Recent developments and strategies for the discovery of TACE inhibitors.

INTRODUCTION: TNF-α plays a central role in certain autoimmune diseases as well as in inflammation. The current strategy for excluding TNF-α from circulation is to selectively inhibit TNF-α converting enzyme (TACE), an enzyme that cleaves mTNF-α to active TNF-α. Various TACE inhibitors have been discovered by using different strategies to control inflammatory diseases, cancer, and cardiac hypertrophy. AREAS COVERED: The present article summarizes the design and discovery of novel TACE inhibitors that have been reported in the literature since 2012 onwards. It also includes some reports concerning the new role that TACE plays in cancer and cardiac hypertrophy. EXPERT OPINION: So far, undertaken studies that have looked to design and develop small TACE inhibitors have been discouraging due to the failure of any TACE inhibitors to hit the market. However, some of the latest developments, such as with tartrate-based inhibitors, has given hope to the potentiality of a viable novel selective TACE inhibitor therapeutic in the future. Indeed, some of the novel peptidomimetics and monoclonal antibodies have great potential to pave the way for an effective and safe therapy by selectively inhibiting TACE enzyme.

High throughput screening using acoustic droplet ejection to combine protein crystals and chemical libraries on crystallization plates at high density.

We describe a high throughput method for screening up to 1728 distinct chemicals with protein crystals on a single microplate. Acoustic droplet ejection (ADE) was used to co-position 2.5nL of protein, precipitant, and chemicals on a MiTeGen in situ-1 crystallization plate™ for screening by co-crystallization or soaking. ADE-transferred droplets follow a precise trajectory which allows all components to be transferred through small apertures in the microplate lid. The apertures were large enough for 2.5nL droplets to pass through them, but small enough so that they did not disrupt the internal environment created by the mother liquor. Using this system, thermolysin and trypsin crystals were efficiently screened for binding to a heavy-metal mini-library. Fluorescence and X-ray diffraction were used to confirm that each chemical in the heavy-metal library was correctly paired with the intended protein crystal. A fragment mini-library was screened to observe two known lysozyme ligands using both co-crystallization and soaking. A similar approach was used to identify multiple, novel thaumatin binding sites for ascorbic acid. This technology pushes towards a faster, automated, and more flexible strategy for high throughput screening of chemical libraries (such as fragment libraries) using as little as 2.5nL of each component.

Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening.

Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s(-1)) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.

A simple technique to reduce evaporation of crystallization droplets by using plate lids with apertures for adding liquids.

A method is described for using plate lids to reduce evaporation in low-volume vapor-diffusion crystallization experiments. The plate lids contain apertures through which the protein and precipitants were added to different crystallization microplates (the reservoir was filled before fitting the lids). Plate lids were designed for each of these commonly used crystallization microplates. This system minimizes the dehydration of crystallization droplets containing just a few nanolitres of protein and precipitant, and results in more reproducible diffraction from the crystals. For each lid design, changes in the weight of the plates were used to deduce the rate of evaporation under different conditions of temperature, air movement, droplet size and precipitant. For comparison, the state of dehydration was also visually assessed throughout the experiment. Finally, X-ray diffraction methods were used to compare the diffraction of protein crystals that were conventionally prepared against those that were prepared on plates with plate lids. The measurements revealed that the plate lids reduced the rate of evaporation by 63-82%. Crystals grown in 5 nl drops that were set up with plate lids diffracted to higher resolution than similar crystals from drops that were set up without plate lids. The results demonstrate that plate lids can be instrumental for improving few-nanolitre crystallizations.

Co-morbidities in hospitalized children with severe acute malnutrition.

OBJECTIVE: To study the co morbidities in hospitalized children with severe acute malnourishment. METHODS: 104 severe acute malnourished children were included. RESULTS: 54% had diarrhea and 27.8% had acute respiratory tract infections. Tuberculosis was diagnosed in 22% of cases (60.8% cases in children 6-12 mo old). Malaria and Measles were diagnosed in 3.8% each, and HIV infection was seen in 2.9% cases. Signs of vitamin B and vitamin A deficiency were seen in 14.4% and 5.8% cases, respectively. Malaria and HIV were not found to be major co morbid conditions. CONCLUSIONS: Timely identification and treatment of various co-morbidities is likely to break undernutrition-disease cycle, and to decrease mortality and improve outcome.