The potato tuber,maize endosperm and a chimeric maize-potato ADP-glucose pyrophosphorylase exhibit fundamental differences in Pi inhibition

ADP-glucose pyrophosphorylase (AGPase) is highly regulated by allosteric effectors acting both positively and negatively. Enzymes from various sources differ, however, in the mechanism of allosteric regulation. Here, we determined how the effector, inorganic phosphate (Pi), functions in the presence and absence of saturating amounts of the activator, 3-phosphoglyceric acid (3-PGA). This regulation was examined in the maize endosperm enzyme, the oxidized and reduced forms of the potato tuber enzyme as well as a small subunit chimeric AGPase (MP), which contains both maize endosperm and potato tuber sequences paired with a wild-type maize large subunit. These data, combined with our previous kinetic studies of these enzymes led to a model of Pi inhibition for the various enzymes. The Pi inhibition data suggest that while the maize enzyme contains a single effector site that binds both 3-PGA and Pi, the other enzymes exhibit more complex behavior and most likely have at least two separate interacting binding sites for Pi. The possible physiological implications of the differences in Pi inhibition distinguishing the maize endosperm and potato tuber AGPases are discussed.     

Dynamic modulation of FGFR1–5-HT1A heteroreceptor complexes. Agonist treatment enhances participation of FGFR1 and 5-HT1A homodimers and recruitment of β-arrestin2

New findings show that neurotrophic and antidepressant effects of 5-HT in brain can, in part, be mediated by activation of the 5-HT1A receptor protomer in the hippocampal and raphe FGFR1–5-HT1A heteroreceptor complexes enhancing the FGFR1 signaling. The dynamic agonist modulation of the FGFR1–5-HT1A heteroreceptor complexes and their recruitment of β-arrestin is now determined in cellular models with focus on its impact on 5-HT1AR and FGFR1 homodimerization in the heteroreceptor complexes based on BRET² assays. The findings show that coagonist treatment with 8-OH-DPAT and FGF2 but not treatment with the 5-HT1A agonist alone markedly increases the BRETmax values and significantly reduces the BRET50 values of 5HT1A homodimerization. The effects of FGF2 or FGF20 with or without the 5-HT1A agonist were also studied on the FGFR1 homodimerization of the heteroreceptor complexes. FGF2 produced a marked and rapid increase in FGFR1 homodimerization which partially declined over a 10 min period. Cotreatment with FGF2 and 5-HT1A agonist blocked this decline in FGFR1 homodimerization. Furthermore, FGF2 alone produced a small increase in the BRET² signal from the 5-HT1A-β-arrestin2 receptor–protein complex which was additive to the marked effect of 8-OH-DPAT alone. Taken together, the participation of 5-HT1A and FGFR1 homodimers and recruitment of β-arrestin2 was demonstrated in the FGFR1–5-HT1A heteroreceptor complexes upon agonist treatments.     

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.     

OX133, a monoclonal antibody recognizing protein-bound N-ethylmaleimide for the identification of reduced disulfide bonds in proteins

In vivo, enzymatic reduction of some protein disulfide bonds, allosteric disulfide bonds, provides an important level of structural and functional regulation. The free cysteine residues generated can be labeled by maleimide reagents, including biotin derivatives, allowing the reduced protein to be detected or purified. During the screening of monoclonal antibodies for those specific for the reduced forms of proteins, we isolated OX133, a unique antibody that recognizes polypeptide resident, N-ethylmaleimide (NEM)-modified cysteine residues in a sequence-independent manner. OX133 offers an alternative to biotin-maleimide reagents for labeling reduced/alkylated antigens and capturing reduced/alkylated proteins with the advantage that NEM-modified proteins are more easily detected in mass spectrometry, and may be more easily recovered than is the case following capture with biotin based reagents.     

Design and synthesis of novel tellurodibenzoic acid compounds as kidney-type glutaminase (KGA) inhibitors

Organotellurium compounds have been reported as an immune-modulator sensitizing chemotherapeutics. Herein, we report the design and synthesis of a series of novel tellurodibenzoic acids as mimics of diphenylarsenic acid (DPAA) and potential selective KGA inhibitors. Representative compound 3B exhibited potent inhibition of KGA and glutamine-dependent HCT-116 cells. Stability experiments indicated that 3B has excellent stability under acidic (HCOOH), basic (NH₃·H₂O) and oxidative (H₂O₂) conditions, but reacts with β-ME, DTT and lysine which suggested that compound 3B may interact with cysteine or lysine residues. Moreover, molecular docking disclosed that compound 3B binds to the allosteric site of the GAC tetramer containing Arg³¹⁷-Lys³²⁰-Leu³²¹-Phe³²²-Tyr³⁹⁴-Glu³²⁵, which helped to rationalize the SAR and further design and optimization. Taken together, compound 3B could be used as a starting point for the development of new KGA inhibitors.     

