Development of an Image-Based HCS-Compatible Method for Endothelial Barrier Function Assessment.

The recent renascence of phenotypic drug discovery (PDD) is catalyzed by its ability to identify first-in-class drugs and deliver results when the exact molecular mechanism is partially obscure. Acute respiratory distress syndrome (ARDS) is a severe, life-threatening condition with a high mortality rate that has increased in frequency due to the COVID-19 pandemic. Despite decades of laboratory and clinical study, no efficient pharmacological therapy for ARDS has been found. An increase in endothelial permeability is the primary event in ARDS onset, causing the development of pulmonary edema that leads to respiratory failure. Currently, the detailed molecular mechanisms regulating endothelial permeability are poorly understood. Therefore, the use of the PDD approach in the search for efficient ARDS treatment can be more productive than classic target-based drug discovery (TDD), but its use requires a new cell-based assay compatible with high-throughput (HTS) and high-content (HCS) screening. Here we report the development of a new plate-based image cytometry method to measure endothelial barrier function. The incorporation of image cytometry in combination with digital image analysis substantially decreases assay variability and increases the signal window. This new method simultaneously allows for rapid measurement of cell monolayer permeability and cytological analysis. The time-course of permeability increase in human pulmonary artery endothelial cells (HPAECs) in response to the thrombin and tumor necrosis factor α treatment correlates with previously published data obtained by transendothelial resistance (TER) measurements. Furthermore, the proposed image cytometry method can be easily adapted for HTS/HCS applications.

Structures of human plasma ß-factor XIIa cocrystallized with potent inhibitors.

Activated factor XIIa (FXIIa) is a serine protease that has received a great deal of interest in recent years as a potential target for the development of new antithrombotics. Despite the strong interest in obtaining structural information, only the structure of the FXIIa catalytic domain in its zymogen conformation is available. In this work, reproducible experimental conditions found for the crystallization of human plasma ß-FXIIa and crystal growth optimization have led to determination of the first structure of the active form of the enzyme. Two crystal structures of human plasma ß-FXIIa complexed with small molecule inhibitors are presented herein. The first is the noncovalent inhibitor benzamidine. The second is an aminoisoquinoline containing a boronic acid-reactive group that targets the catalytic serine. Both benzamidine and the aminoisoquinoline bind in a canonical fashion typical of synthetic serine protease inhibitors, and the protease domain adopts a typical chymotrypsin-like serine protease active conformation. This novel structural data explains the basis of the FXII activation, provides insights into the enzymatic properties of ß-FXIIa, and is a great aid toward the further design of protease inhibitors for human FXIIa.

Combating multidrug-resistant Gram-negative bacterial infections.

INTRODUCTION: Multidrug-resistant (MDR) bacterial infections, especially those caused by Gram-negative pathogens, have emerged as one of the world's greatest health threats. The development of novel antibiotics to treat MDR Gram-negative bacteria has, however, stagnated over the last half century. AREAS COVERED: This review provides an overview of recent R&D activities in the search for novel antibiotics against MDR Gram-negatives. It provides emphasis in three key areas. First, the article looks at new analogs of existing antibiotic molecules such as ß-lactams, tetracyclines, and aminoglycoside as well as agents against novel bacterial targets such as aminoacyl-tRNA synthetase and peptide deformylase. Second, it also examines alternative strategies to conventional approaches including cationic antimicrobial peptides, siderophores, efflux pump inhibitors, therapeutic antibodies, and renewed interest in abandoned treatments or those with limited indications. Third, the authors aim to provide an update on the current clinical development status for each drug candidate. EXPERT OPINION: The traditional analog approach is insufficient to meet the formidable challenge brought forth by MDR superbugs. With the disappointing results of the genomics approach for delivering novel targets and drug candidates, alternative strategies to permeate the bacterial cell membrane, enhance influx, disrupt efflux, and target specific pathogens via therapeutic antibodies are attractive and promising. Coupled with incentivized business models, governmental policies, and a clarified regulatory pathway, it is hoped that the antibiotic pipeline will be filled with an effective armamentarium to safeguard global health.

Efficacy of post exposure administration of doxycycline in a murine model of inhalational melioidosis.

Burkholderia pseudomallei is the causative agent of melioidosis. Treatment of melioidosis is suboptimal and developing improved melioidosis therapies requires animal models. In this report, we exposed male BALB/c mice to various amounts of aerosolized B. pseudomallei 1026b to determine lethality. After establishing a median lethal dose (LD(50)) of 2,772 colony forming units (cfu)/animal, we tested the ability of doxycycline administered 6 hours after exposure to a uniformly lethal dose of ~20 LD(50) to prevent death and eliminate bacteria from the lung and spleens. Tissue bacterial burdens were examined by PCR analysis. We found that 100% of mice treated with doxycycline survived and B. pseudomallei DNA was not amplified from the lungs or spleens of most surviving mice. We conclude the BALB/c mouse is a useful model of melioidosis. Furthermore, the data generated in this mouse model indicate that doxycycline is likely to be effective in post-exposure prophylaxis of melioidosis.

