Protease Stability Correlates with Efficacy for High-Affinity, Cell Penetrant MDM2-Binding Stapled Peptides

2020 ◽  
Author(s):  
Lydia Atangcho ◽  
Tejas Navaratna ◽  
Mukesh Mahajan ◽  
Marshall Case ◽  
Kirsten Deprey ◽  
...  
Keyword(s):  
Stapled Peptides ◽  
High Affinity ◽  
Protease Stability

scholarly journals Computational Design of Macrocyclic Binders of S100B(ββ): Novel Peptide Theranostics

Molecules ◽  
2021 ◽  
Vol 26 (3) ◽  
pp. 721
Author(s):  
Srinivasaraghavan Kannan ◽  
Pietro G. A. Aronica ◽  
Thanh Binh Nguyen ◽  
Jianguo Li ◽  
Chandra S. Verma
Keyword(s):  
Molecular Dynamics ◽  
Computational Design ◽  
Diagnostic Tools ◽  
Stapled Peptides ◽  
Altered Expression ◽  
Cellular Processes ◽  
High Affinity ◽  
Limited Success ◽  
Dynamics Simulations ◽  
Partner Proteins

S100B(ββ) proteins are a family of multifunctional proteins that are present in several tissues and regulate a wide variety of cellular processes. Their altered expression levels have been associated with several human diseases, such as cancer, inflammatory disorders and neurodegenerative conditions, and hence are of interest as a therapeutic target and a biomarker. Small molecule inhibitors of S100B(ββ) have achieved limited success. Guided by the wealth of available experimental structures of S100B(ββ) in complex with diverse peptides from various protein interacting partners, we combine comparative structural analysis and molecular dynamics simulations to design a series of peptides and their analogues (stapled) as S100B(ββ) binders. The stapled peptides were subject to in silico mutagenesis experiments, resulting in optimized analogues that are predicted to bind to S100B(ββ) with high affinity, and were also modified with imaging agents to serve as diagnostic tools. These stapled peptides can serve as theranostics, which can be used to not only diagnose the levels of S100B(ββ) but also to disrupt the interactions of S100B(ββ) with partner proteins which drive disease progression, thus serving as novel therapeutics.

scholarly journals Incorporation of Putative Helix-Breaking Amino Acids in the Design of Novel Stapled Peptides: Exploring Biophysical and Cellular Permeability Properties

Molecules ◽  
2019 ◽  
Vol 24 (12) ◽  
pp. 2292 ◽  
Author(s):  
Anthony W. Partridge ◽  
Hung Yi Kristal Kaan ◽  
Yu-Chi Juang ◽  
Ahmad Sadruddin ◽  
Shuhui Lim ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Binding Affinity ◽  
Amino Acid Substitutions ◽  
Cellular Activity ◽  
Stapled Peptides ◽  
Helical Peptides ◽  
High Affinity ◽  
Cellular Permeability ◽  
Target Binding

