Discovery and Characterisation of Highly Cooperative FAK-Degrading PROTACs.

Focal adhesion kinase (FAK) is a key mediator of tumour progression and metastasis. To date, clinical trials of FAK inhibitors have reported disappointing efficacy for oncology indications. We report the design and characterisation of GSK215, a potent, selective, FAK-degrading Proteolysis Targeting Chimera (PROTAC) based on a binder for the VHL E3 ligase and the known FAK inhibitor VS-4718. X-ray crystallography revealed the molecular basis of the highly cooperative FAK-GSK215-VHL ternary complex, and GSK215 showed differentiated in-vitro pharmacology compared to VS-4718. In mice, a single dose of GSK215 induced rapid and prolonged FAK degradation, giving a long-lasting effect on FAK levels (≈96 h) and a marked PK/PD disconnect. This tool PROTAC molecule is expected to be useful for the study of FAK-degradation biology in vivo, and our results indicate that FAK degradation may be a differentiated clinical strategy versus FAK inhibition for the treatment of cancer.

A Photoaffinity-Based Fragment-Screening Platform for Efficient Identification of Protein Ligands.

Advances in genomic analyses enable the identification of new proteins that are associated with disease. To validate these targets, tool molecules are required to demonstrate that a ligand can have a disease-modifying effect. Currently, as tools are reported for only a fraction of the proteome, platforms for ligand discovery are essential to leverage insights from genomic analyses. Fragment screening offers an efficient approach to explore chemical space. Presented here is a fragment-screening platform, termed PhABits (PhotoAffinity Bits), which utilizes a library of photoreactive fragments to covalently capture fragment-protein interactions. Hits can be profiled to determine potency and the site of crosslinking, and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. The PhABit platform is envisioned to be widely applicable to novel protein targets, identifying starting points in the development of therapeutics.

A New Miniature Respirable Sampler for In-mask Sampling: Part 2-Tests Performed Inside the Mask.

This paper is the second in a series of two describing the performance of a miniature and low-weight respirable sampler designed to fit inside filtering facepiece (FFP) and half-mask type respirators. The first paper described the design of the miniature sampler and evaluated the particle and collection performance of the miniature sampler. This paper assesses its comparability with the traditional inward leakage measurement technique, and its safe use. Simultaneous mass measurements of a respirable sodium chloride aerosol were taken inside a total inward leakage chamber by a miniature sampler and by sodium flame photometry. Direct side-by-side comparison of the two methods yielded excellent correlation (R 2 = 0.99), as did comparison when sampling from inside four different masks when worn by a breathing Sheffield dummy head. In addition, comparison tests were carried out using three models of FFP worn by human volunteers both with and without the miniature sampler, in order to test whether or not the presence of the miniature sampler negatively affected the protection offered. The difference between the tests carried out with the miniature sampler and without the miniature sampler was not statistically significant (P = 0.3). In all cases, the masks performed within their protection class, whether the miniature sampler was fitted or not. We therefore conclude that the miniature sampler does not significantly affect the protection offered by the masks. The miniature sampler may prove a viable option for in-mask measurements of respirable dust where low air concentrations of hazardous material are expected.

Performance of High Flow Rate Personal Respirable Samplers When Challenged with Mineral Aerosols of Different Particle Size Distributions.

