MOLECULAR DOCKING ANALYSES OF SOME CYCLOHEXADIENE DERIVATIVES

The molecular docking study was performed with aim to examine the inhibitory potency of two selected cyclohexadiene derivatives (cis-(1S)-3-Fluoro-3,5-cyclohexadiene-1,2-diol (1), and 1,1'-(3,5-Cyclohexadiene-1,3-diyl)dibenzene (2)). The inhibitory potency of compounds 1 and 2 was investigated toward Urokinase Type Plasminogen Activator (uPa). For this purpose AutoDock 4.0 software was used. The thermodynamic parameters achieved from molecular docking simulations, free energy of binding (ΔGbind) and inhibition constant (Ki), are analyzed and discussed. The compound 2 shows better inhibitory potency through uPa, than compound 1.Publishe

Uncharged isocoumarin-based inhibitors of urokinase-type plasminogen activator

BACKGROUND: Urokinase-type plasminogen activator (uPA) plays a major role in extracellular proteolytic events associated with tumor cell growth, migration and angiogenesis. Consequently, uPA is an attractive target for the development of small molecule active site inhibitors. Most of the recent drug development programs aimed at nonpeptidic inhibitors targeted at uPA have focused on arginino mimetics containing amidine or guanidine functional groups attached to aromatic or heterocyclic scaffolds. There is a general problem of limited bioavailability of these charged inhibitors. In the present study, uPA inhibitors were designed on an isocoumarin scaffold containing uncharged substituents. RESULTS: 4-Chloro-3-alkoxyisocoumarins were synthesized in which the 3-alkoxy group contained a terminal bromine; these were compared with similar inhibitors that contained a charged terminal functional group. Additional variations included functional groups attached to the seven position of the isocoumarin scaffold. N- [3-(3-Bromopropoxy)-4-chloro-1-oxo-1H-isochromen-7-yl]benzamide was identified as an uncharged lead inhibitor of uPA, K(i )= 0.034 μM. Molecular modeling of human uPA with these uncharged inhibitors suggests that the bromine occupies the same position as positively charged arginino mimetic groups. CONCLUSION: This study demonstrates that potent uncharged inhibitors of uPA can be developed based upon the isocoumarin scaffold. A tethered bromine in the three position and an aromatic group in the seven position are important contributors to binding. Although the aim was to develop compounds that act as mechanism-based inactivators, these inhibitors are competitive reversible inhibitors

Assessing the similarity of ligand binding conformations with the Contact Mode Score

© 2016 Elsevier Ltd Structural and computational biologists often need to measure the similarity of ligand binding conformations. The commonly used root-mean-square deviation (RMSD) is not only ligand-size dependent, but also may fail to capture biologically meaningful binding features. To address these issues, we developed the Contact Mode Score (CMS), a new metric to assess the conformational similarity based on intermolecular protein-ligand contacts. The CMS is less dependent on the ligand size and has the ability to include flexible receptors. In order to effectively compare binding poses of non-identical ligands bound to different proteins, we further developed the eXtended Contact Mode Score (XCMS). We believe that CMS and XCMS provide a meaningful assessment of the similarity of ligand binding conformations. CMS and XCMS are freely available at http://brylinski.cct.lsu.edu/content/contact-mode-score and http://geaux-computational-bio.github.io/contact-mode-score/

A Computational Investigation of Small-Molecule Engagement of Hot Spots at Protein–Protein Interaction Interfaces

The binding affinity of a protein–protein interaction is concentrated at amino acids known as hot spots. It has been suggested that small molecules disrupt protein–protein interactions by either (i) engaging receptor protein hot spots or (ii) mimicking hot spots of the protein ligand. Yet, no systematic studies have been done to explore how effectively existing small-molecule protein–protein interaction inhibitors mimic or engage hot spots at protein interfaces. Here, we employ explicit-solvent molecular dynamics simulations and end-point MM-GBSA free energy calculations to explore this question. We select 36 compounds for which high-quality binding affinity and cocrystal structures are available. Five complexes that belong to three classes of protein–protein interactions (primary, secondary, and tertiary) were considered, namely, BRD4•H4, XIAP•Smac, MDM2•p53, Bcl-xL•Bak, and IL-2•IL-2Rα. Computational alanine scanning using MM-GBSA identified hot-spot residues at the interface of these protein interactions. Decomposition energies compared the interaction of small molecules with individual receptor hot spots to those of the native protein ligand. Pharmacophore analysis was used to investigate how effectively small molecules mimic the position of hot spots of the protein ligand. Finally, we study whether small molecules mimic the effects of the native protein ligand on the receptor dynamics. Our results show that, in general, existing small-molecule inhibitors of protein–protein interactions do not optimally mimic protein–ligand hot spots, nor do they effectively engage protein receptor hot spots. The more effective use of hot spots in future drug design efforts may result in smaller compounds with higher ligand efficiencies that may lead to greater success in clinical trials

