: Human body fluids and materials were evaluated using attenuated total reflectance Fourier transform infrared spectroscopy. Purified proteins, cosmetics, and foodstuffs were also assayed with the ...method. The results of this study show that the sampled fluids and materials vary in the fingerprint region and locations of the amide I peaks because of the secondary structure of the composite proteins although the C=O stretch is always present. The distinct 1016 cm−1 peak serves as a signature for semen. The lipid‐containing materials (e.g., fingerprints, earwax, tears, and skin) can also be easily separated from the aqueous materials because of the strong CH3 asymmetric stretch of the former. Blood–saliva and blood–urine mixtures were also successfully differentiated using combinations of peaks. Crime scene investigators employing rapid, portable, or handheld infrared spectroscopic instruments may be able to reduce their need for invasive, destructive, and consumptive presumptive test reagents in evaluating trace evidence.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Protein kinases are highly tractable targets for drug discovery. However, the biological function and therapeutic potential of the majority of the 500+ human protein kinases remains unknown. We have ...developed physical and virtual collections of small molecule inhibitors, which we call chemogenomic sets, that are designed to inhibit the catalytic function of almost half the human protein kinases. In this manuscript we share our progress towards generation of a comprehensive kinase chemogenomic set (KCGS), release kinome profiling data of a large inhibitor set (Published Kinase Inhibitor Set 2 (PKIS2)), and outline a process through which the community can openly collaborate to create a KCGS that probes the full complement of human protein kinases.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The highly diverse Numb-associated kinase (NAK) family has been linked to broad cellular functions including receptor-mediated endocytosis, Notch pathway modulation, osteoblast differentiation, and ...dendrite morphogenesis. Consequently, NAK kinases play a key role in a diverse range of diseases from Parkinson's and prostate cancer to HIV. Due to the plasticity of this kinase family, NAK kinases are often inhibited by approved or investigational drugs and have been associated with side effects, but they are also potential drug targets. The presence of cysteine residues in some NAK family members provides the possibility for selective targeting via covalent inhibition. Here we report the first high-resolution structures of kinases AAK1 and BIKE in complex with two drug candidates. The presented data allow a comprehensive structural characterization of the NAK kinase family and provide the basis for rational design of selective NAK inhibitors.
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•First crystal structures of AAK1 and BIKE solved, completing the NAK family•Structural analysis of NAKs performed, revealing unusual family architecture•144 clinical kinase inhibitors screened against AAK1, BIKE, GAK, and MPSK1•Nanomolar and covalent inhibitors discovered from clinical kinase library
The diverse Numb-associated kinases have broad cellular functions and consequently are linked with diverse-ranging diseases as well as off-target effects of clinical drugs. The first structures of AAK1 and BIKE reveal that all members of the family share unusual activation segment architecture and are highly druggable.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Activated RAS GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small ...molecules, such as SCH51344, but their molecular mechanism of action remains generally unknown. Here, using a chemical proteomic approach, we identify the target of SCH51344 as the human mutT homologue MTH1 (also known as NUDT1), a nucleotide pool sanitizing enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells, whereas MTH1 overexpression mitigated sensitivity towards SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys and MTH1 co-crystal structures of both enantiomers provide a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single-strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation, and small-molecule inhibitors of MTH1 in general as a promising novel class of anticancer agents.
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Background
Positioning of total hip bearings involves tradeoffs, because cup orientations most favorable in terms of stability are not necessarily ideal in terms of reduction of contact stress and ...wear potential. Previous studies and models have not addressed these potentially competing considerations for optimal total hip arthroplasty (THA) function.
Questions/purposes
We therefore asked if component positioning in total hips could be addressed in terms of balancing bearing surface wear and stability. Specifically, we sought to identify acetabular component inclination and anteversion orientation, which simultaneously resulted in minimal wear while maximizing construct stability, for several permutations of femoral head diameter and femoral stem anteversion.
Methods
A validated metal-on-metal THA finite element (FE) model was used in this investigation. Five dislocation-prone motions as well as gait were considered as were permutations of femoral anteversion (0°–30°), femoral head diameter (32–48 mm), cup inclination (25°–75°), and cup anteversion (0°–50°), resulting in 4320 distinct FE simulations. A novel metric was developed to identify a range of favorable cup orientations (so-called “landing zone”) by considering both surface wear and component stability.
Results
When considering both wear and stability with equal weight, ideal cup position was more restrictive than the historically defined safe zone and was substantially more sensitive to cup anteversion than to inclination. Ideal acetabular positioning varied with both femoral head diameter and femoral version. In general, ideal cup inclination decreased with increased head diameter (approximately 0.5° per millimeter increase in head diameter). Additionally, ideal inclination increased with increased values of femoral anteversion (approximately 0.3° per degree increase in stem anteversion). Conversely, ideal cup anteversion increased with increased femoral head diameter (0.3° per millimeter increase) and decreased with increased femoral stem anteversion (approximately 0.3° per degree increase). Regressions demonstrated strong correlations between optimal cup inclination versus head diameter (Pearson’s r = −0.88), between optimal cup inclination versus femoral anteversion (r = 0.96), between optimal cup anteversion versus head diameter (r = 0.99), and between optimal cup anteversion and femoral anteversion (r = −0.98). For a 36-mm cup with a 20° anteverted stem, the ideal cup orientation was 46° ± 12° inclination and 15° ± 4° anteversion.