It is thought that the performance of respirable samplers may vary when exposed to dust aerosols with different particle sizes and wind speeds. This study investigated the performance of the GK 4.16 (RASCAL), GK 2.69, PPI 8, and FSP 10, high flow rate personal samplers when exposed to aerosols of mineral dust in a wind tunnel at two different wind speeds (1 and 2 m s(-1)) and orientations (towards and side-on to the source of emission). The mass median aerodynamic diameter of four aerosolized test dusts ranged from 8 to 25 µm with geometric standard deviations from 1.6 to 2 µm. The performance of each sampler type was compared with that of the SIMPEDS (Higgins-Dewell design) sampler. There was slight evidence to suggest that the performance of the FSP 10 is affected by the direction of the inlet relative to the air flow, although this was not significant when most respirable dust concentrations were compared, possibly due to the variability of paired dust concentration results. The GK 2.69, RASCAL, and PPI 8 samplers had similar performances, although the results when side-on to the emission source were generally slightly lower than the SIMPEDS. Despite slight differences between respirable dust concentrations the respirable crystalline silica values were not significantly different from the SIMPEDS. The GK family of cyclones obtained most precise results and more closely matched the SIMPEDS. A comparison with dust concentration results from previous calm air chamber studies (where wind speeds were < 0.4 m s(-1)) found that the relative performance between samplers was similar to those observed in this work indicating consistent performance relative to the SIMPEDS in both calm and moving air.

A New Miniature Respirable Sampler for In-mask Sampling: Part 1-Particle Size Selection Performance.

The Health and Safety Laboratory has developed a miniature respirable sampler to gain a better understanding of the exposure of workers to hazardous substances when they are wearing respiratory protective equipment (RPE) or helmets with visors in the workplace. The study was in two parts and the first part, described herein, was to develop the sampler and test its collection characteristics. Assessment of the impact of the sampler on RPE safety and its comparability with traditional laboratory-based approaches to measure protection factors was discussed in a second article. The miniature sampler (weight-5.4g, length-13mm) was designed to fit into the space available between the nose and chin of an individual inside a filtering facepiece type mask and has a radially omnidirectional inlet with a porous foam particle selector that allows the collection of the respirable fraction on a downstream filter. The sampling efficiency was compared with the respirable convention. A close match with the respirable convention was obtained at a flow rate of 1 l min-1 and the 50% penetration cut off value (d 50) was 4.08 µm. After 3 hours sampling in high humidity (95%), the penetration curve had shifted towards smaller particle sizes (d 50 = 3.81 µm) with 88% of the calculated bias values within 10%. The miniature sampler measured respirable dust and crystalline silica mass concentrations comparable with performance of the Safety In Mines Personal Dust Sampler (SIMPEDS), commonly used in Great Britain, at a flow rate of 0.8 l min-1 The d 50 for the miniature sampler at 0.8 l min-1 (4.4 µm) is within 5% of the d 50 of the SIMPEDS at its prescribed flow rate of 2.2 l min-1 (4.2 µm). These results indicated that the miniature sampler was a good candidate to proceed with tests with RPE described in the second part of this series of two papers.

Collection efficiencies of high flow rate personal respirable samplers when measuring Arizona road dust and analysis of quartz by x-ray diffraction.

Prolonged exposure to respirable crystalline silica (RCS) causes silicosis and is also considered a cause of cancer. To meet emerging needs for precise measurements of RCS, from shorter sampling periods (<4 h) and lower air concentrations, collaborative work was done to assess the differences between personal respirable samplers at higher flow rates. The performance of FSP10, GK2.69, and CIP 10 R samplers were compared with that of the Safety In Mines Personal Dust Sampler (SIMPEDS) sampler as a reference, which is commonly used in the UK for the measurement of RCS. In addition, the performance of the FSP10 and GK 2.69 samplers were compared; at the nominal flow rates recommended by the manufacturers of 10 and 4.2 l · min(-1) and with flow rates proposed by the National Institute for Occupational Safety and Health of 11.2 and 4.4 l · min(-1). Samplers were exposed to aerosols of ultrafine and medium grades of Arizona road dust (ARD) generated in a calm air chamber. All analyses for RCS in this study were performed at the Health and Safety Laboratory. The difference in flow rates for the GK2.69 is small and does not result in a substantial difference in collection efficiency for the dusts tested, while the performance of the FSP10 at 11.2 l · min(-1) was more comparable with samples from the SIMPEDS. Conversely, the GK2.69 collected proportionately more crystalline silica in the respirable dust than other samplers, which then produced RCS results most comparable with the SIMPEDS. The CIP 10 R collected less ultrafine ARD than other samplers, as might be expected based on earlier performance evaluations. The higher flow rate for the FSP10 should be an added advantage for task-specific sampling or when measuring air concentrations less than current occupational exposure limits.

