Kinetic Barrier to Enzyme Inhibition Is Manipulated by Dynamical Local Interactions in E. coli DHFR.

Dihydrofolate reductase (DHFR) is an important drug target and a highly studied model protein for understanding enzyme dynamics. DHFR's crucial role in folate synthesis renders it an ideal candidate to understand protein function and protein evolution mechanisms. In this study, to understand how a newly proposed DHFR inhibitor, 4'-deoxy methyl trimethoprim (4'-DTMP), alters evolutionary trajectories, we studied interactions that lead to its superior performance over that of trimethoprim (TMP). To elucidate the inhibition mechanism of 4'-DTMP, we first confirmed, both computationally and experimentally, that the relative binding free energy cost for the mutation of TMP and 4'-DTMP is the same, pointing the origin of the characteristic differences to be kinetic rather than thermodynamic. We then employed an interaction-based analysis by focusing first on the active site and then on the whole enzyme. We confirmed that the polar modification in 4'-DTMP induces additional local interactions with the enzyme, particularly, the M20 loop. These changes are propagated to the whole enzyme as shifts in the hydrogen bond networks. To shed light on the allosteric interactions, we support our analysis with network-based community analysis and show that segmentation of the loop domain of inhibitor-bound DHFR must be avoided by a successful inhibitor.

Oligomerization and cooperativity in GPCRs from the perspective of the angiotensin AT1 and dopamine D2 receptors.

G Protein-Coupled Receptors (GPCRs) can form homo- and heterodimers or constitute higher oligomeric clusters with other heptahelical GPCRs. In this article, multiscale molecular modeling approaches as well as experimental techniques which are used to study oligomerization of GPCRs are reviewed. In particular, the effect of dimerization/oligomerization to the ligand binding affinity of individual protomers and also on the efficacy of the oligomer are discussed by including diverse examples from the literature. In addition, possible allosteric effects that may emerge upon interaction of GPCRs with membrane components, like cholesterol, is also discussed. Investigation of these above-mentioned interactions may greatly contribute to the candidate molecule screening studies and development of novel therapeutics with fewer adverse effects.

Application of Multiscale Simulation Tools on GPCRs. An Example with Angiotensin II Type 1 Receptor.

G protein-coupled receptors (GPCRs) represent the biggest class of membrane proteins included in signal transduction cascade across the biological lipid bilayers. They are essential target structures for cell signaling and are of great commercial interest to the pharmaceutical industry (~50% of marketed drugs and ~25% of top-selling drugs targeting this receptor family). Recent advances made in molecular biology and computational chemistry open new avenues for the design of new therapeutic compounds. Molecular biology has recently provided the crystal structures of a few ligand-bound GPCRs in active and inactive states, which can be used as accurate templates in modeling studies. Computational chemistry offers a range of simulation, multiscale modeling with ligand- and structure-based approaches, and virtual screening tools for definition and analysis of protein-ligand, protein-protein, and protein-DNA interactions. Development of new approaches and algorithms on statistical methods and free energy simulations help to predict novel optimal compounds. Integrated approach to drug discovery that combines quantum mechanics calculations, molecular docking, molecular dynamics (MD) simulations, quantitative structure-activity relationships (QSAR), and de novo design studies under a single umbrella can be used for decreasing the risk of false-positive results. Each method has its own pros and cons and, when used alone, is not likely to yield very useful results. However, when these methods are combined with positive feedback loops, they may enhance each other and successful drug leads may be obtained. Moreover, investigating the activation mechanisms and atomistic determinants of ligand binding to GPCR targets would allow greater safety in the human life.

Structure-based design of hERG-neutral antihypertensive oxazalone and imidazolone derivatives.

