Prioritizing Small Sets of Molecules for Synthesis through in-silico Tools: A Comparison of Common Ranking Methods.

Prioritizing molecules for synthesis is a key role of computational methods within medicinal chemistry. Multiple tools exist for ranking molecules, from the cheap and popular molecular docking methods to more computationally expensive molecular-dynamics (MD)-based methods. It is often questioned whether the accuracy of the more rigorous methods justifies the higher computational cost and associated calculation time. Here, we compared the performance on ranking the binding of small molecules for seven scoring functions from five docking programs, one end-point method (MM/GBSA), and two MD-based free energy methods (PMX, FEP+). We investigated 16 pharmaceutically relevant targets with a total of 423 known binders. The performance of docking methods for ligand ranking was strongly system dependent. We observed that MD-based methods predominantly outperformed docking algorithms and MM/GBSA calculations. Based on our results, we recommend the application of MD-based free energy methods for prioritization of molecules for synthesis in lead optimization, whenever feasible.

KinFragLib: Exploring the Kinase Inhibitor Space Using Subpocket-Focused Fragmentation and Recombination.

Protein kinases play a crucial role in many cell signaling processes, making them one of the most important families of drug targets. In this context, fragment-based drug design strategies have been successfully applied to develop novel kinase inhibitors. These strategies usually follow a knowledge-driven approach to optimize a focused set of fragments to a potent kinase inhibitor. Alternatively, KinFragLib explores and extends the chemical space of kinase inhibitors using data-driven fragmentation and recombination. The method builds on available structural kinome data from the KLIFS database for over 2500 kinase DFG-in structures cocrystallized with noncovalent kinase ligands. The computational fragmentation method splits the ligands into fragments with respect to their 3D proximity to six predefined functionally relevant subpocket centers. The resulting fragment library consists of six subpocket pools with over 7000 fragments, available at https://github.com/volkamerlab/KinFragLib. KinFragLib offers two main applications: on the one hand, in-depth analyses of the chemical space of known kinase inhibitors, subpocket characteristics, and connections, and on the other hand, subpocket-informed recombination of fragments to generate potential novel inhibitors. The latter showed that recombining only a subset of 624 representative fragments generated 6.7 million molecules. This combinatorial library contains, besides some known kinase inhibitors, more than 99% novel chemical matter compared to ChEMBL and 63% molecules compliant with Lipinski's rule of five.

Identification of Small Molecules that Modulate Mutant p53 Condensation.

Structural mutants of p53 induce global p53 protein destabilization and misfolding, followed by p53 protein aggregation. First evidence indicates that p53 can be part of protein condensates and that p53 aggregation potentially transitions through a condensate-like state. We show condensate-like states of fluorescently labeled structural mutant p53 in the nucleus of living cancer cells. We furthermore identified small molecule compounds that interact with the p53 protein and lead to dissolution of p53 structural mutant condensates. The same compounds lead to condensation of a fluorescently tagged p53 DNA-binding mutant, indicating that the identified compounds differentially alter p53 condensation behavior depending on the type of p53 mutation. In contrast to p53 aggregation inhibitors, these compounds are active on p53 condensates and do not lead to mutant p53 reactivation. Taken together our study provides evidence for structural mutant p53 condensation in living cells and tools to modulate this process.

Computationally Empowered Workflow Identifies Novel Covalent Allosteric Binders for KRASG12C.

Due to its frequent mutations in multiple lethal cancers, KRAS is one of the most-studied anticancer targets nowadays. Since the discovery of the druggable allosteric binding site containing a G12C mutation, KRASG12C has been the focus of attention in oncology research. We report here a computationally driven approach aimed at identifying novel and selective KRASG12C covalent inhibitors. The workflow involved initial enumeration of virtual molecules tailored for the KRAS allosteric binding site. Tools such as pharmacophore modeling, docking, and free-energy perturbations were deployed to prioritize the compounds with the best profiles. The synthesized naphthyridinone scaffold showed the ability to react with G12C and inhibit KRASG12C . Analogues were prepared to establish structure-activity relationships, while molecular dynamics simulations and crystallization of the inhibitor-KRASG12C complex highlighted an unprecedented binding mode.

