scholarly journals Re-Evaluating the Mechanism of Action of α,β-Unsaturated Carbonyl DUB Inhibitors B-AP15 and VLX1570: A Paradigmatic Example of Unspecific Protein Crosslinking with Michael Acceptor Motif-Containing Drugs

2019 ◽  
Author(s):  
Jennifer Ward ◽  
Adán Pinto-Fernández ◽  
LoÏc Cornelissen ◽  
Sarah Bonham ◽  
Laura Diaz Saez ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Specific Protein ◽  
Deubiquitinating Enzymes ◽  
Target Class ◽  
Intact Cells ◽  
High Molecular Weight Complex ◽  
Diverse Range ◽  
Disease States ◽  
Proteomic Approach ◽  
Clinical Hold

<div>Deubiquitinating enzymes are a growing target class across multiple disease states, with several inhibitors now reported. b-AP15 and VLX1570 are two structurally related USP14/UCH-37 inhibitors with a shared α,β-unsaturated carbonyl substructure motif. Initially taken forward into a phase I/II clinical trial for refractory multiple myeloma, VLX1570 has since been put on full clinical hold due to dose limiting toxicity. Through a proteomic approach, here we demonstrate that these compounds target a diverse range of proteins, resulting in the formation of higher molecular weight complexes. Activity-based proteome profiling identified CIAPIN1 as a sub-micromolar covalent target of VLX1570, and further analysis demonstrated that high molecular weight complex formation leads to aggregation of CIAPIN1 in intact cells. Our results suggest that in addition to DUB inhibition, these compounds induce non-specific protein aggregation through cross-linking, providing a molecular explanation for general cellular toxicity.</div>

scholarly journals Re-Evaluating the Mechanism of Action of α,β-Unsaturated Carbonyl DUB Inhibitors B-AP15 and VLX1570: A Paradigmatic Example of Unspecific Protein Crosslinking with Michael Acceptor Motif-Containing Drugs

2019 ◽  
Author(s):  
Jennifer Ward ◽  
Adán Pinto-Fernández ◽  
LoÏc Cornelissen ◽  
Sarah Bonham ◽  
Laura Diaz Saez ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Specific Protein ◽  
Deubiquitinating Enzymes ◽  
Target Class ◽  
Intact Cells ◽  
High Molecular Weight Complex ◽  
Diverse Range ◽  
Disease States ◽  
Proteomic Approach ◽  
Clinical Hold

<div>Deubiquitinating enzymes are a growing target class across multiple disease states, with several inhibitors now reported. b-AP15 and VLX1570 are two structurally related USP14/UCH-37 inhibitors with a shared α,β-unsaturated carbonyl substructure motif. Initially taken forward into a phase I/II clinical trial for refractory multiple myeloma, VLX1570 has since been put on full clinical hold due to dose limiting toxicity. Through a proteomic approach, here we demonstrate that these compounds target a diverse range of proteins, resulting in the formation of higher molecular weight complexes. Activity-based proteome profiling identified CIAPIN1 as a sub-micromolar covalent target of VLX1570, and further analysis demonstrated that high molecular weight complex formation leads to aggregation of CIAPIN1 in intact cells. Our results suggest that in addition to DUB inhibition, these compounds induce non-specific protein aggregation through cross-linking, providing a molecular explanation for general cellular toxicity.</div>

Investigation by HPLC of the Catabolism of Recombinant Tissue Plasminogen Activator in the Rat

1988 ◽  
Vol 60 (01) ◽  
pp. 107-112 ◽  
Author(s):  
Roy Harris ◽  
Louis Garcia Frade ◽  
Lesley J Creighton ◽  
Paul S Gascoine ◽  
Maher M Alexandroni ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Half Life ◽  
Recombinant Tissue Plasminogen Activator ◽  
Rat Plasma ◽  
High Molecular Weight Complex ◽  
Molecular Weights ◽  
Major Activity ◽  
Tissue Plasminogen

