Discovery of very late antigen-4 (VLA-4, alpha4beta1 integrin) allosteric antagonists

Integrins are cell adhesion receptors that mediate cell-to-cell, or cell-to-extracellular matrix adhesion. They represent an attractive target for treatment of multiple diseases. Two classes of small molecule integrin inhibitors have been developed. Competitive antagonists bind directly to the integrin ligand binding pocket and thus disrupt the ligand-receptor interaction. Allosteric antagonists have been developed primarily for α(L)β(2)- integrin (LFA-1, lymphocyte function-associated antigen-1). Here we present the results of screening the Prestwick Chemical Library using a recently developed assay for the detection of α(4)β(1)-integrin allosteric antagonists. Secondary assays confirmed that the compounds identified: 1) do not behave like competitive (direct) antagonists; 2) decrease ligand binding affinity for VLA-4 ～2 orders of magnitude; 3) exhibit antagonistic properties at low temperature. In a cell based adhesion assay in vitro, the compounds rapidly disrupted cellular aggregates. In accord with reports that VLA-4 antagonists in vivo induce mobilization of hematopoietic progenitors into the peripheral blood, we found that administration of one of the compounds significantly increased the number of colony-forming units in mice. This effect was comparable to AMD3100, a well known progenitor mobilizing agent. Because all the identified compounds are structurally related, previously used, or currently marketed drugs, this result opens a range of therapeutic possibilities for VLA-4-related pathologies.     

SIRT1 Activation by Small Molecules: KINETIC AND BIOPHYSICAL EVIDENCE FOR DIRECT INTERACTION OF ENZYME AND ACTIVATOR?

SIRT1 is a protein deacetylase that has emerged as a therapeutic target for the development of activators to treat diseases of aging. SIRT1-activating compounds (STACs) have been developed that produce biological effects consistent with direct SIRT1 activation. At the molecular level, the mechanism by which STACs activate SIRT1 remains elusive. In the studies reported herein, the mechanism of SIRT1 activation is examined using representative compounds chosen from a collection of STACs. These studies reveal that activation of SIRT1 by STACs is strongly dependent on structural features of the peptide substrate. Significantly, and in contrast to studies reporting that peptides must bear a fluorophore for their deacetylation to be accelerated, we find that some STACs can accelerate the SIRT1-catalyzed deacetylation of specific unlabeled peptides composed only of natural amino acids. These results, together with others of this study, are at odds with a recent claim that complex formation between STACs and fluorophore-labeled peptides plays a role in the activation of SIRT1 (Pacholec, M., Chrunyk, B., Cunningham, D., Flynn, D., Griffith, D., Griffor, M., Loulakis, P., Pabst, B., Qiu, X., Stockman, B., Thanabal, V., Varghese, A., Ward, J., Withka, J., and Ahn, K. (2010) J. Biol. Chem. 285, 8340–8351). Rather, the data suggest that STACs interact directly with SIRT1 and activate SIRT1-catalyzed deacetylation through an allosteric mechanism.     

Transformation of Mycobacterium aurum by electroporation: the use of glycine, lysozyme and isonicotinic acid hydrazide in enhancing transformation efficiency

Endoglucanase of Aspergillus niger AS-101 was partially purified by ammonium sulphate fractionation and molecular sieving on Sephadex G-200. The enzyme was found to be unstable on storage, however, it received some protection on the addition of BSA, glycerol or sodium azide. Glycerol also protected the enzyme from inactivation due to freezing and thawing. Studies on the effect of thiol group reagents revealed the involvement of -SH group(s) at the active site of enzyme. The enzyme was a metallo-protein or it required certain metal ions for activation. A variety of modulators such as macroionic compounds and metal ions showed varying effects on the purified endoglucanase.     

