to better understand the structural basis for these differences in NOS inhibitor binding. Computational results show that a conserved tyrosine near the primary inhibitor binding site is anchored more tightly in eNOS than in nNOS, allowing for less flexibility of this residue. We also find that the inefficiency of eNOS to bind a second inhibitor molecule is likely due to the tighter dimer interface in eNOS compared with nNOS. This study provides a better understanding of how subtle structural differences in NOS isoforms can result in substantial dynamic differences that can be exploited in the development of isoform-selective inhibitors.

BCL6 BTB-specific inhibitor reversely represses T-cell activation, Tfh cells differentiation, and germinal center reaction in vivo.

Inhibition of the BCL6 BTB domain results in killing Diffuse Large B-cell Lymphoma (DLBL) cells, reducing the T-cell dependent germinal center (GC) reaction in mice, and reversing GC hyperplasia in nonhuman primates. The available BCL6 BTB-specific inhibitors are poorly water soluble, thus, limiting their absorption in vivo and our understanding of therapeutic strategy targeting GC. We synthesized a prodrug (AP-4-287) from a potent BCL6 BTB inhibitor (FX1) with improved aqueous solubility and pharmacokinetics (PK) in mice. We also evaluated its in vivo biological activity on humoral immune responses using the sheep red blood cells (SRBC)-vaccination mouse model. AP-4-287 had a significant higher aqueous solubility and was readily converted to FX1 in vivo after intraperitoneally (i.p.) administration, but a shorter half-life in vivo. Importantly, AP-4-287 treatment led to a reversible effect on (1) the reduction in the frequency of splenic Ki67+ CD4+ T cells, Tfh cells, and GC B cells; (2) lower GC formation following vaccination; and (3) a decrease in the titers of antigen-specific IgG and IgM antibodies. Our study advances the preclinical development of drug targeting BCL6 BTB domain for the treatment of diseases that are associated with abnormal BCL6 elevation.

Improving the solubility and antileukemia activity of Wnt/ß-catenin signaling inhibitors by disrupting molecular planarity.

Leukemia cells depend on the Wnt/ß-catenin signaling pathway for their growth. Pyrvinium, a known Wnt signaling inhibitor, has demonstrated promising efficacy in the treatment of the aggressive blast phase chronic myeloid leukemia (BP-CML). We previously developed potent inhibitors 1-2 for the Wnt/ß-catenin signaling pathway. However, the further application of these compounds as anti-leukemia agents is limited by their modest anti-leukemia activity in cells and poor aqueous solubility, due to the high molecular planarity of the chemical scaffold. Here, we reported our efforts in the synthesis and in vitro evaluation of 18 new compounds (4a-r) that have been designed to disrupt the molecular planarity of the chemical scaffold. Several compounds of the series showed significantly improved anti-leukemia activity and aqueous solubility. As a highlight, compounds 4c not only maintained excellent inhibitory potency (IC50 = 1.3 nM) for Wnt signaling but also demonstrated good anti-leukemia potency (IC50 = 0.9 µM) in the CML K562 cells. Moreover, compound 4c had an aqueous solubility of 5.9 µg/mL, which is over 50-fold enhanced compared to its parents 1-2.

Regioselective alkylation of 2,4-dihydroxybenzyaldehydes and 2,4-dihydroxyacetophenones.

We report a cesium bicarbonate-mediated alkylation of 2,4-dihydroxybenzyaldehyde and 2,4-dihydroxyacetophenone to generate 4-alkylated products in acetonitrile at 80 °C with excellent regioselectivity, up to 95% isolated yields, and broad substrate scope.

Repurposing Acitretin as an Antipseudomonal Agent Targeting the Pseudomonas aeruginosa Iron-Regulated Heme Oxygenase.