Angiotensin II receptor type 1 (AT1) antagonists are the most recent drug class against hypertension. Recently first crystal structure of AT1 receptor is deposited to the protein data bank (PDB ID: 4YAY). In this work, several molecular screening methods such as molecular docking and de novo design studies were performed and it is found that oxazolone and imidazolone derivatives reveal similar/better interaction energy profiles compared to the FDA approved sartan molecules at the binding site of the AT1 receptor. A database consisting of 3500-fragments were used to enumerate de novo designed imidazolone and oxazolone derivatives and hereby more than 50000 novel small molecules were generated. These derivatives were then used in high throughput virtual screening simulations (Glide/HTVS) to find potent hit molecules. In addition, virtual screening of around 18 million small drug-like compounds from ZINC database were screened at the binding pocket of the AT1 receptor via Glide/HTVS method. Filtered structures were then used in more sophisticated molecular docking simulations protocols (i.e., Glide/SP; Glide/XP; Glide/IFD; Glide/QPLD, and GOLD). However, the K+ ion channel/drug interactions should also be considered in studies implemented in molecular level against their cardiovascular risks. Thus, selected compounds with high docking scores via all diverse docking algorithms are also screened at the pore domain regions of human ether-a-go-go-related gene (hERG1) K+ channel to remove the high affinity hERG1 blocking compounds. High docking scored compounds at the AT1 with low hERG1 affinity is considered for long molecular dynamics (MD) simulations. Post-processing analysis of MD simulations assisted for better understanding of molecular mechanism of studied compounds at the binding cavity of AT1 receptor. Results of this study can be useful for designing of novel and safe AT1 inhibitors.

Integration of multi-scale molecular modeling approaches with experiments for the in silico guided design and discovery of novel hERG-Neutral antihypertensive oxazalone and imidazolone derivatives and analysis of their potential restrictive effects on cell proliferation.

AT1 antagonists is the most recent drug class of molecules against hypertension and they mediate their actions through blocking detrimental effects of angiotensin II (A-II) when acts on type I (AT1) A-II receptor. The effects of AT1 antagonists are not limited to cardiovascular diseases. AT1 receptor blockers may be used as potential anti-cancer agents - due to the inhibition of cell proliferation stimulated by A-II. Therefore, AT1 receptors and the A-II biosynthesis mechanisms are targets for the development of new synthetic drugs and therapeutic treatment of various cardiovascular and other diseases. In this work, multi-scale molecular modeling approaches were performed and it is found that oxazolone and imidazolone derivatives reveal similar/better interaction energy profiles compared to the FDA approved sartan molecules at the binding site of the AT1 receptor. In silico-guided designed hit molecules were then synthesized and tested for their binding affinities to human AT1 receptor in radioligand binding studies, using [125I-Sar1-Ile8] AngII. Among the compounds tested, 19d and 9j molecules bound to receptor in a dose response manner and with relatively high affinities. Next, cytotoxicity and wound healing assays were performed for these hit molecules. Since hit molecule 19d led to deceleration of cell motility in all three cell lines (NIH3T3, A549, and H358) tested in this study, this molecule is investigated in further tests. In two cell lines (HUVEC and MCF-7) tested, 19d induced G2/M cell cycle arrest in a concentration dependent manner. Adherent cells detached from the plates and underwent cell death possibly due to apoptosis at 19d concentrations that induced cell cycle arrest.

Structure-based design of hERG-neutral antihypertensive oxazalone and imidazolone derivatives.

