Interventions Associated With Prospective Pharmacist Evaluation of Scheduled Admissions for Dofetilide Initiation.

Background: Initiation of dofetilide requires hospital admission because of its proarrhythmic risk. To reduce the risk of adverse events associated with dofetilide, our institution has a standard operating protocol for dofetilide initiation. Regardless, patients are sometimes admitted for dofetilide initiation with unaddressed pharmacotherapy concerns that may delay therapy initiation and/or increase the risk for adverse events. Objective: To characterize interventions associated with pharmacist evaluation of scheduled dofetilide admissions prior to hospitalization. Methods: Patients scheduled for dofetilide initiation were evaluated by a pharmacist prior to admission. Identified interventions were categorized into the following recommendations: (1) against the use of dofetilide; (2) dofetilide starting dose adjustment; (3) appropriate washout of previous antiarrhythmic drug; (4) transesophageal echocardiogram prior to dofetilide initiation; (5) discontinuation or dose adjustment of interacting drug; (6) electrolyte supplementation upon discharge; (7) other intervention. The primary outcome measure was the frequency and types of identified and accepted interventions. Results: Twenty-two patients were evaluated during the 9-month study period. Fourteen interventions were identified, 13 of which were accepted by an electrophysiology provider. The most common intervention was for recommendation of a transesophageal echocardiogram prior to initiating dofetilide because of inadequate oral anticoagulation (n = 6). Other accepted interventions were for discontinuation or dose adjustment of interacting drug (n = 3), dofetilide starting dose adjustment (n = 2), electrolyte supplementation upon discharge (n = 2), and remeasurement of interventricular septal wall thickness (n = 1). Conclusion: Pharmacist evaluation of scheduled dofetilide admissions prior to hospitalization can serve to identify and resolve pharmacotherapy concerns related to dofetilide use.

Discovery of the SMYD3 Inhibitor BAY-6035 Using Thermal Shift Assay (TSA)-Based High-Throughput Screening.

SMYD3 (SET and MYND domain-containing protein 3) is a protein lysine methyltransferase that was initially described as an H3K4 methyltransferase involved in transcriptional regulation. SMYD3 has been reported to methylate and regulate several nonhistone proteins relevant to cancer, including mitogen-activated protein kinase kinase kinase 2 (MAP3K2), vascular endothelial growth factor receptor 1 (VEGFR1), and the human epidermal growth factor receptor 2 (HER2). In addition, overexpression of SMYD3 has been linked to poor prognosis in certain cancers, suggesting SMYD3 as a potential oncogene and attractive cancer drug target. Here we report the discovery of a novel SMYD3 inhibitor. We performed a thermal shift assay (TSA)-based high-throughput screening (HTS) with 410,000 compounds and identified a novel benzodiazepine-based SMYD3 inhibitor series. Crystal structures revealed that this series binds to the substrate binding site and occupies the hydrophobic lysine binding pocket via an unprecedented hydrogen bonding pattern. Biochemical assays showed substrate competitive behavior. Following optimization and extensive biophysical validation with surface plasmon resonance (SPR) analysis and isothermal titration calorimetry (ITC), we identified BAY-6035, which shows nanomolar potency and selectivity against kinases and other PKMTs. Furthermore, BAY-6035 specifically inhibits methylation of MAP3K2 by SMYD3 in a cellular mechanistic assay with an IC50 <100 nM. Moreover, we describe a congeneric negative control to BAY-6035. In summary, BAY-6035 is a novel selective and potent SMYD3 inhibitor probe that will foster the exploration of the biological role of SMYD3 in diseased and nondiseased tissues.

Implementation and evaluation of depression screening in patients with recently diagnosed coronary artery disease.

