New Classes of Alanine Racemase Inhibitors Identified by High-Throughput Screening Show Antimicrobial Activity against Mycobacterium tuberculosis

In an effort to discover new drugs to treat tuberculosis (TB) we chose alanine racemase as the target of our drug discovery efforts. In Mycobacterium tuberculosis, the causative agent of TB, alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan. Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity. Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target.To obtain non-substrate alanine racemase inhibitors, we developed a high-throughput screening platform and screened 53,000 small molecule compounds for enzyme-specific inhibitors. We examined the 'hits' for structural novelty, antimicrobial activity against M. tuberculosis, general cellular cytotoxicity, and mechanism of enzyme inhibition. We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors. Seven of these are active against M. tuberculosis and minimally cytotoxic against mammalian cells.This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Improved risk stratification of patients with atrial fibrillation: an integrated GARFIELD-AF tool for the prediction of mortality, stroke and bleed in patients with and without anticoagulation.

OBJECTIVES: To provide an accurate, web-based tool for stratifying patients with atrial fibrillation to facilitate decisions on the potential benefits/risks of anticoagulation, based on mortality, stroke and bleeding risks. DESIGN: The new tool was developed, using stepwise regression, for all and then applied to lower risk patients. C-statistics were compared with CHA2DS2-VASc using 30-fold cross-validation to control for overfitting. External validation was undertaken in an independent dataset, Outcome Registry for Better Informed Treatment of Atrial Fibrillation (ORBIT-AF). PARTICIPANTS: Data from 39 898 patients enrolled in the prospective GARFIELD-AF registry provided the basis for deriving and validating an integrated risk tool to predict stroke risk, mortality and bleeding risk. RESULTS: The discriminatory value of the GARFIELD-AF risk model was superior to CHA2DS2-VASc for patients with or without anticoagulation. C-statistics (95% CI) for all-cause mortality, ischaemic stroke/systemic embolism and haemorrhagic stroke/major bleeding (treated patients) were: 0.77 (0.76 to 0.78), 0.69 (0.67 to 0.71) and 0.66 (0.62 to 0.69), respectively, for the GARFIELD-AF risk models, and 0.66 (0.64-0.67), 0.64 (0.61-0.66) and 0.64 (0.61-0.68), respectively, for CHA2DS2-VASc (or HAS-BLED for bleeding). In very low to low risk patients (CHA2DS2-VASc 0 or 1 (men) and 1 or 2 (women)), the CHA2DS2-VASc and HAS-BLED (for bleeding) scores offered weak discriminatory value for mortality, stroke/systemic embolism and major bleeding. C-statistics for the GARFIELD-AF risk tool were 0.69 (0.64 to 0.75), 0.65 (0.56 to 0.73) and 0.60 (0.47 to 0.73) for each end point, respectively, versus 0.50 (0.45 to 0.55), 0.59 (0.50 to 0.67) and 0.55 (0.53 to 0.56) for CHA2DS2-VASc (or HAS-BLED for bleeding). Upon validation in the ORBIT-AF population, C-statistics showed that the GARFIELD-AF risk tool was effective for predicting 1-year all-cause mortality using the full and simplified model for all-cause mortality: C-statistics 0.75 (0.73 to 0.77) and 0.75 (0.73 to 0.77), respectively, and for predicting for any stroke or systemic embolism over 1 year, C-statistics 0.68 (0.62 to 0.74). CONCLUSIONS: Performance of the GARFIELD-AF risk tool was superior to CHA2DS2-VASc in predicting stroke and mortality and superior to HAS-BLED for bleeding, overall and in lower risk patients. The GARFIELD-AF tool has the potential for incorporation in routine electronic systems, and for the first time, permits simultaneous evaluation of ischaemic stroke, mortality and bleeding risks. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier for GARFIELD-AF (NCT01090362) and for ORBIT-AF (NCT01165710)

Two-year outcomes of patients with newly diagnosed atrial fibrillation: results from GARFIELD-AF.

AIMS: The relationship between outcomes and time after diagnosis for patients with non-valvular atrial fibrillation (NVAF) is poorly defined, especially beyond the first year. METHODS AND RESULTS: GARFIELD-AF is an ongoing, global observational study of adults with newly diagnosed NVAF. Two-year outcomes of 17 162 patients prospectively enrolled in GARFIELD-AF were analysed in light of baseline characteristics, risk profiles for stroke/systemic embolism (SE), and antithrombotic therapy. The mean (standard deviation) age was 69.8 (11.4) years, 43.8% were women, and the mean CHA2DS2-VASc score was 3.3 (1.6); 60.8% of patients were prescribed anticoagulant therapy with/without antiplatelet (AP) therapy, 27.4% AP monotherapy, and 11.8% no antithrombotic therapy. At 2-year follow-up, all-cause mortality, stroke/SE, and major bleeding had occurred at a rate (95% confidence interval) of 3.83 (3.62; 4.05), 1.25 (1.13; 1.38), and 0.70 (0.62; 0.81) per 100 person-years, respectively. Rates for all three major events were highest during the first 4 months. Congestive heart failure, acute coronary syndromes, sudden/unwitnessed death, malignancy, respiratory failure, and infection/sepsis accounted for 65% of all known causes of death and strokes for <10%. Anticoagulant treatment was associated with a 35% lower risk of death. CONCLUSION: The most frequent of the three major outcome measures was death, whose most common causes are not known to be significantly influenced by anticoagulation. This suggests that a more comprehensive approach to the management of NVAF may be needed to improve outcome. This could include, in addition to anticoagulation, interventions targeting modifiable, cause-specific risk factors for death. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Phase Ib dose-finding trial of lapatinib plus pegylated liposomal doxorubicin in advanced HER2-positive breast cancer

