Fibrinogen aggregation is the last step in blood coagulation. Inhibition of fibrinogen aggregation could lead to anticoagulation effects. However, there is no good method for the ready evaluation of fibrinogen coagulation. A commonly used path method is slow and requires an expensive instrument. In this project, we have developed a microplate reader and In evaluating inhibitors of fibrinogen coagulations there is no good method. As an important process in hemostasis, fibrinogen coagulation is often detected by micro-plate reader. In our test of fibrinogen coagulation, we improved the observing and analyzing method by using photograph to see concentration-depended effect of thrombin inhibitors on the coagulation. Three known thrombin inhibitors, AEBSF, APMSF and PMSF, were applied to develop the method for detecting the fibrinogen coagulation. The results showed our method is of accuracy in determination of the amount of fibrin when compared with other types of methods.

The pathogenicity and intractable nature of the microorganism Staphylococcus aureus (SA) has been long documented and highlighted by many health care agencies, with emphasis on its ability to exploit the human coagulation system to deadly effect. Two drugs from a class of inhibitors known as Direct Thrombin Inhibitors (DTI) have been shown to have a substantial effect on the enzyme secreted by SA known as Staphylocoagulase (SC), but up until now the application of this potential treatment has been limited. This paper strives to supply an overview of these clinical studies and propose a novel protocol for testing DTI's on SA in an in vitro setting. Three DTIs have been identified, including two already tested in clinical trials, and computational molecular docking simulations have been applied to elucidate the mechanisms of action for the inhibition. An additional DTI has been developed using these mechanisms as principles and shows promise for future development. After conducting this preliminary protocol, it has been found that running a minimum inhibitory concentration test across several tubes with varying degrees of these DTIs demonstrated varying levels of coagulation consistent with the findings of clinical research papers. It is fair to conclude, then, that after development or discovery of new coagulase inhibitors, they can be quickly and accurately tested against existent DTIs to gauge their efficacy.

Despite the unfavorable pharmacokinetic properties associated with peptides, they are still of great interest in drug development due to a multitude of interesting biological functions. The development of peptidomimetics strives to maintain or improve the biological activity of a peptide concurrently with removing the unwanted properties. This thesis describes two synthetic approaches to peptidomimetics with particular emphasis on secondary structure mimetics. First the design, synthesis and evaluation of two beta-turn mimetics incorporated in the endorphin Leu-enkephalin is presented. The beta-turn mimetics were stabilized by replacement of the intramolecular hydrogen bond with an ethylene bridge, and the amide bond between Tyr and Gly was replaced with an ether linkage. Linear analogues of the two mimetics were also synthesized. The peptidomimetics and their linear analogues were evaluated in a competitive binding assay at two opiate receptors, my and delta. One of the cyclized beta-turn mimetics was found to be a delta receptor antagonist with an IC50 value of 160 nM. Second a synthetic strategy to a beta-strand mimetic using 2-fluoro-4-iodopyridine as scaffold is described. The synthesis involved a Grignard exchange reaction on the pyridine scaffold using an amino acid derivative as electrophile followed by an SNAr reaction using an amine as nucleophile. The synthesis of a tripeptidomimetic of Leu-Gly-Gly and attempts to introduce chiral building blocks at the C-terminal, as well as studies towards elongated mimetics are presented. Two additional studies deal with the synthesis of two classes of potential thrombin inhibitors based on the pyridine scaffold. The first class contain pyridine as central fragment (P2 residue) substituted with a para-amidinobenzylamine group as P1 residue and various benzoyl groups as P3 residues. Three potential thrombin inhibitors were synthesized and found to be microM inhibitors in an enzymatic assay. In the second class, the pyridine ring serves as P3 residue. This class also lacks a strongly basic group in the P1 position. A small library of eight compounds were synthesized and evaluated in the enzymatic assay. Unfortunately, these compounds lacked inhibitory activity.