Iron is an essential micronutrient for the survival and virulence of the bacterial pathogen Pseudomonas aeruginosa. To overcome iron withholding and successfully colonize a host, P. aeruginosa uses a variety of mechanisms to acquire iron, including the secretion of high-affinity iron chelators (siderophores) or the uptake and utilization of heme. P. aeruginosa heme oxygenase (HemO) plays pivotal roles in heme sensing, uptake, and utilization and has emerged as a therapeutic target for the development of antipseudomonal agents. Using a high-throughput fluorescence quenching assay combined with minimum inhibitory concentration measurements, we screened the Selleck Bioactive collection of 2100 compounds and identified acitretin, a Food and Drug Administration-approved oral retinoid, as a potent and selective inhibitor of HemO. Acitretin binds to HemO with a KD value of 0.10 ± 0.02 µM and inhibits the growth of P. aeruginosa PAO1 with an IC50 of 70 ± 18 µg/mL. In addition, acitretin showed good selectivity for HemO, which uniquely generates BVIXß/δ, over human heme oxygenase (hHO1) and other BVIXα-producing homologues such as the heme oxygenases from Neisseria meningitidis (nmHO) and Acinetobacter baumannii (abHO). The binding of acitretin within the HemO active site was confirmed by 1H-15N heteronuclear single-quantum coherence nuclear magnetic resonance, and molecular modeling provided further insight into potential interactions of acitretin with residues specific for orienting heme in the ß/δ selective HemO. Moreover, at 20 µM, acitretin inhibited the enzymatic activity of HemO in P. aeruginosa cells by >60% and effectively blocked the ability of P. aeruginosa to sense and acquire heme as demonstrated in the ß-galactosidase transcriptional reporter assay.

GTP-binding inhibitors increase LRRK2-linked ubiquitination and Lewy body-like inclusions.

Parkinson's disease (PD) is one of the most common movement disorders with loss of dopaminergic neurons and the presence of Lewy bodies in certain brain areas. However, it is not clear how Lewy body (inclusion with protein aggregation) formation occurs. Mutations in leucine-rich repeat kinase 2 (LRRK2) can cause a genetic form of PD and contribute to sporadic PD with the typical Lewy body pathology. Here, we used our recently identified LRRK2 GTP-binding inhibitors as pharmacological probes to study the LRRK2-linked ubiquitination and protein aggregation. Pharmacological inhibition of GTP-binding by GTP-binding inhibitors (68 and Fx2149) increased LRRK2-linked ubiquitination predominantly via K27 linkage. Compound 68- or Fx2149 increased G2019S-LRRK2-linked ubiquitinated aggregates, which occurred through the atypical linkage types K27 and K63. Coexpression of K27R and K63R, which prevented ubiquitination via K27 and K63 linkages, reversed the effects of 68 and Fx2149. Moreover, 68 and Fx2149 also promoted G2019S-LRRK2-linked aggresome (Lewy body-like inclusion) formation via K27 and K63 linkages. These findings demonstrate that LRRK2 GTP-binding activity is critical in LRRK2-linked ubiquitination and aggregation formation. These studies provide novel insight into the LRRK2-linked Lewy body-like inclusion formation underlying PD pathogenesis.

BCL6 Inhibitor-Mediated Downregulation of Phosphorylated SAMHD1 and T Cell Activation Are Associated with Decreased HIV Infection and Reactivation.

Clearance of HIV-infected germinal center (GC) CD4+ follicular helper T cells (Tfh) after combination antiretroviral therapy (ART) is essential to an HIV cure. Blocking B cell lymphoma 6 (BCL6; the master transcription factor for Tfh cells) represses HIV infection of tonsillar CD4+ Tfh ex vivo, reduces GC formation, and limits immune activation in vivo We assessed the anti-HIV activity of a novel BCL6 inhibitor, FX1, in Tfh/non-Tfh CD4+ T cells and its impact on T cell activation and SAMHD1 phosphorylation (Thr592). FX1 repressed HIV-1 infection of peripheral CD4+ T cells and tonsillar Tfh/non-Tfh CD4+ T cells (P < 0.05) and total elongated and multispliced HIV-1 RNA production during the first round of viral life cycle (P < 0.01). Using purified circulating CD4+ T cells from uninfected donors, we demonstrate that FX1 treatment resulted in downregulation pSAMHD1 expression (P < 0.05) and T cell activation (HLA-DR, CD25, and Ki67; P < 0.01) ex vivo corresponding with inhibition of HIV-1 and HIV-2 replication. Ex vivo HIV-1 reactivation using purified peripheral CD4+ T cells from HIV-infected ART-suppressed donors was also blocked by FX1 treatment (P < 0.01). Our results indicate that BCL6 function contributes to Tfh/non-Tfh CD4+ T cell activation and cellular susceptibility to HIV infection. BCL6 inhibition represents a novel therapeutic strategy to potentiate HIV suppression in Tfh/non-Tfh CD4+ T cells without reactivation of latent virus.IMPORTANCE The expansion and accumulation of HIV-infected BCL6+ Tfh CD4+ T cells are thought to contribute to the persistence of viral reservoirs in infected subjects undergoing ART. Two mechanisms have been raised for the preferential retention of HIV within Tfh CD4+ T cells: (i) antiretroviral drugs have limited tissue distribution, resulting in insufficient tissue concentration and lower efficacy in controlling HIV replication in lymphoid tissues, and (ii) cytotoxic CD8+ T cells within lymphoid tissues express low levels of chemokine receptor (CXCR5), thus limiting their ability to enter the GCs to control/eliminate HIV-infected Tfh cells. Our results indicate that the BCL6 inhibitor FX1 can not only repress HIV infection of tonsillar Tfh ex vivo but also suppress HIV infection and reactivation in primary, non-Tfh CD4+ T cells. Our study provides a rationale for targeting BCL6 protein to extend ART-mediated reduction of persistent HIV and/or support strategies toward HIV remission beyond ART cessation.