Preparation and certification of two new bulk welding fume reference materials for use in laboratories undertaking analysis of occupational hygiene samples.

Workers can be exposed to fume, arising from welding activities, which contain toxic metals and metalloids. Occupational hygienists need to assess and ultimately minimize such exposure risks. The monitoring of the concentration of particles in workplace air is one assessment approach whereby fume, from representative welding activities, is sampled onto a filter and returned to a laboratory for analysis. Inductively coupled plasma-atomic emission spectrometry and inductively coupled plasma-mass spectrometry are generally employed as instrumental techniques of choice for the analysis of such filter samples. An inherent difficulty, however, with inductively coupled plasma-based analytical techniques is that they typically require a sample to be presented for analysis in the form of a solution. The efficiency of the required dissolution step relies heavily upon the skill and experience of the analyst involved. A useful tool in assessing the efficacy of this dissolution step would be the availability and subsequent analysis of welding fume reference materials with stated elemental concentrations and matrices that match as closely as possible the matrix composition of welding fume samples submitted to laboratories for analysis. This article describes work undertaken at the Health and Safety Laboratory to prepare and certify two new bulk welding fume reference materials that can be routinely used by analysts to assess the performance of the digestion procedures they employ in their laboratories.

Integrated Direct-to-Biology Platform for the Nanoscale Synthesis and Biological Evaluation of PROTACs.

Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that co-opt the cell's natural proteasomal degradation mechanisms to degrade undesired proteins. A challenge associated with PROTACs is the time and resource-intensive optimization; thus, the development of high-throughput platforms for their synthesis and biological evaluation is required. In this study, we establish an ultra-high-throughput experimentation (ultraHTE) platform for PROTAC synthesis, followed by direct addition of the crude reaction mixtures to cellular degradation assays without any purification. This 'direct-to-biology' (D2B) approach was validated and then exemplified in a medicinal chemistry campaign to identify novel BRD4 PROTACs. Using the D2B platform, the synthesis of 650 PROTACs was carried out in a 1536-well plate, and subsequent biological evaluation was performed by a single scientist in less than 1 month. Due to its ability to hugely accelerate the optimization of new degraders, we anticipate our platform will transform the synthesis and testing of PROTACs.

Multicomponent Measurement of Respirable Quartz, Kaolinite and Coal Dust using Fourier Transform Infrared Spectroscopy (FTIR): A Comparison Between Partial Least Squares and Principal Component Regressions.

Exposure to respirable crystalline silica (RCS) is potentially hazardous to the health of thousands of workers in Great Britain. Both X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy can be used to measure RCS to assess exposures. The current method outlined in the Health and Safety Executive's (HSE) Methods for the Determination of Hazardous Substances (MDHS) guidance series is 'MDHS 101 Crystalline silica in respirable airborne dust - Direct-on-filter analyses by infrared spectroscopy or x-ray'. This describes a procedure for the determination of time-weighted average concentrations of RCS either as quartz or cristobalite in airborne dust. FTIR is more commonly employed because it is less expensive, potentially portable and relatively easy to use. However, the FTIR analysis of RCS is affected by spectral interference from silicates. Chemometric techniques, known as Partial Least Squares Regression (PLSR) and Principal Component Regression (PCR), are two computational processes that have the capability to remove spectral interference from FTIR spectra and correlate spectral features with constituent concentrations. These two common chemometric processes were tested on artificial mixtures of quartz and kaolinite in coal dust using the same commercially available software package. Calibration, validation and prediction samples were prepared by collecting aerosols of these dusts onto polyvinylchloride (PVC) filters using a Safety in Mines Personal Dust Sampler (SIMPEDS) respirable cyclone. PCR and PLSR analyses were compared when processing the same spectra. Good correlations between the target values, measured using XRD, were obtained for both the PCR and PLSR models e.g. 0.98-0.99 (quartz), 0.98-0.98 (kaolinite) and 0.96-0.97 (coal). The level of agreement between PCR and PLSR was within the 95% confidence value for each analyte. Slight differences observed between predicted PCR and PLSR values were due to the number of optimal principal components applied to each chemometric process. The presence of kaolinite in these samples caused an 18% overestimation of quartz, for the FTIR, when following MDHS 101 without a chemometric method. Chemometric methods are a useful approach to obtain interference-free results for the measurement of RCS from some workplace environments and to provide a multicomponent analysis to better characterise exposures of workers.