Conclusions
The range of cup orientations that maximized stability and minimized wear (so-called “landing zone”) was substantially smaller than historical guidelines and specifically did not increase with increased head size, challenging the presumption that larger heads are more forgiving. In particular, when the cup is oriented to improve not only stability, but also wear in the model, there was little or no added stability achieved by the use of larger femoral heads. Additionally, ideal cup positioning was more sensitive to cup anteversion than to inclination.
Clinical Relevance
Positioning THA bearings involves tradeoffs regarding stability and long-term bearing wear. Cup positions most favorable to minimization of wear such as low inclination and elevated anteversion were detrimental in terms of construct stability. Orientations were identified that best balanced the competing considerations of wear and stability.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UL, UM, UPUK, VKSCE, ZAGLJ
Dual-specificity tyrosine-(Y)-phosphorylation-regulated kinases (DYRKs) play key roles in brain development, regulation of splicing, and apoptosis, and are potential drug targets for ...neurodegenerative diseases and cancer. We present crystal structures of one representative member of each DYRK subfamily: DYRK1A with an ATP-mimetic inhibitor and consensus peptide, and DYRK2 including NAPA and DH (DYRK homology) box regions. The current activation model suggests that DYRKs are Ser/Thr kinases that only autophosphorylate the second tyrosine of the activation loop YxY motif during protein translation. The structures explain the roles of this tyrosine and of the DH box in DYRK activation and provide a structural model for DYRK substrate recognition. Phosphorylation of a library of naturally occurring peptides identified substrate motifs that lack proline in the P+1 position, suggesting that DYRK1A is not a strictly proline-directed kinase. Our data also show that DYRK1A wild-type and Y321F mutant retain tyrosine autophosphorylation activity.
•Crystal structures of DYRK1A with ATP-mimetic inhibitor and peptide substrate•Crystal structure of DYRK2 including NAPA and DH box region•Determination of consensus substrate motifs for DYRK1A and DYRK2•Observation of DYRK1A autophosphorylation on tyrosine in vitro
Soundararajan et al. determine structures for Down Syndrome kinases DYRK1A, with an inhibitor and consensus peptide, and DYRK2, including NAPA and DH box regions. The structures explain the roles of the activation loop tyrosine and the DH-box in DYRK activation and provide a model for DYRK substrate recognition.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
NRF2 is emerging as a major regulator of cellular metabolism. However, most studies have been performed in cancer cells, where co-occurring mutations and tumor selective pressures complicate the ...influence of NRF2 on metabolism. Here we use genetically engineered, non-transformed primary murine cells to isolate the most immediate effects of NRF2 on cellular metabolism. We find that NRF2 promotes the accumulation of intracellular cysteine and engages the cysteine homeostatic control mechanism mediated by cysteine dioxygenase 1 (CDO1), which catalyzes the irreversible metabolism of cysteine to cysteine sulfinic acid (CSA). Notably,
is preferentially silenced by promoter methylation in human non-small cell lung cancers (NSCLC) harboring mutations in KEAP1, the negative regulator of NRF2. CDO1 silencing promotes proliferation of NSCLC by limiting the futile metabolism of cysteine to the wasteful and toxic byproducts CSA and sulfite (SO
), and depletion of cellular NADPH. Thus, CDO1 is a metabolic liability for NSCLC cells with high intracellular cysteine, particularly NRF2/KEAP1 mutant cells.
Despite the success of protein kinase inhibitors as approved therapeutics, drug discovery has focused on a small subset of kinase targets. Here we provide a thorough characterization of the Published ...Kinase Inhibitor Set (PKIS), a set of 367 small-molecule ATP-competitive kinase inhibitors that was recently made freely available with the aim of expanding research in this field and as an experiment in open-source target validation. We screen the set in activity assays with 224 recombinant kinases and 24 G protein-coupled receptors and in cellular assays of cancer cell proliferation and angiogenesis. We identify chemical starting points for designing new chemical probes of orphan kinases and illustrate the utility of these leads by developing a selective inhibitor for the previously untargeted kinases LOK and SLK. Our cellular screens reveal compounds that modulate cancer cell growth and angiogenesis in vitro. These reagents and associated data illustrate an efficient way forward to increasing understanding of the historically untargeted kinome.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Crime scene investigators and laboratory analysts use chemical tests to detect and differentiate body fluids. Testing often requires a sample of the stain, and the chemicals may cause degradation of ...the fluid or interfere with subsequent tests. Colorimetric chemical tests do not differentiate between different types of the same fluid, such as venous and menstrual blood, and there is no presumptive test available to simultaneously differentiate several body fluids. In this study, we recorded ATR FT‐IR spectra of venous and menstrual blood, semen, saliva, and breastmilk. Neat and simulated casework body fluid samples were analyzed on cotton, nylon, wood, paper, and glass substrates. Differences in fluid composition, including proteins and small molecules, resulted in spectral differences. Venous and menstrual blood is differentiated by the peak at 1039 cm−1 attributed to phosphoric acid found in menstrual blood. Peak intensity is influenced by the porosity and weave of the substrate fabric.
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