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A little girl is bitten by a kissing bug (also known as a triatomine bug). Trypanosoma cruzi (T. cruzi) parasites from the insect's feces enter the bite wound and start [...]

Author(s): Monya Baker [1] [illus. 1] Stefan Knapp's results made him feel like he was riding a roller coaster. Knapp, a pharmaceutical scientist at Goethe University Frankfurt in Germany, and [...]

Author(s): Mehdi Bouhaddou [1]; Matthew S. DiStefano [1]; Eric A. Riesel [1]; Emilce Carrasco [1]; Hadassa Y. Holzapfel [1]; DeAnalisa C. Jones [1]; Gregory R. Smith [1]; Alan D. Stern [...]

Vaccines are a triumph of science over infection. They have defeated smallpox, which the World Health Organization declared eradicated in 1980, and dramatically lowered the toll of many other infectious [...]

Hepatitis C virus (HCV) remains a major public health problem, affecting approximately 130 million people worldwide. HCV infection can lead to cirrhosis, hepatocellular carcinoma, and end-stage liver disease, as well as extrahepatic complications such as cryoglobulinemia and lymphoma. Preventative and therapeutic options are severely limited; there is no HCV vaccine available, and nonspecific, IFN-based treatments are frequently ineffective. Development of targeted antivirals has been hampered by the lack of robust HCV cell culture systems that reliably predict human responses. Here, we show the entire HCV life cycle recapitulated in micropatterned cocultures (MPCCs) of primary human hepatocytes and supportive stroma in a multiwell format. MPCCs form polarized cell layers expressing all known HCV entry factors and sustain viral replication for several weeks. When coupled with highly sensitive fluorescence- and luminescence-based reporter systems, MPCCs have potential as a high-throughput platform for simultaneous assessment of in vitro efficacy and toxicity profiles of anti-HCV therapeutics. viral hepatitis | liver | tissue engineering | drug development | infection www.pnas.org/cgi/doi/10.1073/pnas.0915130107

Over the past decade, West Nile virus (WNV) has spread to all 48 of the lower United States as well as to parts of Canada, Mexico, the Caribbean, and South America, with outbreaks of neuroinvasive disease occurring annually. At present, no therapeutic or vaccine is available for human use. Epidemics of WNV and other emerging infectious disease threats demand cost-efficient and scalable production technologies that can rapidly transfer effective therapeutics into the clinical setting. We have previously reported that Hu-E16, a humanized anti-WNV mAb, binds to a highly conserved epitope on the envelope protein, blocks viral fusion, and shows promising postexposure therapeutic activity. Herein, we generated a plant-derived Hu-E16 mAb that can be rapidly scaled up for commercial production. Plant Hu-E16 was expressed at high levels within 8 days of infiltration in Nicotiana benthamiana plants and retained high-affinity binding and potent neutralizing activity in vitro against WNV. A single dose of plant Hu-E16 protected mice against WNV-induced mortality even 4 days after infection at rates that were indistinguishable from mammalian-cell-produced HuE16. This study demonstrates the efficacy of a plant-produced mAb against a potentially lethal infection several days after exposure in an animal challenge model and provides a proof of principle for the development of plant-derived mAbs as therapy against emerging infectious diseases. flavivirus | mAb therapeutics | plant-made pharmaceuticals www.pnas.org/cgi/doi/10.1073/pnas.0914503107

Glycoanalysis is important in the manufacture and quality control of protein therapeutics. An emerging method for glycoanalysis is the use of lectin arrays. Critical to the performance of these arrays is the immobilization of lectin molecules. Polydopamine has recently been shown to adsorb to a wide variety of surfaces. In this study, polydopamine (pDA) was used to modify gold, indium, and iridium surfaces and promote the adhesion of the [alpha]-mannose-specific lectin concanavalin A (Con A). The activity of the surface-bound lectin was demonstrated with the [alpha]-mannose-presenting glycoprotein ribonuclease B (RNase B). Surface plasmon resonance spectroscopy (SPRS) was used to demonstrate the selective affinity of RNase B for Con A. Surface-MALDI-TOF MS experiments revealed that the affinity of polydopamine-immobilized Con A for the glycoforms of RNase B is significantly affected by slight variations in oligosaccharide structure and composition. Specifically, surface-bound Con A binds certain Man7, Man8, and Man9 RNase B glycoforms more strongly than Man5 and Man6 glycoforms.