Cethromycin versus clarithromycin for community-acquired pneumonia: comparative efficacy and safety outcomes from two double-blinded, randomized, parallel-group, multicenter, multinational noninferiority studies.

Community-acquired pneumonia (CAP) continues to be a major health challenge in the United States and globally. Factors such as overprescribing of antibiotics and noncompliance with dosing regimens have added to the growing antibacterial resistance problem. In addition, several agents available for the treatment of CAP have been associated with serious side effects. Cethromycin is a new ketolide antibiotic that may provide prescribing physicians with an additional agent to supplement a continually limited armamentarium. Two global phase III noninferiority studies (CL05-001 and CL06-001) to evaluate cethromycin safety and efficacy were designed and conducted in patients with mild to moderate CAP. Study CL05-001 demonstrated an 83.1% clinical cure rate in the cethromycin group compared with 81.1% in the clarithromycin group (95% confidence interval [CI], -4.8%, +8.9%) in the intent to treat (ITT) population and a 94.0% cethromycin clinical cure rate compared with a 93.8% clarithromycin cure rate (95% CI, -4.5%, +5.1%) in the per protocol clinical (PPc) population. Study CL06-001 achieved an 82.9% cethromycin clinical cure rate in the ITT population compared with an 88.5% clarithromycin cure rate (95% CI, -11.9%, +0.6%), whereas the clinical cure rate in the PPc population was 91.5% in cethromycin group compared with 95.9% in clarithromycin group (95% CI, -9.1%, +0.3%). Both studies met the primary endpoints for clinical cure rate based on predefined, sliding-scale noninferiority design. Therefore, in comparison with clarithromycin, these two noninferiority studies demonstrated the efficacy and safety of cethromycin, with encouraging findings of efficacy in subjects with Streptococcus pneumoniae bacteremia. No clinically significant adverse events were observed during the studies. Cethromycin may be a potential oral therapy for the outpatient treatment of CAP.

Methylenedioxy- and ethylenedioxy-fused indolocarbazoles: potent human topoisomerase I inhibitors and antitumor agents.

The indolo[2,3-a]carbazole alkaloids constitute an important class of natural products with interesting and diverse biological activities. A series of novel ring-fused indolocarbazoles were synthesized and evaluated for inhibition of topoisomerase I-mediated relaxation of supercoiled DNA and in vitro antitumor activity. The derivatives bearing a methylenedioxy or an ethylenedioxy ring fused onto the nonglycosylated indole (1a, 1b) demonstrated more potent anti-topoisomerase I activity. The isopropylenedioxy analogue 1c was approximately half as active as 1a, while the O-dimethoxy analogue 1d and the regioisomers 2a and 2b were essentially devoid of measurable activity, implying that the stacking with the intact DNA strand has been impeded by these compounds due to steric hindrance. The newly synthesized indolocarbazoles were screened against the NCI's 60 tumor cell lines. The order of activity, based on the mean GI50 values, is as follows: 1a > 2a ~ 1d > 1b > MCR-47 > 2b. Though in general the analogues that showed potent activity against topoisomerase I (1a, 1b) also showed potent in vitro inhibition of tumor cell growth, the antitumor activity of the anti-topoisomerase I inactive 1d and 2a were intriguing. COMPARE analyses confirmed that the topoisomerase I is the primary target for 1a and 1b; however, other target(s) or pathway(s) may also be involved, with PLD1 and MERTK suggested. Further investigation of these molecular targets against these indolocarbazoles is warranted.

Synthesis, proapoptotic screening, and structure-activity relationships of novel aza-lupane triterpenoids.

Apoptosis is a highly regulated process by which excessive cells are eliminated in order to maintain normal cell development and tissue homeostasis. Resistance to apoptosis often contributes to failure in cancer prevention and treatment. Apoptotic cell death regulators are considered important targets for discovery and development of new therapeutic agents in oncology research. A class of novel aza-lupane triterpenoids were designed, synthesized, and evaluated for antitumor activity against a panel of cancer cell lines of different histogenic origin and for ability to induce apoptosis. 3,30-Bis(aza) derivatives were identified not only to possess improved cytotoxicity compared to the natural product betulinic acid but also to affect cell death predominantly via apoptosis, whereas the mono(aza) derivatives apparently triggered cell death via different, non-apoptotic pathway(s).

Synthesis and cytotoxicity of 2-cyano-28-hydroxy-lup-1-en-3-ones.