Application of a Fourier Transform Infrared (FTIR) Principal Component Regression (PCR) Chemometric Method for the Quantification of Respirable Crystalline Silica (Quartz), Kaolinite, and Coal in Coal Mine Dusts from Australia, UK, and South Africa.

This article describes the approach used to assess the performance of a Fourier transform infrared (FTIR) and principal component regression (PCR) chemometric method when measuring respirable quartz, kaolinite, and coal in samples from a variety of mines from different countries; relative to target assigned values determined using X-ray diffraction (XRD). For comparison, FTIR results using the partial least squares regression (PLSR) method are also available. Bulk dusts from 10 Australian mines were scanned using XRD and grouped into three sets based on the levels of quartz, kaolinite, and feldspar within their crystalline mineral composition. Prediction samples were generated from 5 of these Australian mine dusts, Durrans coal dust, 2 mine dusts from the UK, and a single South African mine dust (71 samples in total) by collecting the aerosolized respirable dust onto 25-mm diameter polyvinylchloride filters using the Safety in Mines Personal Dust Sampler (SIMPEDS) operating at 2.2 l min-1. The predicted values from the FTIR chemometric methods were compared with assigned target values determined using a direct on-aerosol filter XRD analysis method described in Method for the Determination of Hazardous Substances (MDHS) 101. Limits of detection (LOD) and uncertainty values for each analyte were calculated from a linear regression between target and predicted values. The uncertainty was determined using the calibration uncertainty equation for an unweighted regression. FTIR results from PCR and PLSR are very similar. For the PCR method, the LOD for quartz, kaolinite, and coal were 5, 25, and 71 µg, respectively. For quartz, an LOD of 5 µg corresponds to an airborne quartz concentration of 10 µg m-3, assuming a 4-h sampling time and collection flow rate of 2.2 l min-1. The FTIR measurement met the expected performance criteria outlined in ISO 20581 when sampling quartz for more than 4 h using a flow rate of 2.2 l min-1 at a concentration of 0.1 mg m-3 (100 µg m-3), the current workplace exposure limit in Great Britain. This method met the same performance criteria when measuring exposures at the Australian Workplace Exposure Standard (WES) concentration of 0.05 mg m-3, although in this case a sampling period greater than 8 h was needed.

Characterizing and Comparing Emissions of Dust, Respirable Crystalline Silica, and Volatile Organic Compounds from Natural and Artificial Stones.

