UHRF1 is a key mediator of inheritance of epigenetic DNA methylation patterns during cell division and is a putative target for cancer therapy. Recent studies indicate that interdomain interactions ...critically influence UHRF1’s chromatin-binding properties, including allosteric regulation of its histone binding. Here, using an integrative approach that combines small angle X-ray scattering, NMR spectroscopy, and molecular dynamics simulations, we characterized the dynamics of the tandem tudor domain–plant homeodomain (TTD–PHD) histone reader module, including its 20-residue interdomain linker. We found that the apo TTD–PHD module in solution comprises a dynamic ensemble of conformers, approximately half of which are compact conformations, with the linker lying in the TTD peptide–binding groove. These compact conformations are amenable to cooperative, high-affinity histone binding. In the remaining conformations, the linker position was in flux, and the reader adopted both extended and compact states. Using a small-molecule fragment screening approach, we identified a compound, 4-benzylpiperidine-1-carboximidamide, that binds to the TTD groove, competes with linker binding, and promotes open TTD–PHD conformations that are less efficient at H3K9me3 binding. Our work reveals a mechanism by which the dynamic TTD–PHD module can be allosterically targeted with small molecules to modulate its histone reader function for therapeutic or experimental purposes.
Survival of motor neuron (SMN) functions in diverse biological pathways via recognition of symmetric dimethylarginine (Rme2s) on proteins by its Tudor domain, and deficiency of SMN leads to spinal ...muscular atrophy. Here we report a potent and selective antagonist with a 4-iminopyridine scaffold targeting the Tudor domain of SMN. Our structural and mutagenesis studies indicate that both the aromatic ring and imino groups of compound 1 contribute to its selective binding to SMN. Various on-target engagement assays support that compound 1 specifically recognizes SMN in a cellular context and prevents the interaction of SMN with the R1810me2s of RNA polymerase II subunit POLR2A, resulting in transcription termination and R-loop accumulation mimicking SMN depletion. Thus, in addition to the antisense, RNAi and CRISPR/Cas9 techniques, potent SMN antagonists could be used as an efficient tool to understand the biological functions of SMN.
Deacetoxycephalosporin C synthase (DAOCS) catalyzes the oxidative ring expansion of penicillin N (penN) to give deacetoxycephalosporin C (DAOC), which is the committed step in the biosynthesis of the ...clinically important cephalosporin antibiotics. DAOCS belongs to the family of non-heme iron(II) and 2-oxoglutarate (2OG) dependent oxygenases, which have substantially conserved active sites and are proposed to employ a consensus mechanism proceeding via formation of an enzyme·Fe(II)·2OG·substrate ternary complex. Previously reported kinetic and crystallographic studies led to the proposal of an unusual “ping-pong” mechanism for DAOCS, which was significantly different from other members of the 2OG oxygenase superfamily. Here we report pre-steady-state kinetics and binding studies employing mass spectrometry and NMR on the DAOCS-catalyzed penN ring expansion that demonstrate the viability of ternary complex formation in DAOCS catalysis, arguing for the generality of the proposed consensus mechanism for 2OG oxygenases.
The trend of measles in Afghanistan Nishat, Homeira; Aman, Mohammad Iqbal; Ibrahimi, Farahnaz ...
The Pan African medical journal,
2019, Letnik:
33
Journal Article
XIAP and cIAP1 are members of the inhibitor of apoptosis protein (IAP) family and are key regulators of anti-apoptotic and pro-survival signaling pathways. Overexpression of IAPs occurs in various ...cancers and has been associated with tumor progression and resistance to treatment. Structure-based drug design (SBDD) guided by structural information from X-ray crystallography, computational studies, and NMR solution conformational analysis was successfully applied to a fragment-derived lead resulting in AT-IAP, a potent, orally bioavailable, dual antagonist of XIAP and cIAP1 and a structurally novel chemical probe for IAP biology.
Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptosis and pro-survival signaling pathways whose deregulation is often associated with tumor genesis and tumor growth. IAPs have ...been proposed as targets for anticancer therapy, and a number of peptidomimetic IAP antagonists have entered clinical trials. Using our fragment-based screening approach, we identified nonpeptidic fragments binding with millimolar affinities to both cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP). Structure-based hit optimization together with an analysis of protein–ligand electrostatic potential complementarity allowed us to significantly increase binding affinity of the starting hits. Subsequent optimization gave a potent nonalanine IAP antagonist structurally distinct from all IAP antagonists previously reported. The lead compound had activity in cell-based assays and in a mouse xenograft efficacy model and represents a highly promising start point for further optimization.
