Discovery of Novel, Orally Bioavailable, Antileishmanial Compounds Using Phenotypic Screening

Leishmaniasis is a parasitic infection that afflicts approximately 12 million people worldwide. There are several limitations to the approved drug therapies for leishmaniasis, including moderate to severe toxicity, growing drug resistance, and the need for extended dosing. Moreover, miltefosine is currently the only orally available drug therapy for this infection. We addressed the pressing need for new therapies by pursuing a two-step phenotypic screen to discover novel, potent, and orally bioavailable antileishmanials. First, we conducted a high-throughput screen (HTS) of roughly 600,000 small molecules for growth inhibition against the promastigote form of the parasite life cycle using the nucleic acid binding dye SYBR Green I. This screen identified approximately 2,700 compounds that inhibited growth by over 65% at a single point concentration of 10 μM. We next used this 2700 compound focused library to identify compounds that were highly potent against the disease-causing intra-macrophage amastigote form and exhibited limited toxicity toward the host macrophages. This two-step screening strategy uncovered nine unique chemical scaffolds within our collection, including two previously described antileishmanials. We further profiled two of the novel compounds for in vitro absorption, distribution, metabolism, excretion, and in vivo pharmacokinetics. Both compounds proved orally bioavailable, affording plasma exposures above the half-maximal effective concentration (EC50) concentration for at least 12 hours. Both compounds were efficacious when administered orally in a murine model of cutaneous leishmaniasis. One of the two compounds exerted potent activity against trypanosomes, which are kinetoplastid parasites related to Leishmania species. Therefore, this compound could help control multiple parasitic diseases. The promising pharmacokinetic profile and significant in vivo efficacy observed from our HTS hits highlight the utility of our two-step phenotypic screening strategy and strongly suggest that medicinal chemistry optimization of these newly identified scaffolds will lead to promising candidates for an orally available anti-parasitic drug

On the Mechanism of Action of SJ-172550 in Inhibiting the Interaction of MDM4 and p53

SJ-172550 (1) was previously discovered in a biochemical high throughput screen for inhibitors of the interaction of MDMX and p53 and characterized as a reversible inhibitor (J. Biol. Chem. 2010; 285∶10786). Further study of the biochemical mode of action of 1 has shown that it acts through a complicated mechanism in which the compound forms a covalent but reversible complex with MDMX and locks MDMX into a conformation that is unable to bind p53. The relative stability of this complex is influenced by many factors including the reducing potential of the media, the presence of aggregates, and other factors that influence the conformational stability of the protein. This complex mechanism of action hinders the further development of compound 1 as a selective MDMX inhibitor

NALP3 inflammasome upregulation and CASP1 cleavage of the glucocorticoid receptor cause glucocorticoid resistance in leukemia cells

Glucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and resistance to glucocorticoids in leukemia cells confers poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the prednisolone sensitivity of primary leukemia cells from 444 patients newly diagnosed with ALL and found significantly higher expression of CASP1 (encoding caspase 1) and its activator NLRP3 in glucocorticoid-resistant leukemia cells, resulting from significantly lower somatic methylation of the CASP1 and NLRP3 promoters. Overexpression of CASP1 resulted in cleavage of the glucocorticoid receptor, diminished the glucocorticoid-induced transcriptional response and increased glucocorticoid resistance. Knockdown or inhibition of CASP1 significantly increased glucocorticoid receptor levels and mitigated glucocorticoid resistance in CASP1-overexpressing ALL. Our findings establish a new mechanism by which the NLRP3-CASP1 inflammasome modulates cellular levels of the glucocorticoid receptor and diminishes cell sensitivity to glucocorticoids. The broad impact on the glucocorticoid transcriptional response suggests that this mechanism could also modify glucocorticoid effects in other diseases

Combinatorial dapoxyl dye library and its application to site selective probe for human serum albumin

The combinatorial fluorescent dapoxyl dye library was prepared by both solution- and solid-phase synthesis, generating 80 unique dapoxyl derivatives. A fluorescence-based screening toward human serum albumin (HSA) found one highly sensitive HSA binder (A41-S) with over 55-fold intensity,change. Displacement assay showed the selective binding of A41-S to the site I of HSA, addressing its potential to be 4 highly selective and sensitive HSA probe.1145sciescopu

A high-throughput screen for compounds that inhibit aggregation of the Alzheimer's peptide