INTRODUCTION: Patients with coronary artery disease (CAD) are at an increased risk for depression. Additionally, comorbid depression in patients with CAD is associated with increased mortality and worse cardiac outcomes. Screening this patient population for depression is recommended but is not routinely done in practice. The purpose of this quality improvement initiative was to implement a protocol to screen patients with CAD for depression using the Patient Health Questionnaire (PHQ-9). Primary objectives were to determine the frequency of positive depression screens and the frequency of acceptance of mental health (MH) service referral. METHODS: Patients with CAD were screened for depression using the PHQ-9 during a hospital admission to the inpatient cardiology unit at the Clement J. Zablocki Veterans Affairs Medical Center. All patients were rescreened for depression at 4 and 8 weeks after discharge. Patients with positive screens for depression were offered referral for MH services, and reasons for decline were documented. RESULTS: Of the 36 patients screened for depression, 14 (39%) screened positive for depression, including 10 patients at baseline (28%), 3 additional patients (8%) at week 4 after discharge, and 1 additional patient (3%) at week 8 after discharge. Of the 14 patients who screened positive for depression, 3 patients (21%) accepted MH service referral. The most commonly reported reason for declining referral was no perceived benefit. DISCUSSION: The results of this initiative support the utility of using the PHQ-9 for depression screening in patients with recently diagnosed CAD and offering MH service referral for treatment of comorbid depression.

Selective, Small-Molecule Co-Factor Binding Site Inhibition of a Su(var)3-9, Enhancer of Zeste, Trithorax Domain Containing Lysine Methyltransferase.

The first chemical probe to primarily occupy the co-factor binding site of a Su(var)3-9, enhancer of a zeste, trithorax (SET) domain containing protein lysine methyltransferase (PKMT) is reported. Protein methyltransferases require S-adenosylmethionine (SAM) as a co-factor (methyl donor) for enzymatic activity. However, SAM itself represents a poor medicinal chemistry starting point for a selective, cell-active inhibitor given its extreme physicochemical properties and its role in multiple cellular processes. A previously untested medicinal chemistry strategy of deliberate file enrichment around molecules bearing the hallmarks of SAM, but with improved lead-like properties from the outset, yielded viable hits against SET and MYND domain-containing protein 2 (SMYD2) that were shown to bind in the co-factor site. These leads were optimized to identify a highly biochemically potent, PKMT-selective, and cell-active chemical probe. While substrate-based inhibitors of PKMTs are known, this represents a novel, co-factor-derived strategy for the inhibition of SMYD2 which may also prove applicable to lysine methyltransferase family members previously thought of as intractable.

Identification and characterization of the first fragment hits for SETDB1 Tudor domain.

SET domain bifurcated protein 1 (SETDB1) is a human histone-lysine methyltransferase which is amplified in human cancers and was shown to be crucial in the growth of non-small and small cell lung carcinoma. In addition to its catalytic domain, SETDB1 harbors a unique tandem tudor domain which recognizes histone sequences containing both methylated and acetylated lysines, and likely contributes to its localization on chromatin. Using X-ray crystallography and NMR spectroscopy fragment screening approaches, we have identified the first small molecule fragment hits that bind to histone peptide binding groove of the Tandem Tudor Domain (TTD) of SETDB1. Herein, we describe the binding modes of these fragments and analogues and the biophysical characterization of key compounds. These confirmed small molecule fragments will inform the development of potent antagonists of SETDB1 interaction with histones.

Fragment-based discovery of a chemical probe for the PWWP1 domain of NSD3.

Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains.

Evaluation of the Impact of an Institution-Specific Dofetilide Initiation Protocol on Mean Hospital Length of Stay and Cost for Dofetilide Initiation.

