SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also purportedly plays an ...important role in the programmed cell death pathway (PD-1/PD-L1). Because it is an oncoprotein associated with multiple cancer-related diseases, as well as a potential immunomodulator, controlling SHP2 activity is of significant therapeutic interest. Recently in our laboratories, a small molecule inhibitor of SHP2 was identified as an allosteric modulator that stabilizes the autoinhibited conformation of SHP2. A high throughput screen was performed to identify progressable chemical matter, and X-ray crystallography revealed the location of binding in a previously undisclosed allosteric binding pocket. Structure-based drug design was employed to optimize for SHP2 inhibition, and several new protein–ligand interactions were characterized. These studies culminated in the discovery of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine (SHP099, 1), a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor.
SET and MYND domain-containing protein 2 (SMYD2) is a protein lysine methyltransferase that catalyzes the transfer of methyl groups from S-adenosylmethionine (AdoMet) to acceptor lysine residues on ...histones and other proteins. To understand the kinetic mechanism and the function of individual domains, human SMYD2 was overexpressed, purified, and characterized. Substrate specificity and product analysis studies established SMYD2 as a monomethyltransferase that prefers nonmethylated p53 peptide substrate. Steady-state kinetic and product inhibition studies showed that SMYD2 operates via a rapid equilibrium random Bi Bi mechanism at a rate of 0.048 ± 0.001 s–1, with K Ms for AdoMet and the p53 peptide of 0.031 ± 0.01 μM and 0.68 ± 0.22 μM, respectively. Metal analyses revealed that SMYD2 contains three tightly bound zinc ions that are important for maintaining the structural integrity and catalytic activity of SMYD2. Catalytic activity was also shown to be dependent on the GxG motif in the S-sequence of the split SET domain, as a G18A/G20A double mutant and a sequence deletion within the conserved motif impaired AdoMet binding and significantly decreased enzymatic activity. The functional importance of other SMYD2 domains including the MYND domain, the cysteine-rich post-SET domain, and the C-terminal domain (CTD), were also investigated. Taken together, these results demonstrated the functional importance of distinct domains in the SMYD family of proteins and further advanced our understanding of the catalytic mechanism of this family.
SHP2 is a nonreceptor protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also plays an important role in ...the programed cell death pathway (PD-1/PD-L1). As an oncoprotein as well as a potential immunomodulator, controlling SHP2 activity is of high therapeutic interest. As part of our comprehensive program targeting SHP2, we identified multiple allosteric binding modes of inhibition and optimized numerous chemical scaffolds in parallel. In this drug annotation report, we detail the identification and optimization of the pyrazine class of allosteric SHP2 inhibitors. Structure and property based drug design enabled the identification of protein–ligand interactions, potent cellular inhibition, control of physicochemical, pharmaceutical and selectivity properties, and potent in vivo antitumor activity. These studies culminated in the discovery of TNO155, (3S,4S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-2-oxa-8-azaspiro4.5decan-4-amine (1), a highly potent, selective, orally efficacious, and first-in-class SHP2 inhibitor currently in clinical trials for cancer.
Background: Aurora kinases (A, B and C) play a critical role in regulating mitosis and cell division. Aurora kinase A is required for correct spindle assembly while aurora kinase B is involved in ...histone H3 phosphorylation, chromosome segregation, and cytokinesis. Emerging data suggest these proteins are implicated in the survival and proliferation of both haematologic and solid malignancies, and therefore represent novel therapeutic targets. Several aurora kinase inhibitors have been described, mainly inhibiting both A and B kinases, although inhibition of aurora kinase B is more closely associated with the effects seen with these compounds. We have therefore investigated the activity of AZD1152, a novel, specific inhibitor of aurora kinase activity, with selectivity for aurora B.
Methods: Drug activity was studied in a panel of leukaemic cell lines (HL-60, MV411, THP-1, U937) and in primary AML cells (n=4) at concentrations of 0–1000 nM AZD1152 for up to 168 h. Cell number and percent viability were determined using a Viacount assay on a Guava PCA-96 analyser, cell cycle distribution (including apoptotic and polyploid (>4n) cells) by PI staining with flow cytometry, and the phosphorylation of an aurora kinase B substrate histone H3 by fluorescence immunocytochemistry using a phospho-specific antibody.