BCL6 BTB-specific inhibition via FX1 treatment reduces Tfh cells and reverses lymphoid follicle hyperplasia in Indian rhesus macaque (Macaca mulatta).

BACKGROUND: The BTB domain of B-cell lymphoma 6 (BCL6) protein was identified as a therapeutic target for B-cell lymphoma. This study compared the pharmacokinetics (PK) of the BCL6 BTB inhibitor (FX1) between mice and macaques, as well as evaluating its lymphoid suppressive effect in uninfected macaques with lymphoid hyperplasia. MATERIALS AND METHODS: Eight uninfected adult Indian rhesus macaques (Macaca mulatta) were used in the study, four animals carrying lymphoid tissue hyperplasia. Plasma FX1 levels were measured by HPLC-MS/MS. Lymph node biopsies were used for H&E and immunohistochemistry staining, as well as mononuclear cell isolation for flow cytometry analysis. RESULTS: Inhibition of the BCL6 BTB domain with FX1 led to a reduction in the frequency of GC, Tfh CD4+ , and Tfh precursor cells, as well as resolving lymphoid hyperplasia, in rhesus macaques. CONCLUSIONS: B-cell lymphoma 6 inhibition may represent a novel strategy to reduce hyperplastic lymphoid B-cell follicles and decrease Tfh cells.

The effects of novel heme oxygenase inhibitors on the growth of Pseudomonas aeruginosa.

OBJECTIVES: This study was aimed to develop small molecule inhibitors of the P. aeruginosa heme oxygenase (pa-HemO) as potential treatment of infections caused by P. aeruginosa. METHODS: New compounds were designed based on the crystal structure of pa-HemO. The binding affinities (KD) were determined using intrinsic fluorescence quenching assays. The anti-microbial effects of the new compounds was evaluated by minimal inhibitory concentration 50% (MIC50). RESULTS: Eleven compounds were synthesized as potential pa-HemO inhibitors. New compounds demonstrated KD values ranging from 1.5 to 180 µM, and MIC50 values ranging from 26 to 260 µg/mL. The compounds had good affinity with HemO and promising anti-microbial effects on P. aeruginosa. CONCLUSIONS: The new inhibitors described herein can inhibit the growth of P. aeruginosa via the inhibition of pa-HemO. There may be broad prospects for HemO inhibitors to treat P. aeruginosa related infections.

Correction to: The Asp99-Arg188 salt bridge of the Pseudomonas aeruginosa HemO is critical in allowing conformational flexibility during catalysis.

In the original publication, fifth author's name was incorrectly published as Pierre Moenne-Loccoz.

The Asp99-Arg188 salt bridge of the Pseudomonas aeruginosa HemO is critical in allowing conformational flexibility during catalysis.

The P. aeruginosa iron-regulated heme oxygenase (HemO) is required within the host for the utilization of heme as an iron source. As iron is essential for survival and virulence, HemO represents a novel antimicrobial target. We recently characterized small molecule inhibitors that bind to an allosteric site distant from the heme pocket, and further proposed binding at this site disrupts a nearby salt bridge between D99 and R188. Herein, through a combination of site-directed mutagenesis and hydrogen-deuterium exchange mass spectrometry (HDX-MS), we determined that the disruption of the D99-R188 salt bridge leads to significant decrease in conformational flexibility within the distal and proximal helices that form the heme-binding site. The RR spectra of the resting state Fe(III) and reduced Fe(II)-deoxy heme-HemO D99A, R188A and D99/R188A complexes are virtually identical to those of wild-type HemO, indicating no significant change in the heme environment. Furthermore, mutation of D99 or R188 leads to a modest decrease in the stability of the Fe(II)-O2 heme complex. Despite this slight difference in Fe(II)-O2 stability, we observe complete loss of enzymatic activity. We conclude the loss of activity is a result of decreased conformational flexibility in helices previously shown to be critical in accommodating variation in the distal ligand and the resulting chemical intermediates generated during catalysis. Furthermore, this newly identified allosteric binding site on HemO represents a novel alternative drug-design strategy to that of competitive inhibition at the active site or via direct coordination of ligands to the heme iron.

