In vitro kinetic measurements allow mechanistic characterization of binding interactions and are particularly valuable throughout drug discovery, from confirmation of on-target binding in early ...discovery to fine-tuning of drug-binding properties in pre-clinical development. Early chemical matter often exhibits transient kinetics, which remain challenging to measure in a routine drug discovery setting. For example, characterization of irreversible inhibitors has classically relied on the alkylation rate constant, yet this metric fails to resolve its fundamental constituent rate constants, which drive reversible binding kinetics and affinity complex inactivation. In other cases, extremely rapid association processes, which can approach the diffusion limit, also remain challenging to measure. To address these limitations, a practical kinetic rebinding assay is introduced that may be applied for kinetic screening and characterization of compounds. The new capabilities afforded by this probe-based assay emerge from mixed-phase partitioning in a flow-injection configuration and have been implemented using label-free biosensing. A finite element analysis-based biosensor model, simulating inhibition of rebinding within a crowded hydrogel milieu, provided surrogate test data that enabled development and validation of an algebraic model for estimation of kinetic interaction constants. An experimental proof-of-principle demonstrating estimation of the association rate constant, decoupled from the dissociation process, provided further validation.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts no new class of antibiotic with activity against Gram-negative bacteria has been approved in over fifty ...years. Natural products and their derivatives have a key role in combating Gram-negative pathogens. Here we report chemical optimization of the arylomycins-a class of natural products with weak activity and limited spectrum-to obtain G0775, a molecule with potent, broad-spectrum activity against Gram-negative bacteria. G0775 inhibits the essential bacterial type I signal peptidase, a new antibiotic target, through an unprecedented molecular mechanism. It circumvents existing antibiotic resistance mechanisms and retains activity against contemporary multidrug-resistant Gram-negative clinical isolates in vitro and in several in vivo infection models. These findings demonstrate that optimized arylomycin analogues such as G0775 could translate into new therapies to address the growing threat of multidrug-resistant Gram-negative infections.
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KISLJ, NUK, SBMB, UL, UM, UPUK
In label-free biomolecular interaction analysis, a standard injection provides an injection of uniform analyte concentration. An alternative approach exploiting Taylor dispersion produces a ...continuous analyte titration allowing a full analyte dose response to be recorded in a single injection. The enhanced biophysical characterization that is possible with this new technique is demonstrated using a commercially available surface plasmon resonance-based biosensor. A kinetic interaction model was fitted locally to Taylor dispersion curves for estimation of the analyte diffusion coefficient in addition to affinity/kinetic constants. Statistical confidence in the measured parameters from a single Taylor dispersion injection was comparable to that obtained for global analysis of multiple standard injections. The affinity constants for multisite interactions were resolved with acceptable confidence limits. Importantly, a single analyte injection could be treated as a high-resolution real-time affinity isotherm and was demonstrated using the complex two-site interaction of warfarin with human serum albumin. In all three model interactions tested, the kinetic/affinity constants compared favorably with those obtained from standard kinetic analysis and the estimates of analyte diffusion coefficients were in good agreement with the expected values.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
A new method based on Taylor dispersion has been developed that enables an analyte gradient to be titrated over a ligand-coated surface for kinetic/affinity analysis of interactions from a minimal ...number of injections. Taylor dispersion injections generate concentration ranges in excess of four orders of magnitude and enable the analyte diffusion coefficient to be reliably estimated as a fitted parameter when fitting binding interaction models. A numerical model based on finite element analysis, Monte Carlo simulations, and statistical profiling were used to compare the Taylor dispersion method with standard fixed concentration injections in terms of parameter correlation, linearity of parameter error space, and global versus local model fitting. A dramatic decrease in parameter correlations was observed for TDi curves relative to curves from standard fixed concentration injections when surface saturation was achieved. In FCI the binding progress is recorded with respect to injection time, whereas in TDi the second time dependency encoded in the analyte gradient increases resolving power. This greatly lowers the dependence of all parameters on each other and on experimental interferences. When model parameters were fitted locally, the performance of TDis remained comparable to global model fitting, whereas fixed concentration binding response curves yielded unreliable parameter estimates.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Multidetector computed tomographic angiography (CTA) was evaluated alone and with imaging of the pelvic and thigh veins (CTA–CTV). As compared with a reference standard for the diagnosis of acute ...pulmonary embolism, CTA–CTV had a higher sensitivity than CTA alone, with similar specificity.
Multidetector computed tomographic angiography (CTA) was evaluated alone and with imaging of the pelvic and thigh veins (CTA–CTV). CTA–CTV had a higher sensitivity than CTA alone, with similar specificity.
Uncertainty persists about the accuracy of contrast-enhanced multidetector computed tomographic angiography (CTA) for the diagnosis of pulmonary embolism. The sensitivity of single-slice CTA has ranged from 60
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to 100
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percent, and the specificity has ranged from 81
1
to 100
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percent. A previous review focused on the diagnostic accuracy of single-slice CTA.