Results: AZD1152 displayed time- and concentration-dependent activity in cell lines and primary cells. A clear decrease in phospho histone H3 positive cells was apparent by 48 h, with <1% positive cells at 50 nM AZD1152 in cell lines compared with 8–10% in control cells and <0.5% positive cells in primary cultures at 100 nM AZD1152 compared with 2 - 4.5% in control cultures. In all cell lines studied a cell population with >4n DNA content by flow cytometry was apparent at 48 h (18–60% cells), confirmed by karyotype analysis which showed a variable but marked increase in chromosome number. This population had typically reduced, with a corresponding increase in apoptotic cells, by 96 h. In THP-1 cells, in which little effect on cell viability was observed, this >4n population increased to 80% by 96 h and persisted out to 168 h, with little apoptosis. In primary cells an increase in the G2/M population was evident in some cultures at 48 h, with only small changes in cells with >4n DNA content. In cell lines 10 nM AZD1152 resulted in a 28–65% reduction in cell number and a 6–35% reduction in cell viability after 48 h. At 100 nM these values increased to 65–78% and 6–42%, respectively. Notably little further increase in activity was observed at 1000 nM. By 96 h 100 nM AZD1152 reduced cell number by >80% in all cell lines, and cell viability by >67% in 3 lines (17% reduction in THP-1 cells). In primary AML cells AZD1152 over 48 h had little effect on cell number, but by 120 h this had decreased by >50% at 100 nM AZD1152, with a small effect on cell viability. Activity with 48 h exposure to drug followed by 72 h drug free was similar to that seen with a continuous 120 h exposure.
Conclusions: The specific aurora kinase inhibitor AZD1152 demonstrates antiproliferative and apoptotic activity in leukaemic cell lines and primary cells at concentrations associated with changes in the phosphorylation of an aurora kinase B substrate protein.
Sequence data are presented for the Saccharomyces cerevisiae
TAP1 gene and for a mutant allele, tap1-1, that activates transcription of the promoter-defective yeast SUP4 tRNA
Tyr
allele SUP4A53T61. ...The degree of in vivo activation of this allele by tap1-1 is strongly affected by the nature of the flanking DNA sequences at 5'-flanking DNA sequences as far away as 413 bp from the tRNA gene and by 3'-flanking sequences as well. We considered the possibility that this dependency is related to the nature of the chromatin assembled on these different flanking sequences. TAP1 encodes a protein 1,006 amino acids long. The tap1-1 mutation consists of a thymine-to-cytosine DNA change that changes amino acid 683 from tyrosine to histidine. Recently, Amberg et al. reported the cloning and sequencing of RAT1, a yeast gene identical to TAP1, by complementation of a mutant defect in poly(A) RNA export from the nucleus to the cytoplasm (D. C. Amberg, A. L. Goldstein, and C. N. Cole, Genes Dev. 6:1173-1189, 1992). The RAT1/TAP1 gene product has extensive sequence similarity to a yeast DNA strand transfer protein that is also a riboexonuclease (variously known as KEM1, XRN1, SEP1, DST2, or RAR5; reviewed by Kearsey and Kipling Trends Cell Biol. 1:110-112, 1991). The tap1-1 amino acid substitution affects a region of the protein in which KEM1 and TAP1 are highly similar in sequence.
The liquid and glass states of metal-organic frameworks (MOFs) have recently become of interest due to the potential for liquid-phase separations and ion transport, alongside the fundamental nature ...of the latter as a new, fourth category of melt-quenched glass. Here we show that the MOF liquid state can be blended with another MOF component, resulting in a domain structured MOF glass with a single, tailorable glass transition. Intra-domain connectivity and short range order is confirmed by nuclear magnetic resonance spectroscopy and pair distribution function measurements. The interfacial binding between MOF domains in the glass state is evidenced by electron tomography, and the relationship between domain size and T
investigated. Nanoindentation experiments are also performed to place this new class of MOF materials into context with organic blends and inorganic alloys.