Stapled α-helical peptides represent an emerging superclass of macrocyclic molecules with drug-like properties, including high-affinity target binding, protease resistance, and membrane permeability. As a model system for probing the chemical space available for optimizing these properties, we focused on dual Mdm2/MdmX antagonist stapled peptides related to the p53 N-terminus. Specifically, we first generated a library of ATSP-7041 (Chang et al., 2013) analogs iteratively modified by L-Ala and D-amino acids. Single L-Ala substitutions beyond the Mdm2/(X) binding interfacial residues (i.e., Phe3, Trp7, and Cba10) had minimal effects on target binding, α-helical content, and cellular activity. Similar binding affinities and cellular activities were noted at non-interfacial positions when the template residues were substituted with their d-amino acid counterparts, despite the fact that d-amino acid residues typically ‘break’ right-handed α-helices. d-amino acid substitutions at the interfacial residues Phe3 and Cba10 resulted in the expected decreases in binding affinity and cellular activity. Surprisingly, substitution at the remaining interfacial position with its d-amino acid equivalent (i.e., Trp7 to d-Trp7) was fully tolerated, both in terms of its binding affinity and cellular activity. An X-ray structure of the d-Trp7-modified peptide was determined and revealed that the indole side chain was able to interact optimally with its Mdm2 binding site by a slight global re-orientation of the stapled peptide. To further investigate the comparative effects of d-amino acid substitutions we used linear analogs of ATSP-7041, where we replaced the stapling amino acids by Aib (i.e., R84 to Aib4 and S511 to Aib11) to retain the helix-inducing properties of α-methylation. The resultant analog sequence Ac–Leu–Thr–Phe–Aib–Glu–Tyr–Trp–Gln–Leu–Cba–Aib–Ser–Ala–Ala–NH2 exhibited high-affinity target binding (Mdm2 Kd = 43 nM) and significant α-helicity in circular dichroism studies. Relative to this linear ATSP-7041 analog, several d-amino acid substitutions at Mdm2(X) non-binding residues (e.g., d-Glu5, d-Gln8, and d-Leu9) demonstrated decreased binding and α-helicity. Importantly, circular dichroism (CD) spectroscopy showed that although helicity was indeed disrupted by d-amino acids in linear versions of our template sequence, stapled molecules tolerated these residues well. Further studies on stapled peptides incorporating N-methylated amino acids, l-Pro, or Gly substitutions showed that despite some positional dependence, these helix-breaking residues were also generally tolerated in terms of secondary structure, binding affinity, and cellular activity. Overall, macrocyclization by hydrocarbon stapling appears to overcome the destabilization of α-helicity by helix breaking residues and, in the specific case of d-Trp7-modification, a highly potent ATSP-7041 analog (Mdm2 Kd = 30 nM; cellular EC50 = 600 nM) was identified. Our findings provide incentive for future studies to expand the chemical diversity of macrocyclic α-helical peptides (e.g., d-amino acid modifications) to explore their biophysical properties and cellular permeability. Indeed, using the library of 50 peptides generated in this study, a good correlation between cellular permeability and lipophilicity was observed.

Neuronal and Extraneuronal Uptake of 3H-Norepinephrine in the Heart of the Active Bat: An Electron Microscopic Radioautographic Study

1973 ◽  
Vol 31 ◽  
pp. 522-523
Author(s):  
Martin Hagopian ◽  
Michael D. Gershon ◽  
Eladio A. Nunez
Keyword(s):  
Smooth Muscle ◽  
Monoamine Oxidase ◽  
Vascular Smooth Muscle ◽  
Cardiac Muscle ◽  
Maximum Rate ◽  
External Medium ◽  
Extraneuronal Uptake ◽  
Electron Microscopic ◽  
Cardiac Tissues ◽  
High Affinity

The ability of cardiac tissues to take up norepinephrine from an external medium is well known. Two mechanisms, called Uptake and Uptake respectively by Iversen have been differentiated. Uptake is a high affinity system associated with adrenergic neuronal elements. Uptake is a low affinity system, with a higher maximum rate than that of Uptake. Uptake has been associated with extraneuronal tissues such as cardiac muscle, fibroblasts or vascular smooth muscle. At low perfusion concentrations of norepinephrine most of the amine taken up by Uptake is metabolized. In order to study the localization of sites of norepinephrine storage following its uptake in the active bat heart, tritiated norepinephrine (2.5 mCi; 0.064 mg) was given intravenously to 2 bats. Monoamine oxidase had been inhibited with pheniprazine (10 mg/kg) one hour previously to decrease metabolism of norepinephrine.

Clinical Application and Results of the Assessment of Coronary Blood Flow by the Regional Precordial Xenon Residue Detection Technique

1978 ◽  
Vol 17 (04) ◽  
pp. 161-171
Author(s):  
H.-J. Engel ◽  
H. Hundeshagen ◽  
P. R. Lichtlen
Keyword(s):  
Wall Motion ◽  
High Flow ◽  
Detection Technique ◽  
Fatty Tissue ◽  
Technical Aspects ◽  
Residue Detection ◽  
High Affinity ◽  
Artery Disease ◽  
The Right ◽  
Drug Interventions

Methodological and technical aspects as well as application and results of the precordial Xenon-residue-detection technique are critically reviewed. The results concern mainly normal flow in various regions of the heart esp. in the free wall of the right and left ventricle, poststenotic flow in patients with coronary artery disease in relation to the degree of proximal nar-rowings as well as wall motion of the corresponding LV segment, bypassgraft flow and flow after drug interventions esp. nitrates, betablockers, the calcium-antagonist Nifedipine and the coronary dilator Dipyridamole. In spite of its serious limitations (high affinity of Xenon for fatty tissue, geometrical problems in the assessment of flow and its relation to anatomy, gas exchange in situations of high flow etc.), the technique is found to be a usefull investigatory tool. Due to its technical display and the related high costs routine application is, however, prohibitive.

Binding of 131I-Labeled Tissue-Type Plasminogen Activator on De-Endothelialized Lesions in Rabbits

1987 ◽  
Vol 26 (05) ◽  
pp. 224-228 ◽  
Author(s):  
Y. Isaka ◽  
H. Etani ◽  
K. Kimura ◽  
S. Yoneda ◽  
T. Kamada ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Abdominal Aorta ◽  
Half Life ◽  
Balloon Catheter ◽  
Biochemical Properties ◽  
Tissue Type ◽  
Fibrin Deposition ◽  
Tissue Type Plasminogen Activator ◽  
High Affinity ◽  
Type Plasminogen Activator

Tissue-type plasminogen activator (t-PA) which has a high affinity for fibrin in the clot, was labeled with 131I by the iodogen method, and its binding to de-endothelialized lesions in the rabbit was measured to assess the detectability of thrombi. The de-endothelialized lesion was induced in the abdominal aorta with a Fogarty 4F balloon catheter. Two hours after the de-endothelialization, 131I-labeled t-PA (125 ± 46 μCi) was injected intravenously. The initial half-life of the agent in blood (n = 12) was 2.9 ± 0.4 min. The degree of binding of 131I-labeled t-PA to the de-endothelialized lesion was evaluated at 15 min (n = 6) or at 30 min (n = 6) after injection of the agent. In spite of the retention of the biochemical properties of 131I-labeled t-PA and the presence of fibrin deposition at the de-endothelialized lesion, the binding of t-PA to the lesion was not sufficiently strong. Lesion-to-control ratios (cpm/g/cpm/g) were 1.65 ± 0.40 (at 15 min) and 1.39 ± 1.31 (at 30 min), and lesion-to-blood ratios were 1.39 ± 0.32 (at 15 min) and 1.36 ± 0.23 (at 30 min). These results suggest that radiolabeled t-PA may be inappropriate as a radiopharmaceutical for the scintigraphic detection of a pre-existing thrombotic lesion.

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

1990 ◽  
Vol 63 (02) ◽  
pp. 193-203 ◽  
Author(s):  
John R Shainoff ◽  
Deborah J Stearns ◽  
Patricia M DiBello ◽  
Youko Hishikawa-Itoh
Keyword(s):  
Peritoneal Macrophages ◽  
Affinity Binding ◽  
Macrophage Cell ◽  
High Affinity Binding ◽  
Amino Terminal ◽  
High Affinity ◽  
Terminal Domain ◽  
Weak Binding ◽  
Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

Protein S and C4b-Binding Protein: Components Involved in the Regulation of the Protein C Anticoagulant System

1991 ◽  
Vol 66 (01) ◽  
pp. 049-061 ◽  
Author(s):  
Björn Dahlbäck
Keyword(s):  
Vitamin K ◽  
Complement System ◽  
Protein C ◽  
Protein S ◽  
High Affinity ◽  
Anticoagulant System ◽  
Dependent Protein ◽  
The Complement System ◽  
Β Chain ◽  
Dependent Domain