Anticoagulants, also known as antithrombotics, are among the most commonly prescribed medications in the United States. Understanding how these medications work, the propensity for interactions with other drugs, dietary factors, and disease states is important for clinicians assessing and providing care to patients in all environments. In this review, we seek to provide essential information for the home health care provider for evaluating patients receiving anticoagulants commonly prescribed in the home health care setting. The low-molecular-weight heparins and vitamin K antagonists are the most commonly used agents for outpatient anticoagulation. New agents, such as the direct factor Xa inhibitors and direct thrombin inhibitors have recently been approved with additional new agents in the approval process and development pipeline. We seek to review the most pertinent information for each of these classes of medications providing information on pharmacology, interactions with other drugs, diet, and diseases and important clinical information.

<p>α₁-proteinase inhibitor (α₁-PI) is the most abundant serine protease inhibitor (serpin) in plasma. The α₁-PI M358R mutant exhibits greatly increased rates of thrombin inhibition compared to wild type α₁-PI, which predominantly inhibits neutrophil elastase. M358R (P1) lies at the reactive centre (P1-P1’) bond of the reactive centre loop (RCL) of α₁-PI, cleaved by cognate proteases as they become trapped in the serpin-type inhibitory complex. The relationship between RCL structure and serpin inhibitor function is incompletely understood and has not been subjected to saturation mutagenesis. α₁-PI M358R is a less potent inhibitor of thrombin than natural thrombin-inhibitory serpins, suggesting room for engineered improvement into an antithrombotic protein drug.</p> <p>Phage display is a powerful tool for screening mutant protein libraries, but only one serpin (PAI-1) has previously been mutated and expressed in this manner. In this study the T7Select10-3b (Novagen) phage display system was used to express α₁-PI variants and PAI-1, fused to the first 348 residues of the T7 10B coat protein. Following confirmation that α₁-PI M358R retained inhibitory activity when fused to T7Select10-3b phage, this system was used to express a library of α₁-PI mutant proteins with all possible codon combinations at positions P2 (P357) and P1 (M358) (441 mutants). The library was biopanned using a novel technique in order to amplify only the α₁-PI P2P1 mutants capable of forming stable complexes with thrombin. The P357/M358R mutant was the only P2P1 mutant enriched, indicating that the α₁-PI M358R protein has the optimal P2P1 sequence for thrombin inhibition.</p> <p>A second T7Select10-3b library of α₁-PI mutant proteins was generated to identify the optimal sequence at positions P7 through to P3 (amino acids 352-356) for thrombin inhibition. The P2 and P1 positions were maintained at P357/M358R, while all possible codon combinations at positions P7 through to P3 were represented (>4.08 million mutants). The library was biopanned using the protocol developed for the P2P1 library, before sequences were inserted into an <em>E. coli</em> expression vector and α₁-PI M358R P7-P3 mutants were screened for thrombin inhibitory activity. 80 individual colonies were screened, yielding 22 unique P7-P3 mutants with thrombin inhibitory activity greater than the M358R RCL sequence. The consensus observed in sequences with improved activity matched thrombin’s known substrate specificity and also general RCL trends: P7-Not Aromatic/P6-Hydrophobic/P5-T or S/P4-Hydrophobic/P3-Not Aromatic.</p> <p>Kinetic characterization of selected mutants with improved thrombin inhibitory activity yielded two mutants, P7-P3 sequence DITMA and AAFVS, with a second order rate constant of 1.0 x 10⁶ M^-1s^-1. This represents a >2-fold increase in the rate of thrombin inhibition versus α₁-PI M358R. Both the DITMA and AAFVS mutants were found to have a lower stoichiometry of inhibition compared to α₁-PI M358R, indicating that an improved thrombin inhibitory mechanism was also enriched during biopanning.</p> <p>These findings suggest that based on the scaffold of the α₁-PI protein, improved thrombin inhibitory activity can be engineered and selected via phage display. Additionally, this work represents a proof-of-principle for the application of this system to screen libraries of up to 10 million mutants in order to better engineer serpins towards a desired activity.</p> / Master of Health Sciences (MSc)