Inhibitors of the Plasminogen/Plasmin system for the treatment of Traumatic Brain Injury

La plasmina és un enzim proteolític responsable de la degradació de la fibrina, la proteïna estructural dels coàguls sanguinis. És activada a partir del zimogen plasminogen per l’activador tissular del plasminogen (tPA). La degradació de fibrina, o fibrinòlisi, és un procés natural que actua com a mecanisme de control per la hemostàsia i ajuda a mantenir la adequada fluïdesa de la sang. No obstant, hi ha situacions en que la fibrinòlisi es veu afectada, el que pot resultar en un sagnat excessiu, a més d’altres complicacions. Durant un traumatisme cerebral, es pot produir una activació excessiva de plasmina, que està correlacionada amb un increment en la permeabilitat de la barrera Hematoencefàlica (BHE). La inhibició de la plasmina o de la seva activació és per tant una estratègia interessant per minimitzar els danys a la BHE sota aquestes condicions hiperfibrinolítiques. Un nou compost anomenat LTI-6 amb elevada activitat antifibrinolítica va ser descobert en col·laboració amb l’empresa Alxerion Biotech. Aquest compost combina una piperidina, un 1,2,3-triazol i una 1,3,4- oxadiazolona. Els assaigs en sang i plasma van confirmar la seva activitat antifibrinolítica, amb una potència equivalent a l’àcid tranexàmic (TXA). Després d’estudiar la seva afinitat envers possibles receptors biològics, el mecanisme d’acció proposat per LTI-6 és la interacció amb els centres d’unió a lisina presents al plasminogen. Després de proposar diverses modificacions estructurals, un total de deu nous compostos vas ser sintetitzats i estudiats tant in vitro com in silico. La substitució de piperidina per amines lineals va reduir l’activitat global de la molècula. Per contra, substituir la 1,3,4-oxadiazolona per una 1,2,4-oxadiazolona va millorar l’activitat degut a un major nombre de ponts d’hidrogen. A més, modificar el 1,2,3-triazol per un 1,2,4-triazol va produir molècules sense activitat mesurable. Els compostos amb major activitat antifibrinolítica van ser estudiats en un model in vitro de la BHE sota condicions hiperfibrinolítiques. Concentracions elevades de plasminogen i tPA van incrementar la permeabilitat, a més de reduir l’expressió de proteïnes d’unió estreta. La presència dels nous compostos va demostrar tenir un efecte protector, ja que va esmorteir parcialment el dany sobre la BHE. Aquest treball obra la porta a desenvolupar futurs fàrmacs basats en aquests derivats de 1,2,3-triazol per a dues possibles aplicacions: com a nou fàrmac antifibrinolític que substitueixi al TXA, i com a agent protector de la BHE durant un traumatisme cerebral.La plasmina es una encima proteolítica responsable de la degradación de la fibrina, la proteína estructural de los coágulos sanguíneos. Es activada a partir del zimógeno plasminógeno por el activador de tisular del plasminógeno (tPA). La degradación de fibrina, o fibrinolisis, es un proceso natural que actúa como mecanismo de control para la hemostasis y ayuda a mantener la adecuada fluidez de la sangre. Sin embargo, hay situaciones en que la fibrinolisis se ve alterada, lo que puede resultar en excesivo sangrado y otras complicaciones. Durante un traumatismo cerebral, se puede producir una excesiva activación de plasmina que está correlacionada con un incremento en la permeabilidad de la barrera hematoencefálica (BHE). La inhibición de la plasmina o de su activación es en consecuencia una estrategia interesante para minimizar los daños a la BHE bajo dichas condiciones hiperfibrinolíticas. Un nuevo compuesto llamado LTI-6 con alta actividad antifibrinolítica fue descubierto en colaboración con la empresa Alxerion Biotech. Este compuesto combina una piperidina, un 1,2,3-triazol y una 1,3,4- oxadiazolona. Ensayos en sangre y en plasma confirmaron su actividad antifibrinolítica, con una potencia equivalente al ácido tranexámico (TXA). Tras estudiar su afinidad hacia posibles receptores biológicos, el mecanismo de acción propuesto para LTI-6 es la interacción con los centros de unión a lisina presentes en el plasminógeno. Tras propuesta de varias modificaciones estructurales, un total de diez nuevos compuestos fueron sintetizados i estudiados tanto in vitro como in silico. Sustituir la piperidina por aminas lineales disminuyó la actividad global. Por el contrario, la sustitución de la 1,3,4-oxadiazolona por 1,2,4-oxadiazolona mejoró la actividad debido a un mayor número de puentes de hidrógeno. Además, modificar el 1,2,3-triazol por un 1,2,4-triazol generó moléculas sin actividad detectable. Los compuestos con mayor actividad antifibrinolítica fueron estudiados en un modelo in vitro de BHE bajo condiciones hiperfibrinolíticas. Concentraciones elevadas de plasminógeno y tPA incrementaron la permeabilidad, además de reducir la expresión de proteínas de unión estrecha. La presencia de los nuevos compuestos demostró tener un efecto protector, ya que amortiguó parcialmente el daño a la BHE. Este trabajo abre la puerta a desarrollar futuros fármacos basados en estos derivados de 1,2,3-triazol para dos aplicaciones: como nuevo compuesto antifibrinolítico que sustituta al TXA, y como agente protector de la BHE durante un traumatismo cerebral.Plasmin is a proteolytic enzyme responsible for the degradation of fibrin, the structural protein of blood clots. It is activated from the zymogen plasminogen by tissue plasminogen activator (tPA). The degradation of fibrin, or fibrinolysis, is a natural process which constitutes a control mechanism for hemostasis and helps to ensure blood fluidity. However, there are situations in which the fibrinolytic system is dysregulated, which can result into excessive blood loss and other complications. During traumatic brain injury (TBI), an excessive activation of plasmin has been linked to an increased permeability of the blood-brain barrier (BBB). The inhibition of plasmin or plasmin activation is therefore an interesting strategy to minimize the damage to the BBB under these hyperfibrinolytic conditions during TBI. A novel compound named LTI-6 with high antifibrinolytic activity was discovered in collaboration with the start-up company Alxerion Biotech. This compound combined a piperidine, a 1,2,3-triazole and a 1,3,4- oxadiazolone ring. Blood and plasma assays confirmed its antifibrinolytic activity, with an equivalent potency to the gold standard tranexamic acid (TXA). After studying affinity towards different possible targets, the proposed mechanism of action for LTI-6 is an interaction with the lysine binding sites of plasminogen, hindering its activation into plasmin. After proposing different chemical modifications, a total of ten new compounds were synthesized and studied both in vitro and in silico. Substitution of the piperidine for linear amines hindered the overall activity. In contrast, substituting the 1,3,4-oxadiazolone for a 1,2,4-oxadiazole improved the activity due to a higher number of H-bonds. In addition, modifying the 1,2,3-triazole for a 1,2,4-triazole provided molecules with no detectable activity. The compounds with higher antifibrinolytic activity were studied in a hyperfibrinolytic BBB in vitro model. High concentrations of plasminogen and tPA caused an increased permeability, as well as a reduction of tight junction protein expression. The presence of the new compounds proved to have a protective effect, partially reducing the damage to the BBB. This work opens the door to develop future drugs based on these 1,2,3-triazole derivatives for two purposes: as a novel antifibrinolytic agent to substitute TXA, and as a potential BBB protective agent during TBI