The frequency of cases of accelerated silicosis associated with exposure to dust from processing artificial stones is rapidly increasing globally. Artificial stones are increasingly popular materials, commonly used to fabricate kitchen and bathroom worktops. Artificial stones can contain very high levels of crystalline silica, hence cutting and polishing them without adequate exposure controls represents a significant health risk. The aim of this research was to determine any differences in the emission profiles of dust generated from artificial and natural stones when cutting and polishing. For artificial stones containing resins, the nature of the volatile organic compounds (VOCs) emitted during processing was also investigated. A selection of stones (two natural, two artificial containing resin, and one artificial sintered) were cut and polished inside a large dust tunnel to characterize the emissions produced. The inhalable, thoracic, and respirable mass concentrations of emissions were measured gravimetrically and the amount of crystalline silica in different size fractions was determined by X-ray diffraction. Emissions were viewed using scanning electron microscopy and the particle size distribution was measured using a wide range aerosol spectrometer. VOCs emitted when cutting resin-artificial stones were also sampled. The mass of dust emitted when cutting stones was higher than that emitted when polishing. For each process, the mass of dust generated was similar whether the stone was artificial or natural. The percentage of crystalline silica in bulk stone is likely to be a reasonable, or conservative, estimate of that in stone dust generated by cutting or polishing. Larger particles were produced when cutting compared with when polishing. For each process, normalized particle size distributions were similar whether the stone was artificial or natural. VOCs were released when cutting resin-artificial stones. The higher the level of silica in the bulk material, the higher the level of silica in any dust emissions produced when processing the stone. When working with new stones containing higher levels of silica, existing control measures may need to be adapted and improved in order to achieve adequate control.

A Phenotypic Approach for the Identification of New Molecules for Targeted Protein Degradation Applications.

Targeted protein degradation is an emerging new strategy for the modulation of intracellular protein levels with applications in chemical biology and drug discovery. One approach to enable this strategy is to redirect the ubiquitin-proteasome system to mark and degrade target proteins of interest (POIs) through the use of proteolysis targeting chimeras (PROTACs). Although great progress has been made in enabling PROTACs as a platform, there are still a limited number of E3 ligases that have been employed for PROTAC design. Herein we report a novel phenotypic screening approach for the identification of E3 ligase binders. The key concept underlying this approach is the high-throughput modification of screening compounds with a chloroalkane moiety to generate HaloPROTACs in situ, which were then evaluated for their ability to degrade a GFP-HaloTag fusion protein in a cellular context. As proof of concept, we demonstrated that we could generate and detect functional HaloPROTACs in situ, using a validated Von Hippel-Lindau (VHL) binder that successfully degraded the GFP-HaloTag fusion protein in living cells. We then used this method to prepare and screen a library of approximately 2000 prospective E3 ligase-recruiting molecules.

An indirect Raman spectroscopy method for the quantitative measurement of respirable crystalline silica collected on filters inside respiratory equipment.

This article describes the development of an analytical method to measure respirable crystalline silica (RCS) collected on filters by a miniature sampler placed behind respirators worn by workers to evaluate their 'true' exposure. Test samples were prepared by aerosolising a calibration powder (Quin B) and by pipetting aliquots from suspensions of bulk material (NIST 1878a and Quin B) onto filters. Samples of aerosolised RCS collected onto polyvinyl chloride PVC filters were ashed and their residue was suspended in isopropanol and filtered into a 10 mm diameter area onto silver filters. Samples were also collected by the Health and Safety Executive's (HSE) miniature sampler from within the facepiece of a respirator on a breathing manikin during a simulated work activity. Results obtained using Raman spectroscopy were compared with X-ray diffraction (XRD) measurements, which was used as a reference method and a linear relationship was obtained. Raman has similar estimates of uncertainty when compared with the XRD methods over the measurement range from 5 to 50 µg and obtained the lowest limit of detection (LOD) of 0.26 µg when compared with XRD and Fourier Transform Infrared FTIR methods. A significant intercept and slope coefficient greatly influenced the higher LOD for indirect XRD method. The level of precision and low LOD for Raman spectroscopy will potentially enable workplace measurements at lower concentrations below the Workplace Exposure Limit (WEL) than are achieved using current analytical instrumentation. Different inward leakage ratio (ILR) measurement approaches were compared using six aerosolised sandstone dust tests. For the three highest inward leakage ratios the Portacount® obtained higher values than the RCS mass or the miniWRAS ratios, the latter of which reporting both particle number and quartz mass concentration. However, these limited ILR data were insufficient to establish statistical correlations between the measurement methods.