BACKGROUND: Dofetilide is a class III antiarrhythmic drug indicated for the conversion and maintenance of normal sinus rhythm in symptomatic patients with atrial fibrillation/atrial flutter. Delay in initiation of dofetilide therapy may increase the duration of hospitalization from two nights to three nights to complete US Food and Drug Administration-required monitoring. Therefore, substantial cost savings may be associated with implementation of an institution-specific dofetilide initiation protocol in order to reduce hospitalization to two nights. This could potentially be achieved through protocol-defined utilization of the option for a condensed dosing interval for the first three doses of dofetilide in order to ensure the administration of two doses on the first day of hospitalization. OBJECTIVE: The primary objective of this study was to assess the impact of an institution-specific dofetilide initiation protocol on mean hospital length of stay and cost for dofetilide initiation. METHODS: The study design was a retrospective review of 150 patients admitted to the Clement J. Zablocki Veterans Affairs Medical Center for the purpose of dofetilide initiation. Matching time periods of 18 months before and after implementation of the institution-specific dofetilide initiation protocol were used to randomly select 75 patients from each time period for comparison. RESULTS: A significant reduction in mean hospital length of stay of 0.56 nights post-implementation of the institution-specific dofetilide initiation protocol was identified (95% confidence interval 0.20-0.92; P = 0.0029). Assuming hospital adjusted expenses per inpatient day of US$1831-2413, a reduction in hospital length of stay of 0.56 nights resulted in estimated cost savings of US$1025-1351 per admission for dofetilide initiation. CONCLUSION: Implementation of an institution-specific dofetilide initiation protocol decreases mean hospital length of stay and cost for dofetilide initiation.

Discovery of covalent enzyme inhibitors using virtual docking of covalent fragments.

Here we present a virtual docking screen of 1648 commercially available covalent fragments, and identified covalent inhibitors of cysteine protease cathepsin L. These inhibitors did not inhibit closely related protease cathepsin B. Thus, we have established virtual docking of covalent fragments as an approach to discover covalent enzyme inhibitors.

LLY-283, a Potent and Selective Inhibitor of Arginine Methyltransferase 5, PRMT5, with Antitumor Activity.

Protein arginine methyltransferase 5 (PRMT5) is a type II arginine methyltransferase that catalyzes the formation of symmetric dimethylarginine in a number of nuclear and cytoplasmic proteins. Although the cellular functions of PRMT5 have not been fully unraveled, it has been implicated in a number of cellular processes like RNA processing, signal transduction, and transcriptional regulation. PRMT5 is ubiquitously expressed in most tissues and its expression has been shown to be elevated in several cancers including breast cancer, gastric cancer, glioblastoma, and lymphoma. Here, we describe the identification and characterization of a novel and selective PRMT5 inhibitor with potent in vitro and in vivo activity. Compound 1 (also called LLY-283) inhibited PRMT5 enzymatic activity in vitro and in cells with IC50 of 22 ± 3 and 25 ± 1 nM, respectively, while its diastereomer, compound 2 (also called LLY-284), was much less active. Compound 1 also showed antitumor activity in mouse xenografts when dosed orally and can serve as an excellent probe molecule for understanding the biological function of PRMT5 in normal and cancer cells.

Identification of Novel Mycobacterial Targets for Murine CD4+ T-Cells by IFNγ ELISPOT.

Enzyme-linked immunospot (ELISPOT) is an assay used to detect secretion of cytokines from immune cells. The resolution and sensitivity of ELISPOT allow for the detection of rare T cell specificities and small quantities of molecules produced by individual cells. In this chapter, we describe an epitope screening method that uses CD4+ T cell ELISPOT assays to identify specific novel mycobacterial antigens as potential vaccine candidates. In order to screen a large number of candidate epitopes simultaneously, pools of predicted MHC class II peptides were used to identify mycobacterial specific CD4+ T cells. Using this method, we identified novel mycobacterial antigens as vaccine candidates.

Identification of Mycobacterial Ribosomal Proteins as Targets for CD4+ T Cells That Enhance Protective Immunity in Tuberculosis.

