Mechanism of malarial haem detoxification inhibition by chloroquine

The haem detoxification pathway of the malaria parasite Plasmodiumfalciparum is a potential biochemical target for drug development. Free haem, released after haemoglobin degradation, is polymerized by the parasite to form haemozoin pigment. Plasmodiumfalciparum histidine-rich protein-2 (Pfhrp-2) has been implicated as the catalytic scaffold for detoxification of haem in the malaria parasite. Previously we have shown that a hexapeptide repeat sequence (Ala-His-His-Ala-Ala-Asp), which appears 33 times in Pfhrp-2, may be the major haem binding site in this protein. The haem binding studies carried out by ourselves indicate that up to 18 equivalents of haem could be bound by this protein with an observed Kd of 0.94µM. Absorbance spectroscopy provides evidence that chloroquine is capable of extracting haem bound to Pfhrp-2. This was supported by the Kd value, of 37nM, observed for the haem-chloroquine complex. The native PAGE studies reveal that the formation of the haem-Pfhrp-2 complex is disrupted by chloroquine. These results indicate that chloroquine may be acting by inhibiting haem detoxification/binding to Pfhrp-2. Moreover, the higher affinity of chloroquine for haem than Pfhrp-2 suggests a possible mechanism of action for chloroquine; it may remove the haem bound to Pfhrp-2 and form a complex that is toxic to the parasite.

Download Full-text

Mapping of the fibrinogen-binding site on the staphylocoagulase C-terminal repeat region

10.1101/2021.06.29.450373 ◽

2021 ◽

Author(s):

Ashoka A Maddur ◽

Markus W. Voehler ◽

Peter Panizzi ◽

Jens Meiler ◽

Paul E Bock ◽

...

Keyword(s):

Binding Site ◽

Repeat Sequence ◽

Native Page ◽

Conserved Residues ◽

Nmr Titration ◽

Disordered Structure ◽

Equilibrium Binding ◽

Fibrinogen Binding ◽

Fibrin Networks ◽

Terminal Repeat Region

The N-terminus of S. aureus staphylocoagulase (SC) triggers activation of host prothrombin (ProT), and the SCProT* complex cleaves host fibrinogen (Fbg) to form fibrin (Fbn) deposits, a hallmark of SC-positive endocarditis. The C-terminal domain of the prototypical Newman D2 Tager 104 SC contains 1 pseudo-repeat (PR) and 7 repeats (R1R7) that bind Fbg/Fbn Fragment D (Frag D). This work defines affinities and stoichiometries of Frag D binding to single- and multi-repeat C-terminal constructs, using fluorescence equilibrium binding, NMR titration, Ala scanning, and native PAGE. Constructs containing PR and each single repeat bound Frag D with KD ~50 130 nM and a 1:1 stoichiometry, indicating a conserved binding site shared between PR and each repeat. NMR titration of PR-R7 with Frag D revealed that residues 22-49, bridging PR and R7, constituted the minimal peptide (MP) required for binding, corroborated by Ala scanning, and binding of labeled MP to Frag D. MP alignment with the PR-repeat and inter-repeat junctions identified conserved residues critical for binding. Labeled PR-(R1R7) bound Frag D with KD ~7 32 nM and stoichiometry of 1:5; and PR-R1R2R3, PR-R1R6R7, PR-R3R4R7, and PR-R3R6R7 competed with PR-(R1R7) for Frag D binding, with a 1:3 stoichiometry and KD ~7 42 nM. These findings are consistent with binding at the PR-R junctions with modest inter-repeat sequence variability. Circular dichroism of PR-R7 and PR-(R1R7) suggested a largely disordered structure and conformational flexibility, allowing binding of multiple fibrin(ogen) molecules. This property facilitates pathogen localization on host fibrin networks.