Dissolution testing is a crucial part of pharmaceutical dosage form investigations and is generally performed by analyzing the concentration of the released drug in a defined volume of flowing dissolution medium. As solid-state properties of the components affect dissolution behavior to a large and sometimes even unpredictable extent there is a strong need for monitoring and especially visualizing solid-state properties during dissolution testing. In this study coherent anti-Stokes Raman scattering (CARS) microscopy was used to visualize the solid-state properties of lipid-based oral dosage forms containing the model drug theophylline anhydrate during dissolution in real time. The drug release from the dosage form matrix was monitored with a spatial resolution of about 1.5 [micro]m. In addition, as theophylline anhydrate tends to form the less soluble monohydrate during dissolution, CARS microscopy allowed the solid-state transformation of the drug to be spatially visualized. The results obtained by CARS microscopy revealed that the method used to combine lipid and active ingredient into a sustained release dosage form can influence the physicochemical behavior of the drug during dissolution. In this case, formation of theophylline monohydrate on the surface was visualized during dissolution with tablets compressed from powdered mixtures but not with solid lipid extrudates.

Increasing resistance and decreasing numbers of antibiotics reaching the market point to a growing need for novel antibacterial drugs. Most antibiotics are very inefficient at killing non-multiplying bacteria, which live side by side with multiplying ones of the same strain in a clinical infection. Although non-multiplying bacteria do not usually cause disease, they can revert to the multiplying state that leads to overt disease, at which time resistance can emerge. Here we discuss the concept of developing antibacterial drugs by targeting non-multiplying organisms. We define non-multiplying bacteria, discuss the efficacy of existing antibiotics, and assess whether targeting these bacteria might lead to new antibiotics that will decrease the rate of emergence of resistance. Lastly, we review the potential of new molecular targets and live non-multiplying bacteria as possible routes for the development of novel antimicrobial drugs.

Pteridine reductase (PTR1) is essential for salvage of pterins by parasitic trypanosomatids and is a target for the development of improved therapies. To identify inhibitors of Leishmania major and Trypanosoma cruzi PTR1, we combined a rapid-screening strategy using a folate-based library with structure-based design. Assays were carried out against folate-dependent enzymes including PTR1, dihydrofolate reductase (DHFR), and thymidylate synthase. Affinity profiling determined selectivity and specificity of a series of quinoxaline and 2,4-diaminopteridine derivatives, and nine compounds showed greater activity against parasite enzymes compared with human enzymes. Compound 6a displayed a Ki of 100 nM toward LmPTR1, and the crystal structure of the LmPTRI:NADPH:6a ternary complex revealed a substrate-like binding mode distinct from that previously observed for similar compounds. A second round of design, synthesis, and assay produced a compound (6b) with a significantly improved Ki (37 nM) against LmPTR1, and the structure of this complex was also determined. Biological evaluation of selected inhibitors was performed against the extracellular forms of T. cruzi and L. major, both wild-type and overexpressing PTR1 lines, as a model for PTR1-driven antifolate drug resistance and the intracellular form of T. cruzi. An additive profile was observed when PTR1 inhibitors were used in combination with known DHFR inhibitors, and a reduction in toxicity of treatment was observed with respect to administration of a DHFR inhibitor alone. The successful combination of antifolates targeting two enzymes indicates high potential for such an approach in the development of previously undescribed antiparasitic drugs. antitrypanosomatid agents | antifolates | drug discovery

Functional neuroimaging provides a direct way of investigating the pathophysiology of schizophrenia in vivo. The function of neurotransmitters implicated in schizophrenia, such as dopamine and glutamate, can be assessed using molecular imaging techniques such as positron emission tomography (PET), single-photon emission tomography (SPET) and magnetic resonance spectroscopy (MRS). Regional brain activity, particularly that associated with the cognitive processes and symptoms associated with the disorder, can be studied using functional magnetic resonance imaging (fMRI). Here, we focus on the potential for the use of these techniques in the diagnosis of schizophrenia and in the development of new drugs for its treatment.