Proteolytic Regulation Of Epithelial Sodium Channels By Urokinase Plasminogen Activator: Cutting Edge And Cleavage Sites

Plasminogen activator inhibitor 1 (PAI-1) level is extremely elevated in the edematous fluid of acutely injured lungs and pleurae. Elevated PAI-1 specifically inactivates pulmonary urokinase-type (uPA) and tissue-type plasminogen activators (tPA). We hypothesized that plasminogen activation and fibrinolysis may alter epithelial sodium channel (ENaC) activity, a key player in clearing edematous fluid. Two-chain urokinase (tcuPA) has been found to strongly stimulate heterologous human αβγ ENaC activity in a dose- and time-dependent manner. This activity of tcuPA was completely ablated by PAI-1. Furthermore, a mutation (S195A) of the active site of the enzyme also prevented ENaC activation. By comparison, three truncation mutants of the amino-terminal fragment of tcuPA still activated ENaC. uPA enzymatic activity was positively correlated with ENaC current amplitude prior to reaching the maximal level. In sharp contrast to uPA, neither single-chain tPA nor derivatives, including two-chain tPA and tenecteplase, affected ENaC activity. Furthermore, γ but not α subunit of ENaC was proteolytically cleaved at (177GR↓KR180) by tcuPA. In summary, the underlying mechanisms of urokinase-mediated activation of ENaC include release of self-inhibition, proteolysis of γ ENaC, incremental increase in opening rate, and activation of closed (electrically \ silent\ ) channels. This study for the first time demonstrates multifaceted mechanisms for uPA-mediated up-regulation of ENaC, which form the cellular and molecular rationale for the beneficial effects of urokinase in mitigating mortal pulmonary edema and pleural effusions

Protein-Ligand Interaction Energy-Based Entropy Calculations: Fundamental Challenges For Flexible Systems

Entropy calculations represent one of the most challenging steps in obtaining the binding free energy in biomolecular systems. A novel computationally effective approach (IE) was recently proposed to calculate the entropy based on the computation of protein-ligand interaction energy directly from molecular dynamics (MD) simulations. We present a study focused on the application of this method to flexible molecular systems and compare its performance with well-established normal mode (NM) and quasiharmonic (QH) entropy calculation approaches. Our results raise substantial concerns on the general applicability of IE in terms of reproducibility, reasonable absolute values of the entropy and agreement with NM and QM approaches. IE shows significant variation in the computed entropy values depending on the MD frames chosen for calculations. These deviations render reproducibility of IE calculations to be far from sufficient. We conclude that IE is recommended to be used after substantial modifications with respect to its sampling methodology

Mimicking Intermolecular Interactions of Tight Protein–Protein Complexes for Small-Molecule Antagonists

Tight protein–protein interactions (Kd1000 Å2) are highly challenging to disrupt with small molecules. Historically, the design of small molecules to inhibit protein–protein interactions has focused on mimicking the position of interface protein ligand side chains. Here, we explore mimicry of the pairwise intermolecular interactions of the native protein ligand with residues of the protein receptor to enrich commercial libraries for small-molecule inhibitors of tight protein–protein interactions. We use the high-affinity interaction (Kd=1 nm) between the urokinase receptor (uPAR) and its ligand urokinase (uPA) to test our methods. We introduce three methods for rank-ordering small molecules docked to uPAR: 1) a new fingerprint approach that represents uPA′s pairwise interaction energies with uPAR residues; 2) a pharmacophore approach to identify small molecules that mimic the position of uPA interface residues; and 3) a combined fingerprint and pharmacophore approach. Our work led to small molecules with novel chemotypes that inhibited a tight uPAR⋅uPA protein–protein interaction with single-digit micromolar IC50 values. We also report the extensive work that identified several of the hits as either lacking stability, thiol reactive, or redox active. This work suggests that mimicking the binding profile of the native ligand and the position of interface residues can be an effective strategy to enrich commercial libraries for small-molecule inhibitors of tight protein–protein interactions

Fibrinolytic Regulation of Pulmonary Epithelial Sodium Channels: a Critical Review

Luminal fluid homeostasis in the respiratory system is crucial to maintain the gas- blood exchange in normal lungs and mucociliary clearance in the airways. Epithelial sodium channels (ENaC) govern ~70% of alveolar fluid clearance. Four ENaC subunits have been cloned, namely, ?, ?, ?, and ? ENaC subunits in mammalian cells. This critical review focuses on the expression and function of ENaC in human and murine lungs, and the post-translational regulation by fibrinolysins. Nebulized urokinase was intratracheally delivered for clinical models of lung injury with unknown mechanisms. The central hypothesis is that proteolytically cleaved ENaC channels composed of four subunits are essential pathways to maintain fluid homeostasis in the airspaces, and that fibrinolysins are potential pharmaceutical ENaC activators to resolve edema fluid. This hypothesis is strongly supported by our following observations: 1) ? ENaC is expressed in the apical membrane of human lung epithelial cells; 2) ? ENaC physically interacts with the other three ENaC counterparts; 3) the features of ??? ENaC channels are conferred by ? ENaC; 4) urokinase activates ENaC activity; 5) urokinase deficiency is associated with a markedly distressed pulmonary ENaC function in vivo; 6) ? ENaC is proteolytically cleaved by urokinase; 7) urokinase augments the density of opening channels at the cell surface; and 8) urokinase extends opening time of ENaC channels to the most extent. Our integrated publications laid the groundwork for an innovative concept of pulmonary transepithelial fluid clearance in both normal and diseased lungs

Data-Driven Rational Drug Design

Vast amount of experimental data in structural biology has been generated, collected and accumulated in the last few decades. This rich dataset is an invaluable mine of knowledge, from which deep insights can be obtained and practical applications can be developed. To achieve that goal, we must be able to manage such Big Data\u27\u27 in science and investigate them expertly. Molecular docking is a field that can prominently make use of the large structural biology dataset. As an important component of rational drug design, molecular docking is used to perform large-scale screening of putative associations between small organic molecules and their pharmacologically relevant protein targets. Given a small molecule (ligand), a molecular docking program simulates its interaction with the target protein, and reports the probable conformation of the protein-ligand complex, and the relative binding affinity compared against other candidate ligands. This dissertation collects my contributions in several aspects of molecular docking. My early contribution focused on developing a novel metric to quantify the structural similarity between two protein-ligand complexes. Benchmarks show that my metric addressed several issues associated with the conventional metric. Furthermore, I extended the functionality of this metric to cross different systems, effectively utilizing the data at the proteome level. After developing the novel metric, I formulated a scoring function that can extract the biological information of the complex, integrate it with the physics components, and finally enhance the performance. Through collaboration, I implemented my model into an ultra-fast, adaptive program, which can take advantage of a range of modern parallel architectures and handle the demanding data processing tasks in large scale molecular docking applications