An International comparison of the crystallinity of calibration materials for the analysis of respirable alpha-quartz using X-ray diffraction and a comparison with results from the infrared KBr disc method.

It is important that analytical results, produced to demonstrate compliance with exposure limits are comparable, to ensure controls are monitored to similar standards. Correcting a measurement result of respirable alpha-quartz for the percentage of crystalline material in the calibration dust is good analytical practice and significant changes in the values assigned to calibration materials will affect the interpretation of results by an analyst or occupational hygiene professional. The reissue of the certification for the quartz reference material NIST 1878a in 2005 and differences in comparative values obtained by other work created uncertainty about the values of crystallinity assigned to national calibration dusts for alpha-quartz. Members of an International Organization for Standardization working group for silica measurement ISO/TC146/SC2/WG7 collaborated to investigate the comparability of results by X-ray diffraction (XRD) and to reach a consensus. This paper lists the values recommended by the working group for use with XRD analysis. The values for crystallinity obtained for some of the materials (NIST 1878, Min-U-Sil5 and A9950) were 6-7% lower than the original certification or estimates reported in other comparisons. Crystallinity values obtained by XRD gave a good correlation with BET surface area measurements (r2 = 0.91) but not with mean aerodynamic particle size (r2 = 0.31). Subsamples of two of the materials (A9950 Respirable and Quin 1 Respirable) with smaller particle size distribution than their parent material did not show any significant change in their values for crystallinity, suggesting that the area XRD measurement of these materials within the particle size range collected is more dependent on how the quartz is formed geologically or how it is processed for use. A comparison of results from laboratories using the infrared (IR) and KBr disc method showed that this method is more dependent than XRD on differences in the particle size within the respirable size range, whereas the XRD values were more consistent between the different measurement values obtained on each material. It was not possible to assign a value for percentage purity to each material for users of IR analysis. This work suggests that differences are likely to exist between the results from XRD and IR analysis when measuring 'real' workplace samples and highlights the importance of matching the particle size of the calibration material to the particle size of the workplace dust for measurements of crystalline quartz.

The Measurement of Wood in Construction Dust Samples: A Furnace Based Thermal Gravimetric Approach.

A furnace-based thermal gravimetric method was developed to measure wood in inhalable construction dust. The application of this method showed that reliance on the inhalable concentrations alone may substantially overestimate carpenters' exposures to wood dust at construction worksites. Test samples were prepared by collecting aerosols of gypsum, calcite, quartz, concrete, and wood dust onto quartz fibre filters using the Button inhalable sampler. The average difference between the measured and loaded mass of wood is 2% over the whole analytical range. Ninety percent of thermogravimetric measurements on all test samples (n = 35) were 13% or less. The limit of detection was measured as 0.065 mg. The thermal gravimetric method was applied to samples collected from four new build construction sites and one shop fitting worksite. The workplace inhalable wood dust results ranged from 15% to 104% of the total inhalable dust values. In addition, an x-ray diffraction (XRD) Rietveld method was applied as a complimentary approach to explain the composition of the remaining inhalable dust. Most combined thermal gravimetric and XRD measurements were within 10% of the total inhalable dust mass values, determined gravimetrically. Ninety-five percent were within 26%. The median proportion of mineral dust containing gypsum, calcite, quartz, dolomite, or rutile was 30%. The proportion of mineral dust on individual filters varied considerably.

Performance of laboratories analysing welding fume on filter samples: results from the WASP proficiency testing scheme.