Truly Target-Focused Pharmacophore Modeling: A Novel Tool for Mapping Intermolecular Surfaces.

Pharmacophore models are an accurate and minimal tridimensional abstraction of intermolecular interactions between chemical structures, usually derived from a group of molecules or from a ligand-target complex. Only a limited amount of solutions exists to model comprehensive pharmacophores using the information of a particular target structure without knowledge of any binding ligand. In this work, an automated and customable tool for truly target-focused (T²F) pharmacophore modeling is introduced. Key molecular interaction fields of a macromolecular structure are calculated using the AutoGRID energy functions. The most relevant points are selected by a newly developed filtering cascade and clustered to pharmacophore features with a density-based algorithm. Using five different protein classes, the ability of this method to identify essential pharmacophore features was compared to structure-based pharmacophores derived from ligand-target interactions. This method represents an extremely valuable instrument for drug design in a situation of scarce ligand information available, but also in the case of underexplored therapeutic targets, as well as to investigate protein allosteric pockets and protein-protein interactions.

Synthesis, crystallographic characterization, molecular docking and biological activity of isoquinoline derivatives.

The main objective of this work was to synthesize novel compounds with a benzo[de][1,2,4]triazolo[5,1-a]isoquinoline scaffold by employing (dioxo-benzo[de]isoquinolin-2-yl) thiourea as a building block. Molecular docking was conducted in the COX-2 active site to predict the plausible binding mode and rationalize the structure-activity relationship of the synthesized compounds. The structures of the synthesized compounds were confirmed by HREI-MS, and NMR spectra along with X-ray diffraction were collected for products 1 and 5. Thereafter, anti-inflammatory effect of molecules 1-20 was evaluated in vivo using carrageenan-induced paw edema method, revealing significant inhibition potency in albino rats with an activity comparable to that of the standard drugs indomethacin. Compounds 8, 9, 15 and 16 showed the highest anti-inflammatory activity. However, thermal sensitivity-hot plat test, a radiological examination and motor coordination assessment were performed to test the activity against rheumatoid arthritis. The obtained results indicate promising anti-arthritic activity for compounds 9 and 15 as significant reduction of the serum level of interleukin-1ß [IL-1ß], cyclooxygenase-2 [COX-2] and prostaglandin E2 [PGE2] was observed in CFA rats.

Arginase Structure and Inhibition: Catalytic Site Plasticity Reveals New Modulation Possibilities.

Metalloenzyme arginase is a therapeutically relevant target associated with tumor growth. To fight cancer immunosuppression, arginase activity can be modulated by small chemical inhibitors binding to its catalytic center. To better understand molecular mechanisms of arginase inhibition, a careful computer-aided mechanistic structural investigation of this enzyme was conducted. Using molecular dynamics (MD) simulations in the microsecond range, key regions of the protein active site were identified and their flexibility was evaluated and compared. A cavity opening phenomenon was observed, involving three loops directly interacting with all known ligands, while metal coordinating regions remained motionless. A novel dynamic 3D pharmacophore analysis method termed dynophores has been developed that allows for the construction of a single 3D-model comprising all ligand-enzyme interactions occurring throughout a complete MD trajectory. This new technique for the in silico study of intermolecular interactions allows for loop flexibility analysis coupled with movements and conformational changes of bound ligands. Presented MD studies highlight the plasticity of the size of the arginase active site, leading to the hypothesis that larger ligands can enter the cavity of arginase. Experimental testing of a targeted fragment library substituted by different aliphatic groups validates this hypothesis, paving the way for the design of arginase inhibitors with novel binding patterns.

α-Amylase Modulation: Discovery of Inhibitors Using a Multi-Pharmacophore Approach for Virtual Screening.