Angiotensin II receptor type 1 (AT1) antagonists are the most recent drug class against hypertension. Recently first crystal structure of AT1 receptor is deposited to the protein data bank (PDB ID: 4YAY). In this work, several molecular screening methods such as molecular docking and de novo design studies were performed and it is found that oxazolone and imidazolone derivatives reveal similar/better interaction energy profiles compared to the FDA approved sartan molecules at the binding site of the AT1 receptor. A database consisting of 3500-fragments were used to enumerate de novo designed imidazolone and oxazolone derivatives and hereby more than 50000 novel small molecules were generated. These derivatives were then used in high throughput virtual screening simulations (Glide/HTVS) to find potent hit molecules. In addition, virtual screening of around 18 million small drug-like compounds from ZINC database were screened at the binding pocket of the AT1 receptor via Glide/HTVS method. Filtered structures were then used in more sophisticated molecular docking simulations protocols (i.e., Glide/SP; Glide/XP; Glide/IFD; Glide/QPLD, and GOLD). However, the K+ ion channel/drug interactions should also be considered in studies implemented in molecular level against their cardiovascular risks. Thus, selected compounds with high docking scores via all diverse docking algorithms are also screened at the pore domain regions of human ether-a-go-go-related gene (hERG1) K+ channel to remove the high affinity hERG1 blocking compounds. High docking scored compounds at the AT1 with low hERG1 affinity is considered for long molecular dynamics (MD) simulations. Post-processing analysis of MD simulations assisted for better understanding of molecular mechanism of studied compounds at the binding cavity of AT1 receptor. Results of this study can be useful for designing of novel and safe AT1 inhibitors.

Identification of novel serotonin reuptake inhibitors targeting central and allosteric binding sites: A virtual screening and molecular dynamics simulations study.

The serotonin (5-hydroxytryptamine, 5HT) transporter (SERT) is a member of neurotransmitter sodium symporter (NSS) family, which maintains neurotransmitter by reuptaking 5HT into synapses. Decrease in serotonin concentrations in synaptic clefts have been reported to cause psychological and neurological disorders. Therefore, inhibition of SERT is a potent strategy for the treatment of related diseases such as depression. In this study, approximately 260,000 small molecules from an available chemical database have been virtually screened both at central and allosteric binding sites of SERT to identify potent novel candidate SERT inhibitors. A set of docking algorithms were used to predict binding modes and energies of compounds. Screening analyses led three top-ranked hit compounds (160234, Otava ID: 7118020138; 159166, Otava ID: 7117171303; and 69419, Otava ID: 118671819) for central binding site (S1) and one compound (93507, Otava ID: 6248262) for allosteric binding site (S2). These promising compounds are then subjected to long multiple molecular dynamics (MD) simulations to elucidate their structural and dynamical profiles at the binding cavities of SERT. Higher predicted binding affinities of identified compounds were also confirmed with binding free energy calculations (MM/GBSA) in comparison with the reference central and allosteric binding site inhibitors, paroxetine (8PR) and escitalopram (68P), respectively. To the best of our knowledge, the present work is the first structure-based high throughput virtual screening study reported using recently revealed crystal structure of SERT for screening inhibitors from chemical databases on S1 and S2 binding sites. Small molecule library screening study yielded candidate compounds both at central and allosteric binding site of SERT, and further experimentation may pave the way for developing novel strong inhibitors.

Analysis of the Glutamate Agonist LY404,039 Binding to Nonstatic Dopamine Receptor D2 Dimer Structures and Consensus Docking.

Dopamine receptor D2 (D2R) plays an important role in the human central nervous system and is a focal target of antipsychotic agents. The D2HighR and D2LowR dimeric models previously developed by our group are used to investigate the prediction of binding affinity of the LY404,039 ligand and its binding mechanism within the catalytic domain. The computational data obtained using molecular dynamics simulations fit well with the experimental results. The calculated binding affinities of LY404,039 using MM/PBSA for the D2HighR and D2LowR targets were -12.04 and -9.11 kcal/mol, respectively. The experimental results suggest that LY404,039 binds to D2HighR and D2LowR with binding affinities (Ki) of 8.2 and 1640 nM, respectively. The high binding affinity of LY404,039 in terms of binding to [3H]domperidone was inhibited by the presence of a guanine nucleotide, indicating an agonist action of the drug at D2HighR. The interaction analysis demonstrated that while Asp114 was among the most critical amino acids for D2HighR binding, residues Ser193 and Ser197 were significantly more important within the binding cavity of D2LowR. Molecular modeling analyses are extended to ensemble docking as well as structure-based pharmacophore model (E-pharmacophore) development using the bioactive conformation of LY404,039 at the binding pocket as a template and screening of small-molecule databases with derived pharmacophore models.