An established paradigm in current drug development is (i) to identify a single protein target whose inhibition is likely to result in the successful treatment of a disease of interest; (ii) to assay experimentally large libraries of small-molecule compounds in vitro and in vivo to identify promising inhibitors in model systems; and (iii) to determine whether the findings are extensible to humans. This complex process, which is largely based on trial and error, is risk-, time- and cost-intensive. Computational (virtual) screening of drug-like compounds simultaneously against the atomic structures of multiple protein targets, taking into account protein-inhibitor dynamics, might help to identify lead inhibitors more efficiently, particularly for complex drug-resistant diseases. Here we discuss the potential benefits of this approach, using HIV-1 and Plasmodium falciparum infections as examples. We propose a virtual drug discovery 'pipeline' that will not only identify lead inhibitors efficiently, but also help minimize side-effects and toxicity, thereby increasing the likelihood of successful therapies.

Malaria is a global disease, causing at least 500 million clinical cases and more than one million deaths each year. Moreover, drug-resistant Plasmodium falciparum, the organism that causes most malaria-associated deaths, has become a major problem. Therefore, discovery and investigation of novel targets for anti-malarial drug design is essential to combat this disease. The malarial genome has been sequenced, revealing ~5500 genes. The current post-genomic challenge is functionally to evaluate the essential genes and validate them for therapeutic design. Unfortunately, standard genetics techniques are limited in scope because of low transfection efficiency and a lack of knockdown techniques, thereby rendering the analysis of essential genes difficult.

Author(s): Sara Reardon The anaesthetic ketamine - a hallucinogenic club drug also known as Special K - has tantalized researchers who are seeking new ways to treat depression. The drug [...]

ABSTRACT Background Published pharmaceutical industry-sponsored trials are more likely than non- industry-sponsored trials to report results and conclusions that favor drug over placebo. Little is known about potential biases in [...]

We have investigated the effect of sequence-specific antisense phosphorothioate-modified oligodeoxyribonucleotides (PS-ODNs) targeting different regions of each of the 30/32-kDa protein complex (antigen 85 complex) encoding genes on the multiplication of Mycobacterium tuberculosis. Single PS-ODNs to one of the three mycolyl transferase transcripts, added either once or weekly over the 6-wk observation period, inhibited bacterial growth by up to 1 log unit. A combination of three PS-ODNs specifically targeting all three transcripts inhibited bacterial growth by [approximately equal to] 2 logs; the addition of these PS-ODNs weekly for 6 wk was somewhat more effective than a one-time addition. Targeting the 5' end of the transcripts was more inhibitory than targeting internal sites; the most effective PS-ODNs and target sites had minimal or no secondary structure. The effect of the PS-ODNs was specific, as mismatched PS-ODNs had little or no inhibitory activity. The antisense PS-ODNs, which were highly stable in M. tuberculosis cultures, specifically blocked protein expression by their gene target. PS-ODNs targeting the transcript of a related 24-kDa protein (mpt51) had little inhibitory effect by themselves and did not increase the effect of PS-ODNs against the three members of the 30/32-kDa protein complex. The addition of PS-ODNs against the transcripts of glutamine synthetase I (glnA1) and alanine racemase (alr) modestly increased the inhibitory efficacy of the 30/32-kDa protein complex-specific PS-ODNs to [approximately equal to] 2.5 logs. This study shows that the three mycolyl transferases are highly promising targets for antituberculous therapy by using antisense or other antimicrobial technologies. phosphorothioate oligodeoxyribonucleotides | mRNA secondary structure | inhibition of translation initiation | drug design