Download Full-text

Towards understanding the mechanism of action of mithramycin and its analogues: dimerization and DNA binding studies

Planta Medica ◽

10.1055/s-0036-1596246 ◽

2016 ◽

Vol 81 (S 01) ◽

pp. S1-S381

Author(s):

S Weidenbach ◽

C Hou ◽

JM Chen ◽

OV Tsodikov ◽

J Rohr

Keyword(s):

Dna Binding ◽

Mechanism Of Action ◽

Binding Studies ◽

Dna Binding Studies

Download Full-text

Binding Studies Reveal Phospholipid Specificity and Its Role in the Calcium-Dependent Mechanism of Action of Daptomycin

ACS Infectious Diseases ◽

10.1021/acsinfecdis.1c00316 ◽

2021 ◽

Author(s):

Ioli Kotsogianni ◽

Thomas M. Wood ◽

Francesca M. Alexander ◽

Stephen A. Cochrane ◽

Nathaniel I. Martin

Keyword(s):

Mechanism Of Action ◽

Binding Studies ◽

Calcium Dependent ◽

Dependent Mechanism

Download Full-text

Engineered occluded apo-intermediate of LacY

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816267115 ◽

2018 ◽

Vol 115 (50) ◽

pp. 12716-12721 ◽

Cited By ~ 1

Author(s):

Irina Smirnova ◽

Vladimir Kasho ◽

H. Ronald Kaback

Keyword(s):

Binding Site ◽

Lactose Permease ◽

Binding Studies ◽

X Ray Crystallography ◽

Sugar Binding ◽

Transport Cycle ◽

Open Structures ◽

Unrestricted Access ◽

Tight Association ◽

Alternating Access

The lactose permease of Escherichia coli (LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward (periplasmic)-open structures have been characterized by X-ray crystallography. It is demonstrated here with sugar-binding studies that cross-linking paired-Cys replacements across the closed cytoplasmic cavity stabilize an occluded conformer with an inaccessible sugar-binding site. In addition, a nanobody (Nb) that stabilizes a periplasmic-open conformer with an easily accessible sugar-binding site in WT LacY fails to cause the cytoplasmic cross-linked mutants to become accessible to galactoside, showing that the periplasmic cavity is closed. These results are consistent with tight association of the periplasmic ends in two pairs of helices containing clusters of small residues in the packing interface between N- and C-terminal six-helix bundles of the symporter. However, after reduction of the disulfide bond, the Nb markedly increases the rate of galactoside binding, indicating unrestricted access to the Nb epitope and the galactoside-binding site from the periplasm. The findings indicate that the cross-linked cytoplasmic double-Cys mutants resemble an occluded apo-intermediate in the transport cycle.

Download Full-text

Multiscale simulations examining glycan shield effects on drug binding to influenza neuraminidase

10.1101/2020.08.12.248690 ◽

2020 ◽

Cited By ~ 1

Author(s):

Christian Seitz ◽

Lorenzo Casalino ◽

Robert Konecny ◽

Gary Huber ◽

Rommie E. Amaro ◽

...

Keyword(s):

Drug Development ◽

Ligand Binding ◽

Binding Site ◽

Brownian Dynamics ◽

Drug Binding ◽

Binding Mechanism ◽

Drug Effectiveness ◽

Future Drug ◽

Dynamics Simulations ◽

Inhibit Antibody

AbstractInfluenza neuraminidase is an important drug target. Glycans are present on neuraminidase, and are generally considered to inhibit antibody binding via their glycan shield. In this work we studied the effect of glycans on the binding kinetics of antiviral drugs to the influenza neuraminidase. We created all-atom in silico systems of influenza neuraminidase with experimentally-derived glycoprofiles consisting of four systems with different glycan conformations and one system without glycans. Using Brownian dynamics simulations, we observe a two- to eight-fold decrease in the rate of ligand binding to the primary binding site of neuraminidase due to the presence of glycans. These glycans are capable of covering much of the surface area of neuraminidase, and the ligand binding inhibition is derived from glycans sterically occluding the primary binding site on a neighboring monomer. Our work also indicates that drugs preferentially bind to the primary binding site (i.e. the active site) over the secondary binding site, and we propose a binding mechanism illustrating this. These results help illuminate the complex interplay between glycans and ligand binding on the influenza membrane protein neuraminidase.Statement of SignificanceThe influenza glycoprotein neuraminidase is the target for three FDA-approved influenza drugs in the US. However, drug resistance and low drug effectiveness merits further drug development towards neuraminidase, which is hindered by our limited understanding of glycan effects on ligand binding. Generally, drug developers do not include glycans in their development pipelines. Here, we show that even though glycans can reduce drug binding towards neuraminidase, we recommend future drug development work to focus on strong binders with a long lifetime. Furthermore, we examine the binding competition between the primary and secondary binding sites on neuraminidase, leading us to propose a new, to the best of our knowledge, multivalent binding mechanism.