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SET domain bifurcated protein 1 (SETDB1) is a human histone-lysine methyltransferase which is amplified in human cancers and was shown to be crucial in the growth of non-small and ...small cell lung carcinoma. In addition to its catalytic domain, SETDB1 harbors a unique tandem tudor domain which recognizes histone sequences containing both methylated and acetylated lysines, and likely contributes to its localization on chromatin. Using X-ray crystallography and NMR spectroscopy fragment screening approaches, we have identified the first small molecule fragment hits that bind to histone peptide binding groove of the Tandem Tudor Domain (TTD) of SETDB1. Herein, we describe the binding modes of these fragments and analogues and the biophysical characterization of key compounds. These confirmed small molecule fragments will inform the development of potent antagonists of SETDB1 interaction with histones.
Background User fees for primary care tend to suppress utilization, and many countries are experimenting with fee removal. Studies show that additional inputs are needed after removing fees, although ...well-documented experiences are lacking. This study presents data on the effects of fee removal on facility quality and utilization in Afghanistan, based on a pilot experiment and subsequent nationwide ban on fees. Methods Data on utilization and observed structural and perceived overall quality of health care were compared from before-and-after facility assessments, patient exit interviews and catchment area household surveys from eight facilities where fees were removed and 14 facilities where fee levels remained constant, as part of a larger health financing pilot study from 2005 to 2007. After a national user fee ban was instituted in 2008, health facility administrative data were analysed to assess subsequent changes in utilization and quality. Results The pilot study analysis indicated that observed and perceived quality increased across facilities but did not differ by fee removal status. Difference-in-difference analysis showed that utilization at facilities previously charging both service and drug fees increased by 400% more after fee removal, prompting additional inputs from service providers, compared with facilities that previously only charged service fees or had no change in fees (P = 0.001). Following the national fee ban, visits for curative care increased significantly (P < 0.001), but institutional deliveries did not. Services typically free before the ban—immunization and antenatal care—had immediate increases in utilization but these were not sustained. Conclusion Both pilot and nationwide data indicated that curative care utilization increased following fee removal, without differential changes in quality. Concerns raised by non-governmental organizations, health workers and community leaders over the effects of lost revenue and increased utilization require continued effort to raise revenues, monitor health worker and patient perceptions, and carefully manage health facility performance.
The DDR1 and DDR2 receptor tyrosine kinases are activated by extracellular collagen and have been implicated in a number of human diseases including cancer. We performed a fragment-based screen ...against DDR1 and identified fragments that bound either at the hinge or in the back pocket associated with the DFG-out conformation of the kinase. Modeling based on crystal structures of potent kinase inhibitors facilitated the “back-to-front” design of potent DDR1/2 inhibitors that incorporated one of the DFG-out fragments. Further optimization led to low nanomolar, orally bioavailable inhibitors that were selective for DDR1 and DDR2. The inhibitors were shown to potently inhibit DDR2 activity in cells but in contrast to unselective inhibitors such as dasatinib, they did not inhibit proliferation of mutant DDR2 lung SCC cell lines.
Structural and biochemical studies of the orf12 gene product (ORF12) from the clavulanic acid (CA) biosynthesis gene cluster are described. Sequence and crystallographic analyses reveal two domains: ...a C‐terminal penicillin‐binding protein (PBP)/β‐lactamase‐type fold with highest structural similarity to the class A β‐lactamases fused to an N‐terminal domain with a fold similar to steroid isomerases and polyketide cyclases. The C‐terminal domain of ORF12 did not show β‐lactamase or PBP activity for the substrates tested, but did show low‐level esterase activity towards 3′‐O‐acetyl cephalosporins and a thioester substrate. Mutagenesis studies imply that Ser173, which is present in a conserved SXXK motif, acts as a nucleophile in catalysis, consistent with studies of related esterases, β‐lactamases and D‐Ala carboxypeptidases. Structures of wild‐type ORF12 and of catalytic residue variants were obtained in complex with and in the absence of clavulanic acid. The role of ORF12 in clavulanic acid biosynthesis is unknown, but it may be involved in the epimerization of (3S,5S)‐clavaminic acid to (3R,5R)‐clavulanic acid.
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