SummaryThe catabolism of recombinant tissue plasminogen activator (rt-PA) was investigated after injection of radiolabelled material into rats. Both Iodogen and Chloramine T iodination procedures yielded similar biological activity loss in the resultant labelled rt-PA and had half lives in the rat circulation of 1 and 3 min respectively. Complex formation of rt-PA was investigated by HPLC gel exclusion (TSK G3000 SW) fractionation of rat plasma samples taken 1-2 min after 125I-rt-PA injection. A series of radiolabelled complexes of varying molecular weights were found. However, 60% of the counts were associated with a single large molecular weight complex (350–500 kDa) which was undetectable by immunologically based assays (ELISA and BIA) and showed only low activity with a functional promoter-type t-PA assay. Two major activity peaks in the HPLC fractions were associated with Tree t-PA and a complex having a molecular weight of ̴ 180 kDa. HPLC fractionation to produce these three peaks at various timed intervals after injection of 125I-rt-PA showed each to have a similar initial rate half life in the rat circulation of 4-5 min. The function of these complexes as yet is unclear but since a high proportion of rt-PA is associated with a high molecular weight complex with a short half life in the rat, we suggest that the formation of this complex may be a mechanism by which t-PA activity is initially regulated and finally cleared from the rat circulation.

Novel Pharmacotherapeutics for Stress-Related Disorders

2019 ◽  
pp. 642-672
Author(s):  
Jamie E. Mondello ◽  
Jenny E. Pak ◽  
Dennis F. Lovelock ◽  
Terrence Deak
Keyword(s):  
Mental Health Problems ◽  
Drug Efficacy ◽  
Careful Consideration ◽  
Pharmacological Intervention ◽  
Pharmacological Interventions ◽  
Diverse Range ◽  
Disease States ◽  
Efficacy Trials ◽  
Participant Selection ◽  
The Relationship

Most mental health problems associated with psychological distress originate with activation of centrally regulated stress pathways, yet a diverse range of central nervous system and somatic disease states can be influenced by exposure to severe or unrelenting stress. The goal of this chapter is to provide a conceptual framework to guide the development of pharmacological intervention strategies. We propose that careful consideration of the relationship between the timing of stressful life experiences, pharmacological intervention, and the ultimate expression of disease symptomatology is critical for the development of pharmacological interventions to treat stress-related disorders. We review a range of physiological systems that are known to be activated by stress, offering potentially new targets for drug development efforts, and argue that participant selection is a key predictor of drug efficacy trials. In doing so, we point toward inflammatory signaling pathways as a potential final common mediator of multiple stress-related disease states.

scholarly journals A “Target Class” Screen to Identify Activators of Two-Pore Domain Potassium (K2P) Channels

2020 ◽  
pp. 247255522097612
Author(s):  
David McCoull ◽  
Emma Ococks ◽  
Jonathan M. Large ◽  
David C. Tickle ◽  
Alistair Mathie ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Point Of View ◽  
Resting Membrane Potential ◽  
Small Molecule Activation ◽  
Target Class ◽  
Index Set ◽  
Diverse Range ◽  
K2p Channels ◽  
Additional Approach ◽  
Pore Domain

Two-pore domain potassium (K2P) channels carry background (or leak) potassium current and play a key role in regulating resting membrane potential and cellular excitability. Accumulating evidence points to a role for K2Ps in human pathophysiologies, most notably in pain and migraine, making them attractive targets for therapeutic intervention. However, there remains a lack of selective pharmacological tools. The aim of this work was to apply a “target class” approach to investigate the K2P superfamily and identify novel activators across all the described subclasses of K2P channels. Target class drug discovery allows for the leveraging of accumulated knowledge and maximizing synergies across a family of targets and serves as an additional approach to standard target-based screening. A common assay platform using baculovirus (BacMam) to transiently express K2P channels in mammalian cells and a thallium flux assay to determine channel activity was developed, allowing the simultaneous screening of multiple targets. Importantly, this system, by allowing precise titration of channel function, allows optimization to facilitate the identification of activators. A representative set of channels (THIK-1, TWIK-1, TREK-2, TASK-3, and TASK-2) were screened against a library of Food and Drug Administration (FDA)-approved compounds and the LifeArc Index Set. Activators were then analyzed in concentration–response format across all channels to assess selectivity. Using the target class approach to investigate the K2P channels has enabled us to determine which of the K2Ps are amenable to small-molecule activation, de-risk multiple channels from a technical point of view, and identify a diverse range of previously undescribed pharmacology.