Download Full-text

The Two ADF-H Domains of Twinfilin Play Functionally Distinct Roles in Interactions with Actin Monomers

Molecular Biology of the Cell ◽

10.1091/mbc.e02-03-0157 ◽

2002 ◽

Vol 13 (11) ◽

pp. 3811-3821 ◽

Cited By ~ 55

Author(s):

Pauli J. Ojala ◽

Ville O. Paavilainen ◽

Maria K. Vartiainen ◽

Roman Tuma ◽

Alan G. Weeds ◽

...

Keyword(s):

Binding Site ◽

Binding Activity ◽

Size Exclusion ◽

Actin Dynamics ◽

Actin Binding ◽

Actin Monomer ◽

Wild Type ◽

Native Page ◽

Exclusion Chromatography

Twinfilin is a ubiquitous and abundant actin monomer–binding protein that is composed of two ADF-H domains. To elucidate the role of twinfilin in actin dynamics, we examined the interactions of mouse twinfilin and its isolated ADF-H domains with G-actin. Wild-type twinfilin binds ADP-G-actin with higher affinity (K D = 0.05 μM) than ATP-G-actin (K D = 0.47 μM) under physiological ionic conditions and forms a relatively stable (k off = 1.8 s−1) complex with ADP-G-actin. Data from native PAGE and size exclusion chromatography coupled with light scattering suggest that twinfilin competes with ADF/cofilin for the high-affinity binding site on actin monomers, although at higher concentrations, twinfilin, cofilin, and actin may also form a ternary complex. By systematic deletion analysis, we show that the actin-binding activity is located entirely in the two ADF-H domains of twinfilin. Individually, these domains compete for the same binding site on actin, but the C-terminal ADF-H domain, which has >10-fold higher affinity for ADP-G-actin, is almost entirely responsible for the ability of twinfilin to increase the amount of monomeric actin in cosedimentation assays. Isolated ADF-H domains associate with ADP-G-actin with rapid second-order kinetics, whereas the association of wild-type twinfilin with G-actin exhibits kinetics consistent with a two-step binding process. These data suggest that the association with an actin monomer induces a first-order conformational change within the twinfilin molecule. On the basis of these results, we propose a kinetic model for the role of twinfilin in actin dynamics and its possible function in cells.

Download Full-text

Inhibition of K-RAS4B by a Unique Mechanism of Action: Stabilizing Membrane-Dependent Occlusion of the Effector-Binding Site

Cell Chemical Biology ◽

10.1016/j.chembiol.2018.07.009 ◽

2018 ◽

Vol 25 (11) ◽

pp. 1327-1336.e4 ◽

Cited By ~ 35

Author(s):

Zhenhao Fang ◽

Christopher B. Marshall ◽

Tadateru Nishikawa ◽

Alvar D. Gossert ◽

Johanna M. Jansen ◽

...

Keyword(s):

Binding Site ◽

Mechanism Of Action ◽

Effector Binding ◽

Unique Mechanism

Download Full-text

Enhancement of telomere-plasmid segregation by the X-telomere associated sequence in Saccharomyces cerevisiae involves SIR2, SIR3, SIR4 and ABF1.

Genetics ◽

10.1093/genetics/136.3.757 ◽

1994 ◽

Vol 136 (3) ◽

pp. 757-767 ◽

Cited By ~ 2

Author(s):

S Enomoto ◽

M S Longtine ◽

J Berman

Keyword(s):

Binding Site ◽

Consensus Sequence ◽

Active Role ◽

Repeat Sequence ◽

Dependent Manner ◽

Enhancer Element ◽

Telomere Repeat ◽

Plasmid Segregation ◽

Telomere Function ◽

Associated Sequences

Abstract We have previously shown that circular replicating plasmids that carry yeast telomere repeat sequence (TG1-3) tracts segregate efficiently relative to analogous plasmids lacking the TG1-3 tract and this efficient segregation is dependent upon RAP1. While a long TG1-3 tract is sufficient to improve plasmid segregation, the segregation efficiency of telomere plasmids (TEL-plasmids) is enhanced when the X-Telomere Associated Sequence (X-TAS) is also included on the plasmids. We now demonstrate that the enhancement of TEL-plasmid segregation by the X-TAS depends on SIR2, SIR3, SIR4 and ABF1 in trans and requires the Abf1p-binding site within the X-TAS. Mutation of the Abf1p-binding site within the X-TAS results in TEL-plasmids that are no longer affected by mutations in SIR2, SIR3 or SIR4, despite the fact that other Abf1p-binding sites are present on the plasmid. Mutation of the ARS consensus sequence within the X-TAS converts the X-TAS from an enhancer element to a negative element that interferes with TEL-plasmid segregation in a SIR-dependent manner. Thus, telomere associated sequences interact with TG1-3 tracts on the plasmid, suggesting that the TASs have an active role in modulating telomere function.

Download Full-text

Novel vitamin D analogs that modulate leukemic cell growth and differentiation with little effect on either intestinal calcium absorption or bone mobilization

Blood ◽

10.1182/blood.v74.1.82.82 ◽

1989 ◽

Vol 74 (1) ◽

pp. 82-93 ◽

Cited By ~ 103

Author(s):

JY Zhou ◽

AW Norman ◽

M Lubbert ◽

ED Collins ◽

MR Uskokovic ◽

...

Keyword(s):

Vitamin D ◽

Mechanism Of Action ◽

Vitamin D3 ◽

Clonal Growth ◽

Calcium Absorption ◽

Active Form ◽

Intestinal Calcium Absorption ◽

Leukemic Cells ◽

Binding Studies ◽

Vitamin D Analogs

Abstract Induction of terminal differentiation of leukemic and preleukemic cells is a therapeutic approach to leukemia and preleukemia. The 1 alpha, 25- dihydroxyvitamin D3 [1,25(OH)2D3], the hormonally active form of vitamin D3, can induce differentiation and inhibit proliferation of leukemia cells, but concentrations required to achieve these effects cause life-threatening hypercalcemia. Seven new analogs of 1,25(OH)2D3 were discovered to be either equivalent or more potent than 1,25(OH)2D3 as assessed by: (a) inhibition of clonal proliferation of HL-60, EM-2, U937, and patients' myeloid leukemic cells: and (b) induction of differentiation of HL-60 promyelocytes. Furthermore, these analogs stimulated clonal growth of normal human myeloid stem cells. The most potent analog, 1,25-dihydroxy-16ene-23yne-vitamin D3, was about fourfold more potent than 1,25(OH)2D3. This analog decreased clonal growth and expression of c-myc oncogene in HL-60 cells by 50% within ten hours of exposure. Effects on calcium metabolism of these novel analogs in vivo was assessed by intestinal calcium absorption (ICA) and bone calcium mobilization (BCM). Each of the analogs mediated markedly less (10 to 200-fold) ICA and BCM as compared with 1,25(OH)2D3. To gain insight into the possible mechanism of action of these new analogs, receptor binding studies were done with 1,25(OH)2–16ene-23yne-D3 and showed that it competed only about 60% as effectively as 1,25(OH)2D3 for 1,25(OH)2D3 receptors present in HL-60 cells and 98% as effective as 1,25(OH)2D3 for receptors present in chick intestinal cells. In summary, we have discovered seven novel vitamin D analogs that are more potent than the physiologic 1,25(OH)2D3 as measured by a variety of hematopoietic assays. In contrast, these compounds appear to have the potential to be markedly less toxic (induction of hypercalcemia). These novel vitamin D compounds may be superior to 1,25(OH)2D3 in a number of clinical situations including leukemia/preleukemia; they will provide a tool to dissect the mechanism of action of vitamin D seco-steroids in promoting cellular differentiation.

Download Full-text