Mycobacterium tuberculosis remains a threat to global health, and a more efficacious vaccine is needed to prevent disease caused by M. tuberculosis We previously reported that the mycobacterial ribosome is a major target of CD4+ T cells in mice immunized with a genetically modified Mycobacterium smegmatis strain (IKEPLUS) but not in mice immunized with Mycobacterium bovis BCG. Two specific ribosomal proteins, RplJ and RpsA, were identified as cross-reactive targets of M. tuberculosis, but the breadth of the CD4+ T cell response to M. tuberculosis ribosomes was not determined. In the present study, a library of M. tuberculosis ribosomal proteins and in silico-predicted peptide libraries were used to screen CD4+ T cell responses in IKEPLUS-immunized mice. This identified 24 out of 57 M. tuberculosis ribosomal proteins distributed over both large and small ribosome subunits as specific CD4+ T cell targets. Although BCG did not induce detectable responses against ribosomal proteins or peptide epitopes, the M. tuberculosis ribosomal protein RplJ produced a robust and multifunctional Th1-like CD4+ T cell population when administered as a booster vaccine to previously BCG-primed mice. Boosting of BCG-primed immunity with the M. tuberculosis RplJ protein led to significantly reduced lung pathology compared to that in BCG-immunized animals and reductions in the bacterial burdens in the mediastinal lymph node compared to those in naive and standard BCG-vaccinated mice. These results identify the mycobacterial ribosome as a potential source of cryptic or subdominant antigenic targets of protective CD4+ T cell responses and suggest that supplementing BCG with ribosomal antigens may enhance protective vaccination against M. tuberculosis.

The EED protein-protein interaction inhibitor A-395 inactivates the PRC2 complex.

Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed in vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein-protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.

Identification of Mycobacterial RplJ/L10 and RpsA/S1 Proteins as Novel Targets for CD4+ T Cells.

Tuberculosis (TB) due to Mycobacterium tuberculosis remains a major global infectious disease problem, and a more efficacious vaccine is urgently needed for the control and prevention of disease caused by this organism. We previously reported that a genetically modified strain of Mycobacterium smegmatis called IKEPLUS is a promising TB vaccine candidate. Since protective immunity induced by IKEPLUS is dependent on antigen-specific CD4+ T cell memory, we hypothesized that the specificity of the CD4+ T cell response was a critical feature of this protection. Using in vitro assays of interferon gamma production (enzyme-linked immunosorbent spot [ELISPOT] assays) by splenocytes from IKEPLUS-immunized C57BL/6J mice, we identified an immunogenic peptide within the mycobacterial ribosomal large subunit protein RplJ, encoded by the Rv0651 gene. In a complementary approach, we generated major histocompatibility complex (MHC) class II-restricted T cell hybridomas from IKEPLUS-immunized mice. Screening of these T cell hybridomas against IKEPLUS and ribosomes enriched from IKEPLUS suggested that the CD4+ T cell response in IKEPLUS-immunized mice was dominated by the recognition of multiple components of the mycobacterial ribosome. Importantly, CD4+ T cells specific for mycobacterial ribosomes accumulate to significant levels in the lungs of IKEPLUS-immunized mice following aerosol challenge with virulent M. tuberculosis, consistent with a role for these T cells in protective host immunity in TB. The identification of CD4+ T cell responses to defined ribosomal protein epitopes expands the range of antigenic targets for adaptive immune responses to M. tuberculosis and may help to inform the design of more effective vaccines against tuberculosis.

A Suite of Biochemical Assays for Screening RNA Methyltransferase BCDIN3D.

BCDIN3D is an RNA-methyltransferase that O-methylates the 5' phosphate of RNA and regulates microRNA maturation. To discover small-molecule inhibitors of BCDIN3D, a suite of biochemical assays was developed. A radiometric methyltransferase assay and fluorescence polarization-based S-adenosylmethionine and RNA displacement assays are described. In addition, differential scanning fluorimetry and surface plasmon resonance were used to characterize binding. These assays provide a comprehensive package for the development of small-molecule modulators of BCDIN3D activity.

Suppression of autophagy and antigen presentation by Mycobacterium tuberculosis PE_PGRS47.