Four-site four-electron model for Bi(III)-Bi(V) mixed valence complex

In most quantum models for the four-site four-electron problem the lowest singlet state has two short and two long bonds in the absence of lattice polarization and is called the resonating valence bond (VB) state. It is shown here that if the lattice polarization is large, the ground state is a ‘negative U’ state with valence disproportionation (for example BaBiO₃ with Bi(V) and Bi(III) sites). Furthermore the model shows that the coupling between the pairs on different sites is provided via the VB state.     

Stress eating and tuning out: Cancer cells re-wire metabolism to counter stress

Abstract

Cancer cells reprogram metabolism to maintain rapid proliferation under often stressful conditions. Glycolysis and glutaminolysis are two central pathways that fuel cancer metabolism. Allosteric regulation and metabolite driven post-translational modifications of key metabolic enzymes allow cancer cells glycolysis and glutaminolysis to respond to changes in nutrient availability and the tumor microenvironment. While increased aerobic glycolysis (the Warburg effect) has been a noted part of cancer metabolism for over 80 years, recent work has shown that the elevated levels of glycolytic intermediates are critical to cancer growth and metabolism due to their ability to feed into the anabolic pathways branching off glycolysis such as the pentose phosphate pathway and serine biosynthesis pathway. The key glycolytic enzymes phosphofructokinase-1 (PFK1), pyruvate kinase (PKM2) and phosphoglycerate mutase 1 (PGAM1) are regulated by upstream and downstream metabolites to balance glycolytic flux with flux through anabolic pathways. Glutamine regulation is tightly controlled by metabolic intermediates that allosterically inhibit and activate glutamate dehydrogenase, which fuels the tricarboxylic acid cycle by converting glutamine derived glutamate to α-ketoglutarate. The elucidation of these key allosteric regulatory hubs in cancer metabolism will be essential for understanding and predicting how cancer cells will respond to drugs that target metabolism. Additionally, identification of the structures involved in allosteric regulation will inform the design of anti-metabolism drugs which bypass the off-target effects of substrate mimics. Hence, this review aims to provide an overview of allosteric control of glycolysis and glutaminolysis.     

Modelling of the mechanical and mass transport properties of auxetic molecular sieves: an idealised inorganic (zeolitic) host–guest system

Force field based simulations have been employed to model the structure, and mechanical and mass transport properties of the all-silica zeolite MFI (ZSM5—Si₉₆O₁₉₂). Undeformed and deformed MFI subject to uniaxial loading in each of the three principal directions were investigated. The mechanical properties are predicted to include negative on-axis Poisson's ratios (auxetic behaviour) in the x ₁–x ₃ plane of the undeformed structure, and are strain-dependent. Transformation from positive-to-negative Poisson's ratio behaviour, and vice versa, is predicted for most on-axis Poisson's ratios at critical loading strains. Simulations of the simultaneous sorption of neopentane and benzene guest molecules onto the undeformed host MFI framework indicate a low neopentane-to-benzene loading ratio, consistent with experimental observation. The sorption of these two molecular species onto deformed MFI is Poisson's ratio- and strain-dependent. Uniaxial tensile loading along a direction containing a negative on-axis Poisson's ratio leads to an increase in the loading of the larger neopentane molecules with respect to benzene, strongly correlated with the increase in volume associated with auxetic behaviour.     

Discovery of an intravenous hepatoselective glucokinase activator for the treatment of inpatient hyperglycemia

Glucokinase (hexokinase IV) continues to be a compelling target for the treatment of type 2 diabetes given the wealth of supporting human genetics data and numerous reports of robust clinical glucose lowering in patients treated with small molecule allosteric activators. Recent work has demonstrated the ability of hepatoselective activators to deliver glucose lowering efficacy with minimal risk of hypoglycemia. While orally administered agents require a considerable degree of passive permeability to promote suitable exposures, there is no such restriction on intravenously delivered drugs. Therefore, minimization of membrane diffusion in the context of an intravenously agent should ensure optimal hepatic targeting and therapeutic index. This work details the identification a hepatoselective GKA exhibiting the aforementioned properties.     

A sphingosine 1-phosphate receptor 2 selective allosteric agonist

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound.