Structural basis for the efficient phosphorylation of AZT-MP (3′-azido-3′-deoxythymidine monophosphate) and dGMP by Plasmodium falciparum type I thymidylate kinase

Plasmodium falciparum is the causative agent of malaria, a disease where new drug targets are required due to increasing resistance to current anti-malarials. TMPK (thymidylate kinase) is a good candidate as it is essential for the synthesis of dTTP, a critical precursor of DNA and has been much studied due to its role in prodrug activation and as a drug target. Type I TMPKs, such as the human enzyme, phosphorylate the substrate AZT (3′-azido-3′-deoxythymidine)-MP (monophosphate) inefficiently compared with type II TMPKs (e.g. Escherichia coli TMPK). In the present paper we report that eukaryotic PfTMPK (P. falciparum TMPK) presents sequence features of a type I enzyme yet the kinetic parameters for AZT-MP phosphorylation are similar to those of the highly efficient E. coli enzyme. Structural information shows that this is explained by a different juxtaposition of the P-loop and the azide of AZT-MP. Subsequent formation of the transition state requires no further movement of the PfTMPK P-loop, with no steric conflicts for the azide moiety, allowing efficient phosphate transfer. Likewise, we present results that confirm the ability of the enzyme to uniquely accept dGMP as a substrate and shed light on the basis for its wider substrate specificity. Information resulting from two ternary complexes (dTMP–ADP and AZT-MP–ADP) and a binary complex with the transition state analogue AP5dT [P1-(5′-adenosyl)-P5-(5′-thymidyl) pentaphosphate] all reveal significant differences with the human enzyme, notably in the lid region and in the P-loop which may be exploited in the rational design of Plasmodium-specific TMPK inhibitors with therapeutic potential.

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Ligand-induced activation of human TRPM2 requires the terminal ribose of ADPR and involves Arg1433 and Tyr1349

Biochemical Journal ◽

10.1042/bcj20170091 ◽

2017 ◽

Vol 474 (13) ◽

pp. 2159-2175 ◽

Cited By ~ 19

Author(s):

Ralf Fliegert ◽

Joanna M. Watt ◽

Anja Schöbel ◽

Monika D. Rozewitz ◽

Christelle Moreau ◽

...

Keyword(s):

Hydrogen Bonding ◽

Rational Design ◽

Transient Receptor Potential ◽

Therapeutic Potential ◽

Receptor Potential ◽

Transient Receptor Potential Channel ◽

Site Directed Mutagenesis ◽

Structural Basis ◽

Pharmacological Intervention ◽

Hydroxyl Groups

TRPM2 (transient receptor potential channel, subfamily melastatin, member 2) is a Ca2+-permeable non-selective cation channel activated by the binding of adenosine 5′-diphosphoribose (ADPR) to its cytoplasmic NUDT9H domain (NUDT9 homology domain). Activation of TRPM2 by ADPR downstream of oxidative stress has been implicated in the pathogenesis of many human diseases, rendering TRPM2 an attractive novel target for pharmacological intervention. However, the structural basis underlying this activation is largely unknown. Since ADP (adenosine 5′-diphosphate) alone did not activate or antagonize the channel, we used a chemical biology approach employing synthetic analogues to focus on the role of the ADPR terminal ribose. All novel ADPR derivatives modified in the terminal ribose, including that with the seemingly minor change of methylating the anomeric-OH, abolished agonist activity at TRPM2. Antagonist activity improved as the terminal substituent increasingly resembled the natural ribose, indicating that gating by ADPR might require specific interactions between hydroxyl groups of the terminal ribose and the NUDT9H domain. By mutating amino acid residues of the NUDT9H domain, predicted by modelling and docking to interact with the terminal ribose, we demonstrate that abrogating hydrogen bonding of the amino acids Arg1433 and Tyr1349 interferes with activation of the channel by ADPR. Taken together, using the complementary experimental approaches of chemical modification of the ligand and site-directed mutagenesis of TRPM2, we demonstrate that channel activation critically depends on hydrogen bonding of Arg1433 and Tyr1349 with the terminal ribose. Our findings allow for a more rational design of novel TRPM2 antagonists that may ultimately lead to compounds of therapeutic potential.

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Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1715812114 ◽

2017 ◽

Vol 114 (48) ◽

pp. E10438-E10445 ◽

Cited By ~ 49

Author(s):

David Oyen ◽

Jonathan L. Torres ◽

Ulrike Wille-Reece ◽

Christian F. Ockenhouse ◽

Daniel Emerling ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Malaria Vaccine ◽

Acquired Resistance ◽

Circumsporozoite Protein ◽

Phase Iii ◽

Structural Basis ◽

Parasite Development ◽

Type I ◽

Binding Modes

Acquired resistance against antimalarial drugs has further increased the need for an effective malaria vaccine. The current leading candidate, RTS,S, is a recombinant circumsporozoite protein (CSP)-based vaccine against Plasmodium falciparum that contains 19 NANP repeats followed by a thrombospondin repeat domain. Although RTS,S has undergone extensive clinical testing and has progressed through phase III clinical trials, continued efforts are underway to enhance its efficacy and duration of protection. Here, we determined that two monoclonal antibodies (mAbs 311 and 317), isolated from a recent controlled human malaria infection trial exploring a delayed fractional dose, inhibit parasite development in vivo by at least 97%. Crystal structures of antibody fragments (Fabs) 311 and 317 with an (NPNA)3 peptide illustrate their different binding modes. Notwithstanding, one and three of the three NPNA repeats adopt similar well-defined type I β-turns with Fab311 and Fab317, respectively. Furthermore, to explore antibody binding in the context of P. falciparum CSP, we used negative-stain electron microscopy on a recombinant shortened CSP (rsCSP) construct saturated with Fabs. Both complexes display a compact rsCSP with multiple Fabs bound, with the rsCSP–Fab311 complex forming a highly organized helical structure. Together, these structural insights may aid in the design of a next-generation malaria vaccine.

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Structure of the complex of carboxypeptidase B and N-sulfamoyl-L-arginine

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15016799 ◽

2015 ◽

Vol 71 (10) ◽

pp. 1335-1340 ◽

Cited By ~ 7

Author(s):

Valery Akparov ◽

Nikolay Sokolenko ◽

Vladimir Timofeev ◽

Inna Kuranova

Keyword(s):

Transition State ◽

Rational Design ◽

Enzyme Inhibitor ◽

Structural Basis ◽

Side Chain ◽

Water Molecules ◽

Carboxypeptidase B ◽

Transition State Analogue ◽

Active Site Cleft ◽

Difference Fourier

Porcine pancreatic carboxypeptidase B (EC 3.4.23.6) was complexed with a stable transition-state analogue, N-sulfamoyl-L-arginine, in which an S atom imitates the sp 3-hybridized carbon in the scissile-bond surrogate. Crystals were grown in a form belonging to the same space group, P41212, as the uncomplexed enzyme. X-ray data were collected to a resolution of 1.25 Å. The molecule was refined and the positions of non-H atoms of the inhibitor and water molecules were defined using difference Fourier maps. The enzyme–inhibitor complex and 329 water molecules were further refined to a crystallographic R factor of 0.159. The differences in conformation between the complexed and uncomplexed forms of carboxypeptidase B are shown. The inhibitor is bound in a curved conformation in the active-site cleft, and the sulfamide group is bound to the Zn ion in an asymmetric bidentate fashion. The complex is stabilized by hydrogen bonds between the N1/N2 guanidine group of the inhibitor and the Asp255 carboxyl of the enzyme. The side-chain CH2 groups of the inhibitor are in van der Waals contact with Leu203 and Ile247 in the enzyme. This study provides useful clues concerning how the transition state of arginine may bind to carboxypeptidase B and therefore provides an insight into the structural basis of carboxypeptidase B selectivity, which is useful for the rational design of a carboxypeptidase with improved selectivity for industrial recombinant pro-insulin processing.

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Anticoagulant Activity of Hirulog™, a Direct Thrombin Inhibitor, in Humans

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651573 ◽

1993 ◽

Vol 69 (02) ◽

pp. 157-163 ◽

Cited By ~ 137

Author(s):

Irving Fox ◽

Adrian Dawson ◽

Peter Loynds ◽

Jane Eisner ◽

Kathleen Findlen ◽

...

Keyword(s):

Rational Design ◽

Therapeutic Potential ◽

Anticoagulant Activity ◽

Subcutaneous Administration ◽

Direct Thrombin Inhibitor ◽

Controlled Study ◽

Thrombin Inhibitor ◽

Thrombin Time ◽

Thrombotic Disease ◽

Dose Dependent

SummaryHirulog™ (BG8967) is a direct thrombin inhibitor built by rational design using the protein hirudin as a model (Maraganore et al. [1990]; Biochemistry 29: 7095–101). In order to evaluate the therapeutic potential for hirulog in the management of thrombotic disease, the tolerability and anticoagulant activity of the agent were examined in a study of human volunteers.In a randomized, placebo-controlled study (n = 54), the intravenous infusion of hirulog over 15 min showed a rapid, dose-dependent prolongation of activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). There was a corresponding dose-dependent increase in plasma hirulog levels. The peptide was rapidly cleared with a half-life of 36 min and a total body clearance rate for the peptide of 0.43 1 kg−1 h−1. Similar activity was observed following subcutaneous injection but with sustained pharmacodynamic and pharmacokinetic behavior. There was a significant correlation between pharmacokinetic and pharmacodynamic variables for both intravenous (r = 0.8, p <0.001) and subcutaneous administration (r = 0.7, p = 0.002).To evaluate the possible interactions of aspirin on the tolerability and anticoagulant activity of intravenous hirulog, a cross-over design was employed in eight subjects. Aspirin administration did not modify the peptide’s activity. At the administered dose of 0.6 mg kg−1 h−1 for 2 h, hirulog infusion prolonged APTT from 230 to 260% baseline. The infusion of hirulog in subjects who had received aspirin was not associated with any significant changes in the template bleeding time.The final phase of the study examined the activity and tolerability of hirulog in ten subjects during prolonged intravenous infusions for up to 24 h. The peptide (0.3 mg kg−1 h−1) exhibited sustained anticoagulant activity with no evidence for a cumulative effect. During hirulog infusion, APTT was prolonged from 210 to 250% baseline.In all phases of the study, hirulog administration was generally well-tolerated.Our observations show that hirulog is an active antithrombin agent with excellent tolerability in humans. As a direct thrombin inhibitor, hirulog provides a novel approach for the management of thrombotic disease.

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Isoform-Selective PI3K Inhibitors for Various Diseases

Current Topics in Medicinal Chemistry ◽

10.2174/1568026620666200106141717 ◽

2020 ◽

Vol 20 (12) ◽

pp. 1074-1092 ◽

Cited By ~ 4

Author(s):

Rammohan R.Y. Bheemanaboina

Keyword(s):

Intracellular Signaling ◽

Kinase Inhibitors ◽

Therapeutic Potential ◽

Type I ◽

Selective Inhibitors ◽

Pi3k Inhibitors ◽

Wide Range ◽

Lipid Kinases ◽

Isoform Selectivity

Phosphoinositide 3-kinases (PI3Ks) are a family of ubiquitously distributed lipid kinases that control a wide variety of intracellular signaling pathways. Over the years, PI3K has emerged as an attractive target for the development of novel pharmaceuticals to treat cancer and various other diseases. In the last five years, four of the PI3K inhibitors viz. Idelalisib, Copanlisib, Duvelisib, and Alpelisib were approved by the FDA for the treatment of different types of cancer and several other PI3K inhibitors are currently under active clinical development. So far clinical candidates are non-selective kinase inhibitors with various off-target liabilities due to cross-reactivities. Hence, there is a need for the discovery of isoform-selective inhibitors with improved efficacy and fewer side-effects. The development of isoform-selective inhibitors is essential to reveal the unique functions of each isoform and its corresponding therapeutic potential. Although the clinical effect and relative benefit of pan and isoformselective inhibition will ultimately be determined, with the development of drug resistance and the demand for next-generation inhibitors, it will continue to be of great significance to understand the potential mechanism of isoform-selectivity. Because of the important role of type I PI3K family members in various pathophysiological processes, isoform-selective PI3K inhibitors may ultimately have considerable efficacy in a wide range of human diseases. This review summarizes the progress of isoformselective PI3K inhibitors in preclinical and early clinical studies for anticancer and other various diseases.

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Structural basis for ALK2/BMPR2 receptor complex signaling through kinase domain oligomerization

Nature Communications ◽

10.1038/s41467-021-25248-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Christopher Agnew ◽

Pelin Ayaz ◽

Risa Kashima ◽

Hanna S. Loving ◽

Prajakta Ghatpande ◽

...

Keyword(s):

Md Simulations ◽

Kinase Domain ◽

Structural Basis ◽

Type I ◽

Type Ii ◽

Exchange Mass Spectrometry ◽

Receptor Complexes ◽

Bmp Receptors ◽

Morphogenetic Protein ◽

Smad Proteins

AbstractUpon ligand binding, bone morphogenetic protein (BMP) receptors form active tetrameric complexes, comprised of two type I and two type II receptors, which then transmit signals to SMAD proteins. The link between receptor tetramerization and the mechanism of kinase activation, however, has not been elucidated. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, combined with analysis of SMAD signaling, we show that the kinase domain of the type I receptor ALK2 and type II receptor BMPR2 form a heterodimeric complex via their C-terminal lobes. Formation of this dimer is essential for ligand-induced receptor signaling and is targeted by mutations in BMPR2 in patients with pulmonary arterial hypertension (PAH). We further show that the type I/type II kinase domain heterodimer serves as the scaffold for assembly of the active tetrameric receptor complexes to enable phosphorylation of the GS domain and activation of SMADs.

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Crystal Structures of the Human and Fungal Cytosolic Leucyl-tRNA Synthetase Editing Domains: A Structural Basis for the Rational Design of Antifungal Benzoxaboroles

Journal of Molecular Biology ◽

10.1016/j.jmb.2009.04.073 ◽

2009 ◽

Vol 390 (2) ◽

pp. 196-207 ◽

Cited By ~ 65

Author(s):

Elena Seiradake ◽

Weimin Mao ◽

Vincent Hernandez ◽

Stephen J. Baker ◽

Jacob J. Plattner ◽

...

Keyword(s):

Crystal Structures ◽

Rational Design ◽

Trna Synthetase ◽

Structural Basis

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Abstract 3417: Unselected Human Cardiosphere-derived Cells are Functionally Superior to c-Kit- or CD90-Purified Cardiosphere-derived Cells

Circulation ◽

10.1161/circ.118.suppl_18.s_420-a ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Rachel Ruckdeschel Smith ◽

Isotta Chimenti ◽

Eduardo Marbán

Keyword(s):

Troponin I ◽

Therapeutic Potential ◽

Left Ventricular ◽

Dermal Fibroblasts ◽

Historical Control ◽

Border Zone ◽

Left Ventricular Ejection ◽

Control Group ◽

Type I ◽

Ventricular Ejection Fraction

Cardiosphere-derived cells (CDCs), a naturally heterogeneous mixture of cell sub-populations, were grown from percutaneous endomyocardial adult human biopsy specimens (n=6). c-Kit + and CD90 + CDCs were selected using magnetic-activated cell sorting with excellent purity as determined by flow cytometry. Immunostaining revealed that ~30% of c-Kit + CDCs expressed Nkx2.5, ~100% of CD90 + CDCs expressed procollagen type I, and ~100% of both sub-populations expressed CD105. When placed in co-culture with neonatal myocytes and fibroblasts, c-Kit + CDCs expressed cardiac troponin I, while CD90 + CDCs expressed vimentin. In order to assess the therapeutic potential of purified CDCs, acute myocardial infarcts (MIs) were created in immunodeficient mice and c-Kit + (n=16), CD90 + (n=14), or CD105 + (n=3) CDCs were injected into the border zone. Echocardiograms were performed 3 weeks post-MI to measure left ventricular ejection fraction (LVEF). CD105-injected mice were comparable to an historical control group of mixed CDC-injected mice (LVEF = 41.3±2.9% CD105 vs. 42.8±10.4% CDC [n=11], p=0.60), indicating that the sorting process did not itself impair the therapeutic potential of CDCs. c-Kit- and CD90-injected mice were indistinguishable from one another (LVEF=31.7±8.2% c-Kit vs. 32.1±11.8% CD90, p=0.92), and both groups were significantly outperformed by the CD105-injected mice (p=0.01 and p=0.03, respectively). All groups were then compared to two other historical control groups, mice treated with normal human dermal fibroblasts (NHDFs [n=7]) and mice treated with phosphate-buffered saline (PBS [n=11]). c-Kit-injected mice did significantly outperform both NHDF- (p=0.04) and PBS-injected mice (p=0.03), while more variability in the CD90-injected group resulted in nearly significant comparisons with the NHDF (p=0.08) and PBS groups (p=0.08). While the therapeutic mechanisms of action of these two distinct sub-populations are undoubtedly different, both offer similar global functional benefits in the setting of acute MI. We conclude that the spontaneously-emerging unselected CDC population serves as a therapeutic cell cocktail, and that no functional advantage is conferred by the extra step of sorting for c-Kit + or CD90 + sub-populations.

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An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behaviour

Biochemical Journal ◽

10.1042/bcj20160159 ◽

2016 ◽

Vol 473 (19) ◽

pp. 3269-3290 ◽

Cited By ~ 9

Author(s):

Neda Motamedi-Shad ◽

Alistair M. Jagger ◽

Maximilian Liedtke ◽

Sarah V. Faull ◽

Arjun Scott Nanda ◽

...

Keyword(s):

Rational Design ◽

Conformational Transition ◽

Structural Basis ◽

Linear Polymers ◽

Single Chain ◽

Opposite Face ◽

Induced Changes ◽

Β Sheet ◽

Chain Form ◽

Antiprotease Activity

Serpins are important regulators of proteolytic pathways with an antiprotease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an intermolecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of many pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-antitrypsin (α1-AT) polymerisation in cells. Here, we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39 and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered β-sheet A — on the opposite face of the molecule — more liable to adopt an ‘open’ state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of a preformed serpin–enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the β-sheet A equilibrium towards the ‘open’ state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for a rational design of ligands that is able to dynamically influence α1-AT polymerisation.

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