Better control of postprandial hyperglycemia can be achieved by delaying the absorption of glucose resulting from carbohydrate digestion. Because α-amylase initiates the hydrolysis of polysaccharides, the design of α-amylase inhibitors can lead to the development of new treatments for metabolic disorders such as type II diabetes and obesity. In this study, a rational computer-aided approach was developed to identify novel α-amylase inhibitors. Three-dimensional pharmacophores were developed based on the binding mode analysis of six different families of compounds that bind to this enzyme. In a stepwise virtual screening workflow, seven molecules were selected from a library of 1.4 million. Five out of seven biologically tested compounds showed α-amylase inhibition, and the two most potent compounds inhibited α-amylase with IC50 values of 17 and 27 µm. The scaffold benzylideneacetohydrazide was shared by four of the discovered inhibitors, emerging as a novel drug-like non-carbohydrate fragment and constituting a promising lead scaffold for α-amylase inhibition.

More than a look into a crystal ball: protein structure elucidation guided by molecular dynamics simulations.

The 'form follows function' principle implies that a structural determination of protein structures is indispensable to understand proteins in their biological roles. However, experimental methods still show shortcomings in the description of the dynamic properties of proteins. Therefore, molecular dynamics (MD) simulations represent an essential tool for structural biology to investigate proteins as flexible and dynamic entities. Here, we will give an overview on the impact of MD simulations on structural investigations, including studies that aim at a prediction of protein-folding pathways, protein-assembly processes and the sampling of conformational space by computational means.

Anthocyanin composition, antioxidant efficiency, and α-amylase inhibitor activity of different Hungarian sour cherry varieties (Prunus cerasus L.).

Five Hungarian sour cherry cultivars were studied to determine their anthocyanin contents and their possible inhibitory properties. The water and methanol soluble antioxidant capacities were separately assessed by photoluminescence showing values ranged from 3.4µgmg(-1) to 15.4µgmg(-1), respectively. The "VN1" variety (selected from "Csengodi csokros") showed the highest antioxidant capacity. The anthocyanin content, measured by pH differential method or isolated by solid phase extraction, was the highest also in "VN1". Correlation was found between the anthocyanin content and the high antioxidant capacity. The main anthocyanin components were cyanidin-3-O-rutinoside and cyanidin-3-O-glucoside. The presence of malvidin-3,5-O-diglycoside was verified by MALDI-TOF MS. Sour cherry extracts and selected anthocyanins inhibited the human salivary alpha-amylase catalyzed hydrolysis competitively. The lowest IC50 value, 55µgmL(-1) or 80µM, was measured for malvidin-3,5-O-diglycoside, for which possible binding modes within the alpha-amylase active site could be investigated in silico using molecular docking and molecular dynamics.

From carbohydrates to drug-like fragments: Rational development of novel α-amylase inhibitors.

Starch catabolism leading to high glucose level in blood is highly problematic in chronic metabolic diseases, such as type II diabetes and obesity. α-Amylase catalyzes the hydrolysis of starch, increasing blood sugar concentration. Its inhibition represents a promising therapeutic approach to control hyperglycaemia. However, only few drug-like molecule inhibitors without sugar moieties have been discovered so far, and little information on the enzymatic mechanism is available. This work aims at the discovery of novel small α-amylase binders using a systematic in silico methodology. 3D-pharmacophore-based high throughput virtual screening of small compounds libraries was performed to identify compounds with high α-amylase affinity. Twenty-seven compounds were selected and biologically tested, revealing IC50 values in the micromolar range and ligand efficiency higher than the one of the bound form of acarbose, which is used as a reference for α-amylase inhibition.

Tailored Presentation of Carbohydrates on a Coiled Coil-Based Scaffold for Asialoglycoprotein Receptor Targeting.