New A-ring modified betulin and dihydrobetulin derivatives possessing the 2-cyano-1-en-3-one moiety were prepared and tested for cytotoxicity in seven cancer cell lines. The most active agent 9a synthesized in this account was further demonstrated to induce apoptosis and to activate caspases in malignant melanoma cells.

Pyranocoumarin, a novel anti-TB pharmacophore: synthesis and biological evaluation against Mycobacterium tuberculosis.

Pyranocoumarin compounds were identified to embody a novel and unique pharmacophore for anti-TB activity. A systematic approach was taken to investigate the structural characteristics. Focused libraries of compounds were synthesized and evaluated for their anti-TB activity in primary screening assays. Compounds shown to be active were further determined for MIC and MBC values. Three of the four bactericidal compounds (16, 17c, and 18f) were amino derivatives, with MIC values of 16 microg/mL and respective MBC values of 32, 32, and 64 microg/mL.

Effective attenuation of endotoxin-induced acute lung injury by 2,3-diacetyloxybenzoic acid in two independent animal models.

The pathology of acute lung injury (ALI) is often modeled in animal studies by the administration of lipopolysaccharide (LPS), which results in an endotoxemia with sequelae similar to that seen in acute respiratory distress syndrome (ARDS). Here we report the results of two studies designed to examine the efficacy of a novel agent, 2,3-diacetyloxybenzoic acid (2,3-DABA), in the treatment of LPS-induced ALI. In two separate animal models, 2,3-DABA was effective in significantly reducing lung microvascular permeability, a condition commonly seen in ARDS, which results in pulmonary edema and respiratory insufficiency. In each model, it is demonstrated that the mechanism by which 2,3-DABA exerts this effect occurs subsequent to the recruitment of neutrophils to the site of inflammation. Lung permeability was significantly decreased in both models by treatment with 2,3-DABA, suggesting that this agent, either alone or in combination therapy, may be useful in the treatment of ALI associated with ARDS.

Anti-HIV natural product (+)-calanolide A is active against both drug-susceptible and drug-resistant strains of Mycobacterium tuberculosis.

Naturally occurring anti-HIV-1 agent (+)-calanolide A was found to be active against all of the strains of Mycobacterium tuberculosis tested, including those resistant to the standard antitubercular drugs. Efficacy evaluations in macrophages revealed that (+)-calanolide A significantly inhibited intracellular replication of M. tuberculosis H37Rv at concentrations below the MIC observed in vitro. Preliminary mechanistic studies indicated that (+)-calanolide A rapidly inhibits RNA and DNA synthesis followed by an inhibition of protein synthesis. Compared with known inhibitors, this scenario is more similar to effects observed with rifampin, an inhibitor of RNA synthesis. Since (+)-calanolide A was active against a rifampin-resistant strain, it is believed that these two agents may involve different targets. (+)-Calanolide A and its related pyranocoumarins are the first class of compounds identified to possess antimycobacterial and antiretroviral activities, representing a new pharmacophore for anti-TB activity.

Safety and pharmacokinetic profile of multiple escalating doses of (+)-calanolide A, a naturally occurring nonnucleoside reverse transcriptase inhibitor, in healthy HIV-negative volunteers.

BACKGROUND: (+)-Calanolide A is a naturally occurring nonnucleoside reverse transciptase inhibitor (NNRTI) that exhibits enhanced activity against HIV-1 isolates with the Y181C mutation and retains activity against HIV-1 isolates with dual Y181C and K103N mutations. Previous studies have demonstrated that (+)-calanolide A has a favorable safety profile in both animal and human subjects. METHOD: In this study, the safety and pharmacokinetics of multiple escalating doses of (+)-calanolide A were evaluated in a total of 47 healthy, HIV-seronegative individuals. RESULTS: All adverse events seen in the study were mild to moderate in intensity and were transient. The most common adverse events seen were headache, dizziness, nausea, and taste perversion (oily aftertaste). Laboratory abnormalities were determined to be clinically insignificant or unrelated to (+)-calanolide A administration. No dose-related pattern in adverse event or laboratory abnormality incidence was apparent. In all cohorts examined, administration of (+)-calanolide A produced highly variable plasma levels and absorption profiles. No accumulation of parent compound was seen over the 5-day treatment course, with the day 5 area under the curve (AUC) being approximately one half of that seen on the first day of dosing. Steady-state trough plasma levels were determined in the two highest dose cohorts (600 mg and 800 mg bid for 5 days). Mean elimination half-life in the two highest dosing cohorts combined was 15.5 hours in men and 35.2 hours in women. CONCLUSION: These pharmacokinetic properties, together with the benign safety profile, and unique in vitro resistance pattern warrant the continued development of this potential new antiviral agent.