This paper emphasizes the need for occupational hygiene professionals to require evidence of the quality of welding fume data from analytical laboratories. The measurement of metals in welding fume using atomic spectrometric techniques is a complex analysis often requiring specialist digestion procedures. The results from a trial programme testing the proficiency of laboratories in the Workplace Analysis Scheme for Proficiency (WASP) to measure potentially harmful metals in several different types of welding fume showed that most laboratories underestimated the mass of analyte on the filters. The average recovery was 70-80% of the target value and >20% of reported recoveries for some of the more difficult welding fume matrices were <50%. This level of under-reporting has significant implications for any health or hygiene studies of the exposure of welders to toxic metals for the types of fumes included in this study. Good laboratories' performance measuring spiked WASP filter samples containing soluble metal salts did not guarantee good performance when measuring the more complex welding fume trial filter samples. Consistent rather than erratic error predominated, suggesting that the main analytical factor contributing to the differences between the target values and results was the effectiveness of the sample preparation procedures used by participating laboratories. It is concluded that, with practice and regular participation in WASP, performance can improve over time.

Plate-Based Phenotypic Screening for Pain Using Human iPSC-Derived Sensory Neurons.

Screening against a disease-relevant phenotype to identify compounds that change the outcome of biological pathways, rather than just the activity of specific targets, offers an alternative approach to find modulators of disease characteristics. However, in pain research, use of in vitro phenotypic screens has been impeded by the challenge of sourcing relevant neuronal cell types in sufficient quantity and developing functional end-point measurements with a direct disease link. To overcome these hurdles, we have generated human induced pluripotent stem cell (hiPSC)-derived sensory neurons at a robust production scale using the concept of cryopreserved "near-assay-ready" cells to decouple complex cell production from assay development and screening. hiPSC sensory neurons have then been used for development of a 384-well veratridine-evoked calcium flux assay. This functional assay of neuronal excitability was validated for phenotypic relevance to pain and other hyperexcitability disorders through screening a small targeted validation compound subset. A 2700-compound chemogenomics screen was then conducted to profile the range of target-based mechanisms able to inhibit veratridine-evoked excitability. This report presents the assay development, validation, and screening data. We conclude that high-throughput-compatible pain-relevant phenotypic screening with hiPSC sensory neurons is feasible and ready for application for the identification of new targets, pathways, mechanisms of action, and compounds for modulating neuronal excitability.

Novel selective inhibitors of neutral endopeptidase for the treatment of female sexual arousal disorder. Synthesis and activity of functionalized glutaramides.

Female sexual arousal disorder (FSAD) is a highly prevalent sexual disorder affecting up to 40% of women. We describe herein our efforts to identify a selective neutral endopeptidase (NEP) inhibitor as a potential treatment for FSAD. The rationale for this approach, together with a description of the medicinal chemistry strategy, lead compounds, and SAR investigations are detailed. In particular, the strategy of starting with the clinically precedented selective NEP inhibitor, Candoxatrilat, and targeting low molecular weight and relatively polar mono-carboxylic acids is described. This led ultimately to the prototype development candidate R-13, for which detailed pharmacology and pharmacokinetic parameters are presented.(1)

Environmental Variation and the Persistence of Small Populations.

Understanding the factors that determine the continued survival of small populations is a central problem in conservation biology. The Acorn Woodpecker (Melanerpes formicivorus) naturally occurs in small, isolated populations throughout much of the American Southwest. In spite of this distributional pattern, the species is neither rare nor endangered. Thus it appears to have successfully "solved" the problems to the problems of habitat fragmentation. We used data from a 10-yr field study and simulation models to examine the effects of environmental stochasticity on population survival times. All simulated woodpecker populations went extinct within 49 yr, and the median survival time was only 16 yr. However, when immigration was allowed, persistence times greatly increased; with an immigrant rate of only five individuals per year, most populations lasted >1000 yr. The results of this and other analyses suggest that this population persists only because it is part of a larger "metapopulation," and because it is regularly rescued from extinction by immigration from other, independently varying, populations. This finding has important implications for the development of management strategies designed to preserve small populations that are faced with fragmented distributional patterns and high levels of environmental variation.