Les pentasaccharides sont des inhibiteurs indirects du facteur Xa ayant des profils pharmacocinétiques très prédictibles. En raison de la liaison de forte affinité des pentasaccharides à l'antithrombine, cette pharmacocinétique peut être prédite mais aussi transférée à d'autres molécules qui leur sont liées de manière covalente. L'EP42675 combine dans une seule molécule, une antithrombine directe réversible peptidomimétique (analogue de l'α-NAPAP), et un pentasaccharide inhibiteur indirect du facteur Xa antithrombine dépendant (analogue du fondaparinux). L'EP217609 est le dérivé biotinylé de l'EP42675. Son action anticoagulante peut être neutralisée par l'avidine qui se lie avec une grande affinité et spécificité à la fraction biotine de l'EP217609. La première indication cible de l'EP217609 et de son antidote l'avidine est la chirurgie cardiaque nécessitant une circulation extracorporelle. La deuxième indication cible est le traitement des syndromes coronariens aigus nécessitant ou non une intervention coronarienne percutanée. Les études précliniques et cliniques de phase I et phase IIa résumées ici démontrent l'intérêt d'un tel concept de couplage avec un pentasaccharide : absence de dissociation entre les deux entités, faible variabilité intra et interindividuelle des paramètres pharmacocinétiques et pharmacodynamiques, et une neutralisation de l'activité anticoagulante de l'EP21609 quasi complète et sans effet rebond. / Pentasaccharides are indirect inhibitors of factor Xa with highly predictable pharmacokinetic profiles. Because of the high affinity binding of pentasaccharides to antithrombin, this pharmacokinetics can be predicted but also transferred to other molecules covalently bound to them. EP42675 combines in a single molecule, a reversible direct antithrombin (α-NAPAP peptidomimetic analog), and a pentasaccharide similar to fondaparinux with an indirect anti-factor Xa activity. EP217609 is the biotinylated derivative of EP42675 whose anticoagulant activity can be neutralized by avidin which binds with high affinity and specificity to the biotin moiety of EP217609.The first target indication of EP217609 and its antidote avidin is cardiac surgery requiring extracorporeal circulation. The second target indication is the treatment of acute coronary syndromes requiring or not a percutaneous coronary intervention.The preclinical and clinical Phase I and IIa studies summarized here demonstrate the value of such a coupling concept to the pentasaccharide: absence of dissociation between the two entities, low intra- and interindividual variability of the pharmacokinetic and pharmacodynamic parameters, and an almost complete neutralization of the EP217609 anticoagulant activity with no rebound effect.

This thesis describes different aspects of protein interactions. Initially the function of peptides and their conjugates with small molecule inhibitors on the surface of Human Carbonic Anhydrase isoenzyme II (HCAII) is evaluated. The affinities for HCAII of the flexible, synthetic helix-loop-helix motif conjugated with a series of spacered inhibitors were measured by fluorescence spectroscopy and found in the best cases to be in the low nM range. Dissociation constants show considerable dependence on linker length and vary from 3000 nM for the shortest spacer to 40 nM for the longest with a minimum of 5 nM for a spacer with an intermediate length. A rationale for binding differences based on cooperativity is presented and supported by affinities as determined by fluorescence spectroscopy. Heteronuclear Single Quantum Correlation Nuclear Magnetic Resonance (HSQC) spectroscopic experiments with 15N-labeled HCAII were used for the determination of the site of interaction. The influence of peptide charge and hydrophobicity was evaluated by surface plasmon resonance experiments. Hydrophobic sidechain branching and, more pronounced, peptide charge was demonstrated to modulate peptide – HCAII binding interactions in a cooperative manner, with affinities spanning almost two orders of magnitude. Detailed synthesis of small molecule inhibitors in a general lead discovery library as well as a targeted library for inhibition of α-thrombin is described. For the lead discovery library 160 members emanate from two N4-aryl-piperazine-2-carboxylic acid scaffolds derivatized in two dimensions employing a combinatorial approach on solid support. The targeted library was based on peptidomimetics of the D-Phe-Pro-Arg showing the scaffolds cyclopropane-1R,2R-dicarboxylic acid and (4-amino-3-oxo-morpholin-2-yl)- acetic acid as proline isosters. Employing 4-aminomethyl-benzamidine as arginine mimic and different hydrophobic amines and electrophiles as D-phenylalanine mimics resulted in 34 compounds showing IC50 values for α-thrombin ranging more than three orders of magnitude with the best inhibitor showing an IC50 of 130 nM. Interestingly, the best inhibitors showed reversed stereochemistry in comparison with a previously reported series employing a 3-oxo-morpholin-2-yl-acetic acid scaffold.