Conformational restriction approach to β-secretase (BACE1) inhibitors III: Effective investigation of the binding mode by combinational use of X-ray analysis, isothermal titration calorimetry and theoretical calculations

A high-throughput screen (HTS) of human 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) resulted in several series of compounds with the potential for further optimization. Informatics was used to identify active chemotypes with lead-like profiles and remove compounds that commonly occurred as actives in other HTS screens. The activities were confirmed with IC50 measurements from two orthogonal assay technologies, and further analysis of the Hill slopes and comparison of the ratio of IC50 values at 10 times the enzyme concentration were used to identify artifact compounds. Several series of compounds were rejected as they had both high slopes and poor ratios. A small number of compounds representing the different leading series were assessed using isothermal titration calorimetry, and the X-ray crystal structure of the complex with PFKFB3 was solved. The orthogonal assay technology and isothermal calorimetry were demonstrated to be unreliable in identifying false-positive compounds in this case. Presented here is the discovery of the dihydropyrrolopyrimidinone series of compounds as active and novel inhibitors of PFKFB3, shown by X-ray crystallography to bind to the adenosine triphosphate site. The crystal structures of this series also reveal it is possible to flip the binding mode of the compounds, and the alternative orientation can be driven by a sigma-hole interaction between an aromatic chlorine atom and a backbone carbonyl oxygen. These novel inhibitors will enable studies to explore the role of PFKFB3 in driving the glycolytic phenotype of tumors.

Download Full-text

Investigation of the inclusion of the herbicide cycluron in native cyclodextrins by X-ray diffraction, nuclear magnetic resonance spectroscopy and isothermal titration calorimetry

Supramolecular Chemistry ◽

10.1080/10610278.2012.676180 ◽

2012 ◽

Vol 24 (6) ◽

pp. 406-414

Author(s):

Dyanne L. Cruickshank ◽

Mircea Bogdan ◽

Sorin I. Fărcas ◽

Susan A. Bourne ◽

Mino R. Caira

Keyword(s):

Nuclear Magnetic Resonance ◽

Magnetic Resonance ◽

Magnetic Resonance Spectroscopy ◽

Nuclear Magnetic Resonance Spectroscopy ◽

Isothermal Titration Calorimetry ◽

Titration Calorimetry ◽

Resonance Spectroscopy ◽

X Ray Diffraction ◽

X Ray ◽

Nuclear Magnetic

Download Full-text

X-Ray Crystallography and Isothermal Titration Calorimetry Studies of the Salmonella Zinc Transporter ZntB

Structure ◽

10.1016/j.str.2011.02.011 ◽

2011 ◽

Vol 19 (5) ◽

pp. 700-710 ◽

Cited By ~ 21

Author(s):

Qun Wan ◽

Md Faiz Ahmad ◽

James Fairman ◽

Bonnie Gorzelle ◽

María de la Fuente ◽

...

Keyword(s):

Isothermal Titration Calorimetry ◽

Zinc Transporter ◽

Titration Calorimetry ◽

X Ray ◽

X Ray Crystallography

Download Full-text

Reconciling isothermal titration calorimetry analyses of interactions between complexin and truncated SNARE complexes

eLife ◽

10.7554/elife.30286 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 6

Author(s):

Eric A Prinslow ◽

Chad A Brautigam ◽

Josep Rizo

Keyword(s):

Isothermal Titration Calorimetry ◽

Neurotransmitter Release ◽

Experimental Error ◽

Binding Mode ◽

Snare Complex ◽

Titration Calorimetry ◽

Juxtamembrane Region ◽

C Terminus ◽

Snare Motif ◽

Α Helix

Neurotransmitter release depends on the SNARE complex formed by syntaxin-1, synaptobrevin and SNAP-25, as well as on complexins, which bind to the SNARE complex and play active and inhibitory roles. A crystal structure of a Complexin-I fragment bearing a so-called 'superclamp' mutation bound to a truncated SNARE complex lacking the C-terminus of the synaptobrevin SNARE motif (SNAREΔ60) suggested that an 'accessory' α-helix of Complexin-I inhibits release by inserting into the C-terminus of the SNARE complex. Previously, isothermal titration calorimetry (ITC) experiments performed in different laboratories yielded apparently discrepant results in support or against the existence of such binding mode in solution (Trimbuch et al., 2014; Krishnakumar et al., 2015). Here, ITC experiments performed to solve these discrepancies now show that the region containing the Complexin-I accessory helix and preceding N-terminal sequences does interact with SNAREΔ60, but the interaction requires the polybasic juxtamembrane region of syntaxin-1 and is not affected by the superclamp mutation within the experimental error of these experiments.

Download Full-text

The molecular structure of Schistosoma mansoni PNP isoform 2 provides insights into the nucleotide selectivity of PNPs

10.1101/300533 ◽

2018 ◽

Author(s):

Juliana Roberta Torini ◽

Larissa Romanello ◽

Fernanda Aparecida Heleno Batista ◽

Vitor Hugo Balasco Serrão ◽

Muhammad Faheem ◽

...

Keyword(s):

Catalytic Activity ◽

Schistosoma Mansoni ◽

Binding Site ◽

Kinetic Parameters ◽

Isothermal Titration Calorimetry ◽

Active Site ◽

Binding Mode ◽

Titration Calorimetry ◽

Purine Salvage ◽

Key Enzyme

AbstractPurine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, named as SmPNP2. Screening of different ligands using a thermofluorescence approach indicated cytidine and cytosine as potential ligands. The binding of cytosine was confirmed by isothermal titration calorimetry, with a KD of 27 μM, and kinetic parameters for cytidine catalysis were obtained by ITC resulting in a KM of 76.3 μM. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystallographic structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capability of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site allowing an alternative binding mode for nucleosides, with a 180° rotation from the canonical binding mode. The remarkable plasticity of this binding site deepens the understanding of the correlation between structure and nucleotide selectivity, offering new ways to analyses PNP activity.Author SummarySchistosoma mansoni is a human parasite dependent on purine salvage for purine bases supply. Purine nucleoside phosphorylase (PNP) is a key enzyme in this pathway. It carries two PNP isoforms, one previously characterized (SmPNP1) and one unknown (SmPNP2). Here we present the crystallographic structure of SmPNP2 and its complex with cytosine, cytidine, ribose-l-phosphate, adenine, hypoxanthine, and tubercidin. Cytidine and cytosine were identified as ligands of SmPNP2 using a thermofluorescence approach. Binding of cytosine was proven by Isothermal Titration Calorimetry (ITC) and cytidine, inosine, and adenosine kinetic parameters were also obtained. Purine bases showed different binding in the active site, rotated 180° from the canonical binding mode. It’s the first report showing a Low Molecular Mass PNP capable of catalyzing both types of nucleotide bases. The SmPNP2 odd behavior sheds a new light on the Schistosoma mansoni’s life cycle metabolic adaptation.

Download Full-text