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Visualization of segmental and subsegmental pulmonary arteries is substantially better with four-slice CTA and thin collimation (1.25 mm) than with single-slice CTA.
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,
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In two studies of fewer than 100 patients, sensitivities for the detection of pulmonary embolism with four-slice CTA have been reported to be 96 percent
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and . . .
Abstract Polymyxins are gram-negative antibiotics that target lipid A, the conserved membrane anchor of lipopolysaccharide in the outer membrane. Despite their clinical importance, the molecular ...mechanisms underpinning polymyxin activity remain unresolved. Here, we use surface plasmon resonance to kinetically interrogate interactions between polymyxins and lipid A and derive a phenomenological model. Our analyses suggest a lipid A-catalyzed, three-state mechanism for polymyxins: transient binding, membrane insertion, and super-stoichiometric cluster accumulation with a long residence time. Accumulation also occurs for brevicidine, another lipid A-targeting antibacterial molecule. Lipid A modifications that impart polymyxin resistance and a non-bactericidal polymyxin derivative exhibit binding that does not evolve into long-lived species. We propose that transient binding to lipid A permeabilizes the outer membrane and cluster accumulation enables the bactericidal activity of polymyxins. These findings could establish a blueprint for discovery of lipid A-targeting antibiotics and provide a generalizable approach to study interactions with the gram-negative outer membrane.
The Ras-RAF-MEK-ERK signaling axis, commonly mutated in human cancers, is highly regulated to prevent aberrant signaling in healthy cells. One of the pathway modulators, 14-3-3, a constitutive dimer, ...induces RAF dimerization and activation by binding to a phosphorylated motif C-terminal to the RAF kinase domain. Recent work has suggested that a C-terminal "DTS" region in BRAF is necessary for this 14-3-3-mediated activation. We show that the catalytic activity and ATP binding affinity of the BRAF:14-3-3 complex is insensitive to the presence or absence of the DTS, while the ATP sites of both BRAF molecules are identical and available for binding. We also present a crystal structure of the apo BRAF:14-3-3 complex showing that the DTS is not required to attain the catalytically active conformation of BRAF. Rather, BRAF dimerization induced by 14-3-3 is the key step in activation, allowing the active BRAF:14-3-3 tetramer to achieve catalytic activity comparable to the constitutively active oncogenic BRAF V600E mutant.
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IJS, KILJ, NUK, PNG, UL, UM
The RAS-RAF-MEK-ERK signaling axis is frequently activated in human cancers. Physiological concentrations of ATP prevent formation of RAF kinase-domain (RAF
) dimers that are critical for activity. ...Here we present a 2.9-Å-resolution crystal structure of human BRAF
in complex with MEK and the ATP analog AMP-PCP, revealing interactions between BRAF and ATP that induce an inactive, monomeric conformation of BRAF
. We also determine how 14-3-3 relieves the negative regulatory effect of ATP through a 2.5-Å-resolution crystal structure of the BRAF
-14-3-3 complex, in which dimeric 14-3-3 enforces a dimeric BRAF
assembly to increase BRAF activity. Our data suggest that most oncogenic BRAF mutations alter interactions with ATP and counteract the negative effects of ATP binding by lowering the threshold for RAF dimerization and pathway activation. Our study establishes a framework for rationalizing oncogenic BRAF mutations and provides new avenues for improved RAF-inhibitor discovery.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Osteoclasts are large multinucleated bone-resorbing cells formed by the fusion of monocyte/macrophage-derived precursors that are thought to undergo apoptosis once resorption is complete. Here, by ...intravital imaging, we reveal that RANKL-stimulated osteoclasts have an alternative cell fate in which they fission into daughter cells called osteomorphs. Inhibiting RANKL blocked this cellular recycling and resulted in osteomorph accumulation. Single-cell RNA sequencing showed that osteomorphs are transcriptionally distinct from osteoclasts and macrophages and express a number of non-canonical osteoclast genes that are associated with structural and functional bone phenotypes when deleted in mice. Furthermore, genetic variation in human orthologs of osteomorph genes causes monogenic skeletal disorders and associates with bone mineral density, a polygenetic skeletal trait. Thus, osteoclasts recycle via osteomorphs, a cell type involved in the regulation of bone resorption that may be targeted for the treatment of skeletal diseases.
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•Osteoclasts fission into daughter cells called osteomorphs•Osteomorphs fuse and recycle back into osteoclasts•Osteomorph upregulated genes control bone structure and function in mice•Osteomorph upregulated genes are implicated in rare and common bone diseases in humans
Tracking osteoclasts during cycles of fission and fusion reveals a transcriptionally distinct “osteomorph” population that are fusion competent, motile, and capable of forming osteoclasts that resorb bone.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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