SummaryThe protein C anticoagulant system provides important control of the blood coagulation cascade. The key protein is protein C, a vitamin K-dependent zymogen which is activated to a serine protease by the thrombin-thrombomodulin complex on endothelial cells. Activated protein C functions by degrading the phospholipid-bound coagulation factors Va and VIIIa. Protein S is a cofactor in these reactions. It is a vitamin K-dependent protein with multiple domains. From the N-terminal it contains a vitamin K-dependent domain, a thrombin-sensitive region, four EGF)epidermal growth factor (EGF)-like domains and a C-terminal region homologous to the androgen binding proteins. Three different types of post-translationally modified amino acid residues are found in protein S, 11 γ-carboxy glutamic acid residues in the vitamin K-dependent domain, a β-hydroxylated aspartic acid in the first EGF-like domain and a β-hydroxylated asparagine in each of the other three EGF-like domains. The EGF-like domains contain very high affinity calcium binding sites, and calcium plays a structural and stabilising role. The importance of the anticoagulant properties of protein S is illustrated by the high incidence of thrombo-embolic events in individuals with heterozygous deficiency. Anticoagulation may not be the sole function of protein S, since both in vivo and in vitro, it forms a high affinity non-covalent complex with one of the regulatory proteins in the complement system, the C4b-binding protein (C4BP). The complexed form of protein S has no APC cofactor function. C4BP is a high molecular weight multimeric protein with a unique octopus-like structure. It is composed of seven identical α-chains and one β-chain. The α-and β-chains are linked by disulphide bridges. The cDNA cloning of the β-chain showed the α- and β-chains to be homologous and of common evolutionary origin. Both subunits are composed of multiple 60 amino acid long repeats (short complement or consensus repeats, SCR) and their genes are located in close proximity on chromosome 1, band 1q32. Available experimental data suggest the β-chain to contain the single protein S binding site on C4BP, whereas each of the α-chains contains a binding site for the complement protein, C4b. As C4BP lacking the β-chain is unable to bind protein S, the β-chain is required for protein S binding, but not for the assembly of the α-chains during biosynthesis. Protein S has a high affinity for negatively charged phospholipid membranes, and is instrumental in binding C4BP to negatively charged phospholipid. This constitutes a novel mechanism for control of the complement system on phospholipid surfaces. Recent findings have shown circulating C4BP to be involved in yet another calcium-dependent protein-protein interaction with a protein known as the serum amyloid P-component (SAP). The binding sites on C4BP for protein S and SAP are independent. SAP, which is a normal constituent in plasma and in tissue, is a so-called pentraxin being composed of 5 non-covalently bound 25 kDa subunits. It is homologous to C reactive protein (CRP) but its function is not yet known. The specific high affinity interactions between protein S, C4BP and SAP suggest the regulation of blood coagulation and that of the complement system to be closely linked.

Antithrombin III Binding to Surface Immobilized Heparin and Its Relation to F Xa Inhibition

1987 ◽  
Vol 58 (04) ◽  
pp. 1064-1067 ◽  
Author(s):  
K Kodama ◽  
B Pasche ◽  
P Olsson ◽  
J Swedenborg ◽  
L Adolfsson ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Surface Coating ◽  
Antithrombin Iii ◽  
Lower Class ◽  
Biologically Active ◽  
High Affinity ◽  
Heparin Coating ◽  
Continuous Surface ◽  
Approximate Molecular Weight

SummaryThe mode of F Xa inhibition was investigated on a thromboresistant surface with end-point attached partially depoly-merized heparin of an approximate molecular weight of 8000. Affinity chromatography revealed that one fourth of the heparin used in surface coating had high affinity for antithrombin III (AT). The heparin surface adsorbed AT from both human plasma and solutions of purified AT. By increasing the ionic strength in the AT solution the existence of high and low affinity sites could be shown. The uptake of AT was measured and the density of available high and low affinity sites was found to be in the range of 5 HTid 11 pic.omoles/cmf, respectively Thus the estimated density of biologically active high and low ailmity heparm respectively would be 40 and 90 ng/cm2 The heparin coating did not take up or exert F Xa inhibition by itself. With AT adsorbed on both high and low affinity heparin the surface had the capacity to inhibit several consecutive aliquots of F Xa exposed to the surface. When mainly high affinity sites were saturated with AT the inhibition capacity was considerably lower. Tt was demonstrated that the density of AT on both high and low affinity heparin determines the F Xa inhibition capacity whereas the amount of AT on high affinity sites limits the rate of the reaction. This implies that during the inhibition of F Xa there is a continuous surface-diffusion of AT from sites of a lower class to the high affinity sites where the F Xa/AT complex is formed and leaves the surface. The ability of the immobilized heparin to catalyze inhibition of F Xa is likely to be an important component for the thromboresistant properties of a heparin coating with non-compromized AT binding sequences.

High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

1994 ◽  
Vol 72 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Neelesh Bangalore ◽  
William N Drohan ◽  
Carolyn L Orthner
Keyword(s):  
Binding Sites ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Affinity Binding ◽  
Cell Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gla Domain ◽  
Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

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