Suppression of major histocompatibility complex (MHC) class II antigen presentation is believed to be among the major mechanisms used by Mycobacterium tuberculosis to escape protective host immune responses. Through a genome-wide screen for the genetic loci of M. tuberculosis that inhibit MHC class II-restricted antigen presentation by mycobacteria-infected dendritic cells, we identified the PE_PGRS47 protein as one of the responsible factors. Targeted disruption of the PE_PGRS47 (Rv2741) gene led to attenuated growth of M. tuberculosis in vitro and in vivo, and a PE_PGRS47 mutant showed enhanced MHC class II-restricted antigen presentation during in vivo infection of mice. Analysis of the effects of deletion or over-expression of PE_PGRS47 implicated this protein in the inhibition of autophagy in infected host phagocytes. Our findings identify PE_PGRS47 as a functionally relevant, non-redundant bacterial factor in the modulation of innate and adaptive immunity by M. tuberculosis, suggesting strategies for improving antigen presentation and the generation of protective immunity during vaccination or infection.

Steady-State Spectroscopic Analysis of Proton-Dependent Electron Transfer on Pyrazine-Appended Metal Dithiolenes [Ni(pdt)2], [Pd(pdt)2], and [Pt(pdt)2] (pdt = 2,3-Pyrazinedithiol).

We report the structural, electronic, and acid/base properties of a series of ML2 metal dithiolene complexes, where M = Ni, Pd, Pt and L = 2,3-pyrazinedithiol. These complexes are non-innocent and possess strong electronic coupling between ligands across the metal center. The electronic coupling can be readily quantified in the monoanionic mixed valence state using Marcus-Hush theory. Analysis of the intervalence charge transfer (IVCT) band reveals that that electronic coupling in the mixed valence state is 5800, 4500, and 5700 cm(-1) for the Ni, Pd, and Pt complexes, respectively. We then focus on their response to acid titration in the mixed valence state, which generates the asymmetrically protonated mixed valence mixed protonated state. For all three complexes, protonation results in severe attenuation of the electronic coupling, as measured by the IVCT band. We find nearly 5-fold decreases in electronic coupling for both Ni and Pt, while, for the Pd complex, the electronic coupling is reduced to the point that the IVCT band is no longer observable. We ascribe the reduction in electronic coupling to charge pinning induced by asymmetric protonation. The more severe reduction in coupling for the Pd complex is a result of greater energetic mismatch between ligand and metal orbitals, reflected in the smaller electronic coupling for the pure mixed valence state. This work demonstrates that the bridging metal center can be used to tune the electronic coupling in both the mixed valence and mixed valence mixed protonated states, as well as the magnitude of change of the electronic coupling that accompanies changes in protonation state.

Discovery of a Potent and Selective Coactivator Associated Arginine Methyltransferase 1 (CARM1) Inhibitor by Virtual Screening.

Protein arginine methyltransferases (PRMTs) represent an emerging target class in oncology and other disease areas. So far, the most successful strategy to identify PRMT inhibitors has been to screen large to medium-size chemical libraries. Attempts to develop PRMT inhibitors using receptor-based computational methods have met limited success. Here, using virtual screening approaches, we identify 11 CARM1 (PRMT4) inhibitors with ligand efficiencies ranging from 0.28 to 0.84. CARM1 selective hits were further validated by orthogonal methods. Two structure-based rounds of optimization produced 27 (SGC2085), a CARM1 inhibitor with an IC50 of 50 nM and more than hundred-fold selectivity over other PRMTs. These results indicate that virtual screening strategies can be successfully applied to Rossmann-fold protein methyltransferases.

Global Profiling of Acetyltransferase Feedback Regulation.

Lysine acetyltransferases (KATs) are key mediators of cell signaling. Methods capable of providing new insights into their regulation thus constitute an important goal. Here we report an optimized platform for profiling KAT-ligand interactions in complex proteomes using inhibitor-functionalized capture resins. This approach greatly expands the scope of KATs, KAT complexes, and CoA-dependent enzymes accessible to chemoproteomic methods. This enhanced profiling platform is then applied in the most comprehensive analysis to date of KAT inhibition by the feedback metabolite CoA. Our studies reveal that members of the KAT superfamily possess a spectrum of sensitivity to CoA and highlight NAT10 as a novel KAT that may be susceptible to metabolic feedback inhibition. This platform provides a powerful tool to define the potency and selectivity of reversible stimuli, such as small molecules and metabolites, that regulate KAT-dependent signaling.