scholarly journals A theory relating the action of salts on bacterial respiration to their influence on the solubility of proteins

1947 ◽  
Vol 134 (875) ◽  
pp. 181-201 ◽  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Dehydrogenase Activity ◽  
Halophilic Bacteria ◽  
Convincing Evidence ◽  
Effect Of Temperature ◽  
Bacterial Respiration ◽  
Concentrated Solutions ◽  
Salting Out ◽  
Intact Cells

Evidence has been presented indicating that the action of concentrated solutions of salts on bacterial respiration may be partly explained in terms of salting-out. It has been suggested that the material upon which this action is exerted is probably one of the proteins concerned in respiration, perhaps a dehydrogenating enzyme. This theory provides satisfactory explanations for: ( a ) the relation between salt con­centration and rate of respiration or dehydrogenase activity; ( b ) the effect of temperature on this relation; and ( c ) the effect of pH on this relation, if it is further supposed that only the zwitterionic fraction of the protein is involved. The relative actions of various salts are in fair agreement with this suggestion, but provide no very convincing evidence either for or against it. The chief point of difficulty lies in the range of concentration over which the action is manifest. With halophilic bacteria, the evidence is consonant with the above view if the protein involved is one of high molecular weight. With normal organisms the salt concentra­tions are much lower than those causing salting-out. There is a little evidence that in normal organisms the dehydrogenating enzymes are less sensitive to salts than the intact cells, which may be the source of the discrepancy. No reason for this can yet be suggested, but the property must be absent from the enzymes of halophilic organisms, and whatever it is, its absence must be the foundation of the halophilic character.

P4-129 Genetic study of APH-1 and PEN-2, components of presenilin high molecular weight complex

2004 ◽  
Vol 25 ◽  
pp. S512
Author(s):  
Toshitaka Kawarai ◽  
Antonio Orlacchio ◽  
Ekaterina Rogaeva ◽  
Susan Ling ◽  
Hiroshi Hasegawa ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Genetic Study ◽  
High Molecular Weight Complex

Purification of Human Urokinase and its Topographical Localisation in Renal Parenchyma

1975 ◽  
Author(s):  
K. Andrassy ◽  
E. Ritz ◽  
U. Bleyl ◽  
R. Egbring
Keyword(s):  
Molecular Weight ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Serum Proteins ◽  
Renal Tissue ◽  
Clot Lysis ◽  
Agar Gel ◽  
High Molecular Weight Complex ◽  
Zone Electrophoresis ◽  
Human Serum Proteins

Urokinase Leo was separated by agar zone electrophoresis into an anodic and cathodic fraction. The cathodic fraction, isolated from agar gel by ultracentrifugation, showed two precipitation bands with rabbit Urokinase antibodies. Band I displayed main Urokinase activity, in band II Urokinase was present in a high molecular weight complex with human serum proteins (albumin, a2-macroglobulin, a2HS glycoprotein); with affinity chromatography further separation of Urokinase isoenzymes from serum proteins was possible. The isoelectric point of these two Urokinase isoenzymes were pH 6.8 and pH 8.7 respectively in preliminary results with isoelectric focusing. Purification steps were controlled by disc gel electrophoresis and immunological techniques (Ouchterlony technique, Immunoelectrophoresis, clot lysis test with Urokinase antibodies).Topographic localisation of Urokinase in renal tissue, investigated with antibodies against Urokinase isoenzymes, revealed Urokinase activity both in the iuxtamedullary region (V. arcuatae, V. interlobulares, less V. recta) and in calyceal epithelia of the renal pelvis.

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