Aggregation of the Alzheimer's peptide A beta produces toxic multimeric species that play a key role in the development of Alzheimer's disease. Compounds that inhibit this aggregation may prove useful as therapeutic agents for the prevention or treatment of Alzheimer's disease. Although aggregation inhibitors may already exist in combinatorial libraries, finding these compounds in a cost-effective high-throughput manner poses an enormous challenge. To meet this challenge, we have developed a novel high-throughput screen capable of isolating inhibitors of A beta aggregation from large libraries of inactive candidates. The screen uses a fusion of A beta 42 to GFP. In the absence of inhibition, the rapid misfolding and aggregation of A beta 42 causes the entire fusion protein to misfold, thereby preventing fluorescence. Compounds that inhibit A beta 42 aggregation enable GFP to fold into its native structure and be identified by the resulting fluorescent signal. By implementing the screen on a pilot library of triazine derivatives, we have identified several putative inhibitors. One of the selected compounds was studied in detail by a series of biochemical and biophysical methods. These studies confirmed that the selected compound inhibits aggregation of synthetic A beta 42 peptide. The fluorescence-based method described here is rapid and inexpensive and can be used to screen large libraries for inhibitors of A beta 42 aggregation and/or amyloidogenesis.1188sciescopu

Inhibition of Prostaglandin Transporter Accelerates Wound Healing by Up-Regulation of cAMP-CREB-HIF-1 alpha-VEGF Signaling

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Synthesis and biological evaluation of novel 1,3,5-triazine derivatives as antimicrobial agents

Numerous studies have contributed to the development of natural and synthetic antimicrobial peptides as a prospective source of antibiotic agents. Based on the concept that cationic charge, bulk, and lipophilicity are major factors determining antibacterial activity in these peptides, we designed and screened several combinatorial libraries based on 1,3,5-triazine as a template. A set of compounds were identified to show potent antimicrobial activity together with low hemolytic activity. (c) 2008 Elsevier Ltd. All rights reserved.1169sciescopu

Effects of Long-Distance Running on Cardiac Markers and Biomarkers in Exercise-Induced Hypertension Runners: An Observational Study

Objective To investigate changes of cardiac and muscle damage markers in exercise-induced hypertension (EIH) runners before running (pre-race), immediately after completing a 100-km ultramarathon race, and during the recovery period (24, 72, and 120 hours post-race). Methods In this observational study, volunteers were divided into EIH group (n=11) whose maximum systolic blood pressure was ≥210 mmHg in graded exercise testing and normal exercise blood pressure response (NEBPR) group (n=11). Their blood samples were collected at pre-race, immediately after race, and at 24, 72, and 120 hours post-race. Results Creatine kinase (CK) and cardiac troponin I (cTnI) levels were significantly higher in EIH group than those in the NEBPR group immediately after race and at 24 hours post-race (all p<0.05). However, lactate dehydrogenase (LDH), creatine kinase-myocardial band (CKMB), or CKMB/CK levels did not show any significant differences between the two groups in each period. N-terminal pro-brain natriuretic peptide (NT-proBNP) levels were significantly higher in EIH group than those in NEBPR group immediately after race and at 24 and 72 hours post-race (all p<0.05). A high sensitivity C-reactive protein (hs-CRP) level was significantly higher in EIH group than that in NEBPR group at 24 hours post-race (p<0.05). Conclusion The phenomenon of higher inflammatory and cardiac marker levels in EIH group may exaggerate cardiac volume pressure and blood flow restrictions which in turn can result in cardiac muscle damage. Further prospective studies are needed to investigate the chronic effect of such phenomenon on the cardiovascular system in EIH runners

Styryl-based compounds as potential in vivo imaging agents for beta-amyloid plaques

A group of styryl-based neutral compounds has been synthesized in this study for potential use as in vivo imaging agents for beta-amyloid plaques. Of 56 candidates, 14 compounds were found to label beta-amyloid plaques well on Alzheimer&apos;s disease (AD) human brain sections in vitro. The binding affinity to beta-amyloid fibrils was then determined by measuring the change in fluorescence intensity. Interestingly, we found that a class of quinaldine-styryl scaffold compounds displays specific binding to beta-amyloid fibrils. A representative compound, STB-8, was used in ex vivo and in vivo imaging experiments on an AD transgenic mouse model and demonstrated excellent blood-brain barrier (BBB) permeability and specific staining of the AD beta-amyloid plaques.1150sciescopu