The original version of this Article contained an error in Figure 1b, where the blue '(ZIF-4-Zn)
(ZIF-62)
blend' data curve was omitted from the enthalpy response plot. This has now been corrected in ...both the PDF and HTML versions of the Article.
People with psychosis have high rates of trauma, with a post-traumatic stress disorder (PTSD) prevalence rate of approximately 15%, which exacerbates psychotic symptoms such as delusions and ...hallucinations. Pilot studies have shown that trauma-focused (TF) psychological therapies can be safe and effective in such individuals. This trial, the largest to date, will evaluate the clinical effectiveness of a TF therapy integrated with cognitive behaviour therapy for psychosis (TF-CBTp) on post-traumatic stress symptoms in people with psychosis. The secondary aims are to compare groups on cost-effectiveness; ascertain whether TF-CBTp impacts on a range of other meaningful outcomes; determine whether therapy effects endure; and determine acceptability of the therapy in participants and therapists.
Rater-blind, parallel arm, pragmatic randomised controlled trial comparing TF-CBTp + treatment as usual (TAU) to TAU only. Adults (N = 300) with distressing post-traumatic stress and psychosis symptoms from five mental health Trusts (60 per site) will be randomised to the two groups. Therapy will be manualised, lasting 9 months (m) with trained therapists. We will assess PTSD symptom severity (primary outcome); percentage who show loss of PTSD diagnosis and clinically significant change; psychosis symptoms; emotional well-being; substance use; suicidal ideation; psychological recovery; social functioning; health-related quality of life; service use, a total of four times: before randomisation; 4 m (mid-therapy); 9 m (end of therapy; primary end point); 24 m (15 m after end of therapy) post-randomisation. Four 3-monthly phone calls will be made between 9 m and 24 m assessment points, to collect service use over the previous 3 months. Therapy acceptability will be assessed through qualitative interviews with participants (N = 35) and therapists (N = 5-10). An internal pilot will ensure integrity of trial recruitment and outcome data, as well as therapy protocol safety and adherence. Data will be analysed following intention-to-treat principles using generalised linear mixed models and reported according to Consolidated Standards of Reporting Trials-Social and Psychological Interventions Statement.
The proposed intervention has the potential to provide significant patient benefit in terms of reductions in distressing symptoms of post-traumatic stress, psychosis, and emotional problems; enable clinicians to implement trauma-focused therapy confidently in this population; and be cost-effective compared to TAU through reduced service use.
ISRCTN93382525 (03/08/20).
Osteogenesis imperfecta, fibrous dysplasia/McCune-Albright syndrome and X-linked hypophosphatemia are three rare musculoskeletal diseases characterised by bone deformities, frequent fractures and ...pain. Little high-quality research exists on appropriate treatment and long-term management of these conditions in adults. This is further worsened by limited research funding in rare diseases and a general mismatch between the existing research priorities and those of the patients. This partnership adopted the James Lind Alliance approach to identify the top 10 research priorities for rare musculoskeletal diseases in adults through joint patient, carer and healthcare professional collaboration.
The initial survey for question collection recruited 198 respondents, submitting a total of 988 questions. 77% of the respondents were patients with a rare musculoskeletal disease. Following out-of-scope question exclusion, repeating query grouping and scientific literature check for answers, 39 questions on treatment and long-term management remained. In the second public survey, 220 respondents, of whom 85% were patients with a rare musculoskeletal disease, their carers, relatives or friends, prioritised these uncertainties, which allowed selection of the top 25. In the last stage, patients, carers and healthcare professionals gathered for a priority setting workshop to reach a consensus on the final top 10 research priorities. These focus on the uncertainties surrounding appropriate treatment and holistic long-term disease management, highlighting several aspects indirect to abnormal bone metabolism, such as extra-skeletal symptoms, psychological care of both patients and their families and disease course through ageing.
This James Lind Alliance priority setting partnership is the first to investigate rare bone diseases. The priorities identified here were developed jointly by patients, carers and healthcare professionals. We encourage researchers, funding bodies and other stakeholders to use these priorities in guiding future research for those affected by rare musculoskeletal disorders.
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