Attenuated Accumulation of Novel Fluorine (19F)-Labeled Bile Acid Analogues in Gallbladders of Fibroblast Growth Factor-15 (FGF15)-Deficient Mice.

Our work has focused on defining the utility of fluorine (19F)-labeled bile acid analogues and magnetic resonance imaging (MRI) to identify altered bile acid transport in vivo. In the current study, we explored the ability of this approach to differentiate fibroblast growth factor-15 (FGF15)-deficient from wild-type (WT) mice, a potential diagnostic test for bile acid diarrhea, a commonly misdiagnosed disorder. FGF15 is the murine homologue of human FGF19, an intestinal hormone whose deficiency is an underappreciated cause of bile acid diarrhea. In a pilot and three subsequent pharmacokinetic studies, we treated mice with two 19F-labeled bile acid analogues, CA-lys-TFA and CA-sar-TFMA. After oral dosing, we quantified 19F-labeled bile acid analogue levels in the gallbladder, liver, small and large intestine, and plasma using liquid chromatography mass spectrometry (LC-MS/MS). Both 19F bile acid analogues concentrated in the gallbladders of FGF15-deficient and WT mice, attaining peak concentrations at approximately 8.5 h after oral dosing. However, analogue levels in gallbladders of FGF15-deficient mice were several-fold less compared to those in WT mice. Live-animal 19F MRI provided agreement with our LC-MS/MS-based measures; we detected robust CA-lys-TFA 19F signals in gallbladders of WT mice but no signals in FGF15-deficient mice. Our finding that 19F MRI differentiates FGF15-deficient from WT mice provides additional proof-of-concept for the development of 19F bile acid analogues and 19F MRI as a clinical test to diagnose bile acid diarrhea due to FGF19 deficiency and other disorders.

Incorporation of a Biguanide Scaffold Enhances Drug Uptake by Organic Cation Transporters 1 and 2.

Membrane transporters play a significant role in the transport of many endogenous and exogenous compounds. The knowledge of transporter substrate requirements has allowed further development of drugs that utilize them to ensure tissue permeation. In this study, we demonstrate that inclusion of a biguanide functionality can potentiate uptake by the organic cation transporters 1 and 2 (OCT1 and OCT2). We synthesized 18 pairs of structurally diverse compounds, each pair consisting of a parent amino compound and its biguanide analog; and then assessed their cellular uptake in HEK293 cells overexpressing human OCT1 or OCT2. Our results show that addition of the biguanide significantly improved OCT1- and OCT2-mediated transport for the majority of compounds. The biguanides also inhibited the uptake of prototypical substrates of both transporters, 1-methyl-4-phenylpyridinium (MPP+) and metformin. We found that molecular weight, molecular volume, Log D (pH 7.4), and accessible surface area were important determinants of OCT2 substrates, but none of these parameters was a significant factor for OCT1. More so, the inhibition of MPP+ uptake correlated linearly with that of metformin uptake for the tested biguanides in both cell lines. Taken together, we conclude that the inclusion of the biguanide scaffold in nonsubstrates of OCT1 and OCT2 increase their propensity to become substrates and inhibitors for these transporters.

Iodobenzene-Catalyzed Synthesis of Phenanthridinones via Oxidative C-H Amidation.

We report a novel synthesis of phenanthridinones from N-methoxybenzamides using an oxidative C-H amidation reaction at room temperature in open air with modest to excellent yields. This method demonstrated unprecedented substrate scope. In particular, it solved the long-standing challenge in the synthesis of phenanthridinones with sterically demanding substitutions.

One-pot sequential reaction to 2-substituted-phenanthridinones from N-methoxybenzamides.

The sequential use of a hypervalent iodine reagent leads to the one-pot synthesis of 2-bromo/chloro-phenanthridinones via an amidation of arenes followed by a regioselective halogenation reaction. These consecutive C-H functionalization reactions can be used efficiently to construct 2-substituted-phenanthridinones at room temperature with good to high yields. Application of the current method is highlighted by the concise synthesis of the natural product PJ34.

Novel LRRK2 GTP-binding inhibitors reduced degeneration in Parkinson's disease cell and mouse models.

Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause autosomal-dominant Parkinson's disease (PD) and contribute to sporadic PD. LRRK2 contains Guanosine-5'-triphosphate (GTP) binding, GTPase and kinase activities that have been implicated in the neuronal degeneration of PD pathogenesis, making LRRK2, a potential drug target. To date, there is no disease-modifying drug to slow the neuronal degeneration of PD and no published LRRK2 GTP domain inhibitor. Here, the biological functions of two novel GTP-binding inhibitors of LRRK2 were examined in PD cell and mouse models. Through a combination of computer-aided drug design (CADD) and LRRK2 bio-functional screens, two novel compounds, 68: and 70: , were shown to reduce LRRK2 GTP binding and to inhibit LRRK2 kinase activity in vitro and in cultured cell assays. Moreover, these two compounds attenuated neuronal degeneration in human SH-SY5Y neuroblastoma cells and mouse primary neurons expressing mutant LRRK2 variants. Although both compounds inhibited LRRK2 kinase activity and reduced neuronal degeneration, solubility problems with 70: prevented further testing in mice. Thus, only 68: was tested in a LRRK2-based lipopolysaccharide (LPS)-induced pre-inflammatory mouse model. 68: reduced LRRK2 GTP-binding activity and kinase activity in brains of LRRK2 transgenic mice after intraperitoneal injection. Moreover, LPS induced LRRK2 upregulation and microglia activation in mouse brains. These findings suggest that disruption of GTP binding to LRRK2 represents a potential novel therapeutic approach for PD intervention and that these novel GTP-binding inhibitors provide both tools and lead compounds for future drug development.

Aminoquinoline-assisted vinylic C-H arylation of unsubstituted acrylamide for the selective synthesis of Z olefins.

A method for Pd-catalyzed, aminoquinoline-directed arylation of vinylic C-H bonds with aryl iodides has been developed. This reaction represents a rare example of Pd-catalyzed vinylic C-H functionalization of unsubstituted acrylamide, allowing for the highly regio- and stereoselective preparation of Z-olefins. High tolerance to functional groups is observed with good yields and excellent selectivity. It offers a complementary synthetic method to traditional pathways for Z-olefins.

Inhibitor Bound Crystal Structures of Bacterial Nitric Oxide Synthase.

Nitric oxide generated by bacterial nitric oxide synthase (NOS) increases the susceptibility of Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis to oxidative stress, including antibiotic-induced oxidative stress. Not surprisingly, NOS inhibitors also improve the effectiveness of antimicrobials. Development of potent and selective bacterial NOS inhibitors is complicated by the high active site sequence and structural conservation shared with the mammalian NOS isoforms. To exploit bacterial NOS for the development of new therapeutics, recognition of alternative NOS surfaces and pharmacophores suitable for drug binding is required. Here, we report on a wide number of inhibitor-bound bacterial NOS crystal structures to identify several compounds that interact with surfaces unique to the bacterial NOS. Although binding studies indicate that these inhibitors weakly interact with the NOS active site, many of the inhibitors reported here provide a revised structural framework for the development of new antimicrobials that target bacterial NOS. In addition, mutagenesis studies reveal several key residues that unlock access to bacterial NOS surfaces that could provide the selectivity required to develop potent bacterial NOS inhibitors.

Cyclopropyl-containing positive allosteric modulators of metabotropic glutamate receptor subtype 5.

Positive allosteric modulators (PAMs) binding to the transmembrane (TM) domain of metabotropic glutamate receptor 5 (mGluR5) are promising therapeutic agents for psychiatric disorders and traumatic brain injury (TBI). Novel PAMs based on a trans-2-phenylcyclopropane amide scaffold have been designed and synthesized. Facilitating ligand design and allowing estimation of binding affinities to the mGluR5 TM domain was the novel computational strategy, site identification by ligand competitive saturation (SILCS). The potential protective activity of the new compounds was evaluated using nitric oxide (NO) production in BV2 microglial cell cultures treated with lipopolysaccharide (LPS), and the toxicity of the new compounds tested using a cell viability assay. One of the new compounds, 3a, indicated promising activity with potency of 30 µM, which is 4.5-fold more potent than its lead compound 3,3'-difluorobenzaldazine (DFB), and showed no detectable toxicity with concentrations as high as 1000 µM. Thus this compound represents a new lead for possible development as treatment for TBI and related neurodegenerative disorders.

Cu-mediated direct regioselective C-2 chlorination of indoles.

Cu-mediated C-2 chlorination of indoles was accomplished with copper(ii) chloride through the use of a directing pyrimidyl protection group. A highly regioselective manner can be achieved on a range of indole substrates with excellent functional group tolerance.