Coumarins and pyranocoumarins, potential novel pharmacophores for inhibition of measles virus replication.

A series of coumarin and pyranocoumarin analogues were evaluated in vitro for antiviral efficacy against measles virus (MV), strain Chicago. Of the 22 compounds tested for inhibition, six were found to have selectivity indices greater than 10. These were compounds 5-hydroxy-7-propionyloxy-4-propylcoumarin (2a), 5,7-bis(tosyloxy)-4-propylcoumarin (7); 5-hydroxy-4-propyl-7-tosyloxy-coumarin (8); 6,6-dimethyl-9-propionyloxy-4-propyl-2H,6H-benzo[1,2-b:3,4-b']dipyran-2-one (9); 6,6-dimethyl-9-pivaloyloxy-4-propyl-2H,6H-benzo[1,2-b:3,4-b']dipyran-2-one (10); and 7,8-cis-10,11,12-trans-4-propyl-6,6,10,11-tetramethyl-7,8,9-trihydroxy-2H,6H,12H-benzo[1 ,2-b:3,4-b':5,6-b'']tripyran-2-one (18). Three of the active drugs were propyl coumarin analogues (2a, 7 and 8), two were dipyranone or chromeno-coumarins (9 and 10), and one was a benzotripyranone with a coumarin nucleus (18). Some appeared to be rather specific and potent inhibitors of MV with EC50 values ranging from 0.2 to 50 microg/ml and the majority of the EC50 values being less than 5 pg/ml. The compounds inhibited an additional nine strains of MV, and in virucidal tests the drugs did not physically disrupt the virion to inhibit virus replication. The inhibitory activity for one of the compounds tested (7) was somewhat dependent on virus concentration and it was still active when added to cells up to 24 h after virus exposure. When used in combination with ribavirin, compound 7 appeared not to profoundly affect the antiviral efficacy of ribavirin or its cell-associated toxicity. However, a slightly antagonistic MV-inhibitory effect was observed at the highest concentration of ribavirin used in combination with most concentrations of compound 7 tested. This and related compounds may be valuable leads in the development of a potent and selective class of MV inhibitors that could be used in future in the clinic.

Chalcones and flavonoids as anti-tuberculosis agents.

A series of flavonoids, chalcones and chalcone-like compounds were evaluated for inhibitory activity against Mycobacterium tuberculosis H37Rv. Among them, eight compounds exhibited >90% inhibition on the growth of the bacteria at a concentration of 12.5 microg/mL. Chalcones 1-(2-hydroxyphenyl)-3-(3-chlorophenyl)-2-propen-1-one (22) and 1-(2-hydroxyphenyl)-3-(3-iodophenyl)-2-propen-1-one (37) demonstrated 90 and 92% inhibition, respectively. Chalcone-like compounds (heterocyclic ring-substituted 2-propen-1-one) 1-(4-fluorophenyl)-3-(pyridin-3-yl)-2-propen-1-one (48), 1-(3-hydroxyphenyl)-3-(phenanthren-9-yl)-2-propen-1-one (49), 1-(pyridin-3-yl)-3-(phenanthen-9-yl)-2-propen-1-one (50) and 1-(furan-2-yl)-3-phenyl-2-propen-1-one (51) exhibited 98, 97, 96 and 96% inhibition, respectively. The actual minimum inhibitory concentrations (MIC), defined as the lowest concentration inhibiting 99% of the inoculum, for 22, 37, 48, 49, 50 and 51 were 20.3, 31.5, 48.3, >35.7, 6.8 and 19.2, respectively. A hydrophobic substituent on one aromatic ring, and a hydrogen-bonding group on the other aromatic ring resulted in increased anti-TB activity of the chalcones and chalcone-like compounds. Flavones and flavanones are more geometrically constrained than the corresponding chalcone analogues. The decreased activity of the flavones with respect to the chalcones may be due to the confinement of the terminal aromatic rings to the same plane.

Preparation of New Wittig Reagents and Their Application to the Synthesis of alpha,beta-Unsaturated Phosphonates.

The Wittig reagent [(diethoxyphosphinyl)methylidene]triphenylphosphorane (1b) has been successfully synthesized for the first time via its phosphonium triflate salt (4a), by treating (diethoxyphosphinyl)methyl triflate with triphenylphosphine. The procedure has been applied to the synthesis of other phosphoranes and phosphonium salts. The new Wittig reagents thus synthesized were treated with various aldehydes and an activated ketone, affording the corresponding alpha,beta-unsaturated phosphonates. Triphenylphosphorane 1b and triphenylphosphonium 4a led to both cis and trans isomers with the latter being predominant, while trans isomers were almost exclusively formed when tributyl reagents (1c and 4d) were used.