While it is widely acknowledged by epidemiologists, pharmacologists and physicians that adverse drug reactions (ADRs) occur with considerable frequency, within the realm of medical sociology, drug-induced risk currently receives little critical attention. This paper looks into this medical scientific `black box' to present a comprehensive account of the epistemological and political processes at play in the detection of ADRs. By focusing on the literature generated by pharmacologists, epidemiologists and others working in the field, this paper examines the various techniques and methods used to identify and calculate ADRs both during clinical drug trials and beyond. Although risk associated with drug consumption is often presented as a scientifically calculable objective phenomenon, the attribution of causal mechanisms in determining whether the drug has caused an adverse event is a highly contingent social process, often involving complex clinical judgements. During clinical trials, variables are controlled and exclusions are imposed in order to fulfil scientific protocol requirements. These exclusionary practices mean that major patient population groups such as women and the elderly are often underrepresented during the trial process. At the time a drug product license is granted many uncertainties exist about the risk of ADRs. Once a drug is in widespread use, a more comprehensive profile of risk may begin to emerge. However, given that in everyday use drugs can interact with other drugs, alcohol and even certain foods and that drug reactions can mimic the disease they are supposed to be treating, differentiating between `signal' and `noise' is a messy, contingent complex process. Keywords: Adverse drug reaction; Clinical drug trial; Risk

Keywords: metastasis; surgical orthotopic implantation; immunodeficient mice; green fluorescent protein; tumor imaging; clinically-relevant models Abstract: Currently used rodent tumor models, including transgenictumor models, or subcutaneously-growing human tumors inimmunodeficient mice, do not sufficiently represent clinicalcancer, especially with regard to metastasis and drugsensitivity. In order to obtain clinically accurate models, wehave developed the technique of surgical orthotopic implantation(SOI) to transplant histologically-intact fragments of humancancer, including tumors taken directly from the patient, to thecorresponding organ of immunodeficient rodents. It has beendemonstrated in 70 publications describing 10 tumor types thatSOI allows the growth and metastatic potential of thetransplanted tumors to be expressed and reflects clinical cancer.Unique clinically-accurate and relevant SOI models of humancancer for antitumor and antimetastatic drug discovery include:spontaneous SOI bone metastatic models of prostate cancer, breastcancer and lung cancer spontaneous SOI liver and lymph nodeultra-metastatic model of colon cancer, metastatic models ofpancreatic, stomach, ovarian, bladder and kidney cancer.Comparison of the SOI models with transgenic mouse models ofcancer indicate that the SOI models have more features ofclinical metastatic cancer. Cancer cell lines have been stablytransfected with the jellyfish Aequorea victoriagreenfluorescent protein (GFP) in order to track metastases in freshtissue at ultra-high resolution and externally image metastasesin the SOI models. Effective drugs can be discovered andevaluated in the SOI models utilizing human tumor cell lines andpatient tumors. These unique SOI models have been used forinnovative drug discovery and mechanism studies and serve as abridge linking pre-clinical and clinical research and drugdevelopment. Article History: Registration Date: 02/10/2004

DNA heteroduplexes containing base mismatches were studied for enhanced alkylation by altromycin B to identify the structural and/or dynamics behind enhanced drug reactivity. The results indicate that pluramycin reactivities are determined by the dynamic conformation of the base mismatch as well as by the specific groove interactions between the drug and the DNA. It is determined that the highest pluramycin reactivity with heteroduplex DNA is the result of a thymine:thymine mismatch.

Use of solid-phase strategies for putting together template-based protein mimetics is discussed. Protocols that can extend the potential of solid-phase peptide synthesis for making molecules with great structural complexity are offered. They bring ways to use template-based protein mimetics by combinatorial methods. Template-assembled synthetic protein (TASP) synthesis is expanded in the making of resin-bound cyclic peptides with as many as four orthogonally protected attachment sites.

The need to quantify molecular diversity in the context of pharmainformatics originates from two ends of the drug-discovery spectrum - from the biologist's need to appreciate the inherent complexity of the world of molecular recognition and from the chemist's need to exploit it in novel pharmacophore design. In this article, we survey the applications of molecular diversity principles, from the assessment of biological diversity, through genomics, to drug-design applications in computational chemistry.

Large-scale gene-expression analysis aims to increase the depth of diagnostic and drug-effect profiling, and to accelerate the discovery of key biological processes for therapeutic targeting. Success hinges on the precision and scope of the measurements, and on the integration of computational tools for data mining, visualization and modeling. The ultimate objective is to discover key molecular connections in a disease process by reverse engineering signaling networks from their activity profiles.