The coiled-coil folding motif represents an ideal scaffold for the defined presentation of ligands due to the possibility of positioning them at specific distances along the axis. We created a coiled-coil glycopeptide library to characterize the distances between the carbohydrate-binding sites of the asialoglycoprotein receptors (ASGPR) on hepatocytes. The components of the glycopeptide library vary for the number of displayed ligands (galactose), their position on the peptide sequence, and the space between peptide backbone and carbohydrate. We determined the binding of the glycopeptides to the hepatocytes, and we established the optimal distance and orientation of the galactose moieties for interaction with the ASGPR using flow cytometry. We confirmed that the binding occurs through endocytosis mediated by ASGPR via inhibition studies with cytochalasin D; fluorescence microscopy studies display the uptake of the carrier peptides inside the cell. Thus, this study demonstrates that the coiled-coil motif can be used as reliable scaffold for the rational presentation of ligands.

3D-QSAR, design, synthesis and characterization of trisubstituted harmine derivatives with in vitro antiproliferative properties.

Apolar trisubstituted derivatives of harmine show high antiproliferative activity on diverse cancer cell lines. However, these molecules present a poor solubility making these compounds poorly bioavailable. Here, new compounds were synthesized in order to improve solubility while retaining antiproliferative activity. First, polar substituents have shown a higher solubility but a loss of antiproliferative activity. Second, a Comparative Molecular Field Analysis (CoMFA) model was developed, guiding the design and synthesis of eight new compounds. Characterization has underlined the in vitro antiproliferative character of these compounds on five cancerous cell lines, combining with a high solubility at physiological pH, making these molecules druggable. Moreover, targeting glioma treatment, human intestinal absorption and blood brain penetration have been calculated, showing high absorption and penetration properties.

Coiled-coils in phage display screening: insight into exceptional selectivity provided by molecular dynamics.

Involved in numerous key biological functions, protein helix-helix interactions follow a well-defined intermolecular recognition pattern. The characteristic structure of the α-helical coiled-coil allows for the specific randomization of clearly defined interaction partners within heteromeric systems. In this work, a rationally designed heterodimeric coiled-coil was used to investigate potential factors influencing the sequence selectivity in interhelical interactions.

The impact of molecular dynamics on drug design: applications for the characterization of ligand-macromolecule complexes.

Among all tools available to design new drugs, molecular dynamics (MD) simulations have become an essential technique. Initially developed to investigate molecular models with a limited number of atoms, computers now enable investigations of large macromolecular systems with a simulation time reaching the microsecond range. The reviewed articles cover four years of research to give an overview on the actual impact of MD on the current medicinal chemistry landscape with a particular emphasis on studies of ligand-protein interactions. With a special focus on studies combining computational approaches with data gained from other techniques, this review shows how deeply embedded MD simulations are in drug design strategies and articulates what the future of this technique could be.

Identification of PPARγ Agonists from Natural Sources Using Different In Silico Approaches.

Peroxisome proliferator-activated receptor γ plays an important role in lipid and glucose homeostasis and is the target of many drug discovery investigations because of its role in diseases such as type 2 diabetes. Activation of peroxisome proliferator-activated receptor γ by agonists leads to a conformational change in the ligand-binding domain altering the transcription of several target genes involved in glucose and lipid metabolism, resulting in, for example, facilitation of glucose and lipid uptake and amelioration of insulin resistance, and other effects that are important in the treatment of type 2 diabetes. Peroxisome proliferator-activated receptor γ partial agonists are compounds with diminished agonist efficacy compared to full agonists; however, they maintain the antidiabetic effect of full agonists but do not induce the same magnitude of side effects. This mini-review gives a short introduction to in silico screening methods and recent research advances using computational approaches to identify peroxisome proliferator-activated receptor γ agonists, especially partial agonists, from natural sources and how these ligands bind to the peroxisome proliferator-activated receptor γ in order to better understand their biological effects.

ß- and γ-Amino Acids at α-Helical Interfaces: Toward the Formation of Highly Stable Foldameric Coiled Coils.