Combination of anthracyclines with trastuzumab is hampered by cardiotoxicity. Pegylated liposomal doxorubicin and lapatinib could represent a safer alternative to combination therapy.In this phase Ib study with 3 + 3 dose escalation design, patients with HER2-positive advanced breast cancer received pegylated liposomal doxorubicin 30 mg/m(2) intravenously on day 1 plus lapatinib 1250 (level 1) or 1500 (level 2) mg/day orally on days 1-21 of each 21-day cycle. The aims were to establish the maximum tolerated dose at first cycle, and the activity and safety of multiple cycles.Nine patients out of 11 enrolled were evaluable: 3 at level 1 and 6 at level 2. No dose-limiting toxicities occurred at dose level 1, while 1 (grade 3 diarrhea) occurred at dose level 2, leading to the expansion of this cohort to 6 patients, with no further dose-limiting toxicities. Main grade 1-2 toxicities at first cycle were leucopenia, diarrhea, elevated transaminases, mucositis. Three patients had grade 3 toxicities at subsequent cycles, including colitis, anorexia, stomatitis plus hand-foot syndrome. One partial response, 5 disease stabilizations, and 3 disease progressions were reported.Combination of pegylated liposomal doxorubicin and lapatinib is feasible and potentially active in pretreated HER2-positive advanced breast cancer patients.NCT02131506 (ClinicalTrials.gov identifier)

Characterization of Ixophilin, a thrombin inhibitor from the gut of Ixodes scapularis.

Ixodes scapularis, the black-legged tick, vectors several human pathogens including Borrelia burgdorferi, the agent of Lyme disease in North America. Pathogen transmission to the vertebrate host occurs when infected ticks feed on the mammalian host to obtain a blood meal. Efforts to understand how the tick confronts host hemostatic mechanisms and imbibes a fluid blood meal have largely focused on the anticoagulation strategies of tick saliva. The blood meal that enters the tick gut remains in a fluid state for several days during the process of feeding, and the role of the tick gut in maintaining the blood-meal fluid is not understood. We now demonstrate that the tick gut produces a potent inhibitor of thrombin, a key enzyme in the mammalian coagulation cascade. Chromatographic fractionation of engorged tick gut proteins identified one predominant thrombin inhibitory activity associated with an approximately 18 kDa protein, henceforth referred to as Ixophilin. The ixophilin gene was preferentially transcribed in the guts of feeding nymphs. Expression began after 24 hours of feeding, coincident with the flow of host blood into the tick gut. Immunity against Ixophilin delayed tick feeding, and decreased feeding efficiency significantly. Surprisingly, immunity against Ixophilin resulted in increased Borrelia burgdorferi transmission to the host, possibly due to delayed feeding and increased transmission opportunity. These observations illuminate the potential drawbacks of targeting individual tick proteins in a functional suite. They also underscore the need to identify the "anticoagulome" of the tick gut, and to prioritize a critical subset of anticoagulants that could be targeted to efficiently thwart tick feeding, and block pathogen transmission to the vertebrate host

Expression, purification, crystallization and preliminary X-ray analysis of a truncated soluble domain of human glioma pathogenesis-related protein 1

The expression, purification, crystallization and preliminary X-ray diffraction analysis of the soluble domain of human glioma pathogenesis-related protein 1 (sGLIPR1) is presented

CD169 + macrophages orchestrate innate immune responses by regulating bacterial localization in the spleen

The spleen is an important site for generating protective immune responses against pathogens. After infection, immune cells undergo rapid reorganization to initiate and maintain localized inflammatory responses; however, the mechanisms governing this spatial and temporal cellular reorganization remain unclear. We show that the strategic position of splenic marginal zone CD169+ macrophages is vital for rapid initiation of antibacterial responses. In addition to controlling initial bacterial growth, CD169+ macrophages orchestrate a second phase of innate protection by mediating the transport of bacteria to splenic T cell zones. This compartmentalization of bacteria within the spleen was essential for driving the reorganization of innate immune cells into hierarchical clusters and for local interferon-γ production near sites of bacterial replication foci. Our results show that both phases of the antimicrobial innate immune response were dependent on CD169+ macrophages, and, in their absence, the series of events needed for pathogen clearance and subsequent survival of the host was disrupted. Our study provides insight into how lymphoid organ structure and function are related at a fundamental level.Fil: Perez, Oriana A.. University of Connecticut; Estados UnidosFil: Yeung, Stephen T.. University of Connecticut; Estados UnidosFil: Vera Licona, Paola. University of Connecticut; Estados UnidosFil: Romagnoli, Pablo Alberto. University of Connecticut; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Samji, Tasleem. University of Connecticut; Estados UnidosFil: Ural, Basak B.. University of Connecticut; Estados UnidosFil: Maher, Leigh. University of Connecticut; Estados UnidosFil: Tanaka, Masato. Tokyo University of Pharmacy and Life Sciences; JapónFil: Khanna, Kamal M.. University of Connecticut; Estados Unido