SAR inspired by aldehyde oxidase (AO) metabolism: Discovery of novel,CNS penetrant tricyclic M4 PAMs

This letter describes progress towards an M₄ PAM preclinical candidate inspired by an unexpected aldehyde oxidase (AO) metabolite of a novel, CNS penetrant thieno[2,3-c]pyridine core to an equipotent, non-CNS penetrant thieno[2,3-c]pyrdin-7(6H)-one core. Medicinal chemistry design efforts yielded two novel tricyclic cores that enhanced M₄ PAM potency, regained CNS penetration, displayed favorable DMPK properties and afforded robust in vivo efficacy in reversing amphetamine-induced hyperlocomotion in rats.     

Allosteric Inhibition as a New Mode of Action for BAY 1213790, a Neutralizing Antibody Targeting the Activated Form of Coagulation Factor XI

Factor XI (FXI), the zymogen of activated FXI (FXIa), is an attractive target for novel anticoagulants because FXI inhibition offers the potential to reduce thrombosis risk while minimizing the risk of bleeding. BAY 1213790, a novel anti-FXIa antibody, was generated using phage display technology. Crystal structure analysis of the FXIa–BAY 1213790 complex demonstrated that the tyrosine-rich complementarity-determining region 3 loop of the heavy chain of BAY 1213790 penetrated deepest into the FXIa binding epitope, forming a network of favorable interactions including a direct hydrogen bond from Tyr102 to the Gln451 sidechain (2.9 Å). The newly discovered binding epitope caused a structural rearrangement of the FXIa active site, revealing a novel allosteric mechanism of FXIa inhibition by BAY 1213790. BAY 1213790 specifically inhibited FXIa with a binding affinity of 2.4 nM, and in human plasma, prolonged activated partial thromboplastin time and inhibited thrombin generation in a concentration-dependent manner.     

Phosphoenolpyruvate carboxylase: three-dimensional structure and molecular mechanisms

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) catalyzes the irreversible carboxylation of phosphoenolpyruvate (PEP) to form oxaloacetate and Pi using Mg2+ or Mn2+ as a cofactor. PEPC plays a key role in photosynthesis by C4 and Crassulacean acid metabolism plants, in addition to its many anaplerotic functions. Recently, three-dimensional structures of PEPC from Escherichia coli and the C4 plant maize (Zea mays) were elucidated by X-ray crystallographic analysis. These structures reveal an overall square arrangement of the four identical subunits, making up a "dimer-of-dimers" and an eight-stranded beta barrel structure. At the C-terminal region of the beta barrel, the Mn2+ and a PEP analog interact with catalytically essential residues, confirmed by site-directed mutagenesis studies. At about 20A from the beta barrel, an allosteric inhibitor (aspartate) was found to be tightly bound to down-regulate the activity of the E. coli enzyme. In the case of maize C4-PEPC, the putative binding site for an allosteric activator (glucose 6-phosphate) was also revealed. Detailed comparison of the various structures of E. coli PEPC in its inactive state with maize PEPC in its active state shows that the relative orientations of the two subunits in the basal "dimer" are different, implicating an allosteric transition. Dynamic movements were observed for several loops due to the binding of either an allosteric inhibitor, a metal cofactor, a PEP analog, or a sulfate anion, indicating the functional significance of these mobile loops in catalysis and regulation. Information derived from these three-dimensional structures, combined with related biochemical studies, has established models for the reaction mechanism and allosteric regulation of this important C-fixing enzyme.     

Allosteric interactions among pyrethroid,brevetoxin, and scorpion toxin receptors on insect sodium channels raise an alternative approach for insect control

Intensive pyrethroid use in insect control has led to resistance buildup among various pests. One alternative to battle this problem envisions the combined use of synergistically acting insecticidal compounds. Pyrethroids, scorpion - and β-toxins, and brevetoxins bind to distinct receptor sites on voltage-gated sodium channels (NaChs) and modify their function. The binding affinity of scorpion -toxins to locust, but not rat-brain NaChs, is allosterically increased by pyrethroids and by brevetoxin-1. Brevetoxin-1 also increases the binding of an excitatory β-toxin to insect NaChs. These results reveal differences between insect and mammalian NaChs and may be exploited in new strategies of insect control.