Since peptides are vital for cellular and pathogenic processes, much effort has been put into the design of unnatural oligomers that mimic natural peptide structures, also referred to as foldamers. However, to enable the specific application of foldamers, a thorough characterization of their interaction profiles in native protein environments is required. We report here the application of phage display for the identification of suitable helical environments for a sequence comprising an alternating set of ß- and γ-amino acids. In vitro selected sequences show that an increase in the hydrophobic surface area at the helical interface as well as the incorporation of a polar H-bond donor functionality can significantly improve interhelical interactions involving backbone-extended amino acids. Thus, our data provide insight into the principles of the rational design of foldameric inhibitors for protein-protein interactions.

Impact of fluorination on proteolytic stability of peptides: a case study with α-chymotrypsin and pepsin.

Protease stability is a key consideration in the development of peptide-based drugs. A major approach to increase the bioavailability of pharmacologically active peptides is the incorporation of non-natural amino acids. Due to the unique properties of fluorine, fluorinated organic molecules have proven useful in the development of therapeutically active small molecules as well as in materials and crop science. This study presents data on the ability of fluorinated amino acids to influence proteolytic stability when present in peptide sequences that are based on ideal protease substrates. Different model peptides containing fluorinated amino acids or ethylglycine in the P2, P1'or P2' positions were designed according to the specificities of the serine protease, α-chymotrypsin (EC 3.4.21.1) or the aspartic protease, pepsin (EC 3.4.23.1). The proteolytic stability of the peptides toward these enzymes was determined by an analytical RP-HPLC assay with fluorescence detection and compared to a control sequence. Molecular modeling was used to support the interpretation of the structure-activity relationship based on the analysis of potential ligand-enzyme interactions. Surprisingly, an increase in proteolytic stability was observed only in a few cases. Thus, this systematic study shows that the proteolytic stability of fluorinated peptides is not predictable, but rather is a very complex phenomenon that depends on the particular enzyme, the position of the substitution relative to the cleavage site and the fluorine content of the side chain.

Design, synthesis and molecular docking study of novel quinoxalin-2(1H)-ones as anti-tumor active agents with inhibition of tyrosine kinase receptor and studying their cyclooxygenase-2 activity.

On continuation to our work, new quinoxalin-2(1H)-ones were synthesized to study their cytotoxic effect against HepG-2 and MCF-7 with their effect on the human tyrosine kinase (TRK). Compounds 12, 18, 15, 13, 11a, 20 and 16, respectively, were found to be more potent than cisplatin against HepG2 and selective to TRK. Also, compounds 12, 18, 20, 13, 14, and 22, respectively, exhibited decidedly activity against MCF-7 and selectivity against human TRK compared to cisplatin. A molecular docking study was also performed to gain comprehensive understanding into plausible binding modes and to conclude the structure activity relationships of the synthesized compounds. Moreover, anti-inflammatory activity was studied. Compounds 12, 15, 18 and 22 were found to be potent and selective against COX-2.

Inhibitory potency of flavonoid derivatives on influenza virus neuraminidase.

The constant risk of emerging new influenza virus strains that are resistant to established inhibitors like oseltamivir leaves influenza neuraminidase (NA) a prominent target for drug design. The inhibitory activity of several flavonoid derivatives was experimentally tested in comparison to oseltamivir for the NA expressed by the seasonal influenza virus strains A/California/7/09 (A(H1N1)pdm09), A/Perth/16/09 (A(H3N2)), and B/Brisbane/60/08. IC50 values of polyphenols confirmed moderate inhibition in the µM range. Structurally, the amount and site of glycosylation of tested flavonoids have no significant influence on their inhibitory potency. In a pharmacophore-based docking approach the structure-activity relationship was evaluated. Molecular dynamics simulations revealed highly flexible parts of the enzyme and the contribution of salt bridges to the structural stability of NA. The findings of this study elucidate the impact of flavonoids on viral neuraminidase activity and the analysis of their modes of action provide valuable information about the mechanism of NA inhibition.