Hanoch Senderowitz

The Cystic Fibrosis-causing Mutation ΔF508 Affects Multiple Steps in Cystic Fibrosis Transmembrane Conductance Regulator Biogenesis

The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations. However, the specific steps of folding and assembly of full-length cystic fibrosis transmembrane conductance regulator (CFTR) directly altered by the disease-causing mutation are unclear. To elucidate the effects of the ΔF508 mutation, on various steps in CFTR folding, a series of misfolding and suppressor mutations in the nucleotide binding and transmembrane domains were evaluated for effects on the folding and maturation of the protein. The results indicate that the isolated NBD1 responds to both the ΔF508 mutation and intradomain suppressors of this mutation. In addition, identification of a novel second site suppressor of the defect within the second transmembrane domain suggests that ΔF508 also effects interdomain interactions critical for later steps in the biosynthesis of CFTR.

Small molecule correctors of F508del-CFTR discovered by structure-based virtual screening

Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, ~tenfold higher than hit rates obtained in corresponding high-throughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of our knowledge this is the first report of structure-based discovery of CFTR modulators.

Diagnostic structural criteria for the anomeric effect in carbohydrates and inferences of general significance on their scope and limitations

Abstract A systematic analysis of 529 carbohydrate structures that contain OCO units, retrieved from the Cambridge Structural Database, was performed with regard to the bond lengths (CO) and bond (COC and OCO) angles as they depend on the dihedral angles in the sequence COCOC. This dependence was then interpreted in terms of the anomeric effect. Known and new concepts that concern the manifestations of the anomeric and exo -anomeric effects were thus reassessed. A set of structural criteria of diagnostic value was defined which, together with qualifying arguments, allow evaluation of the scope and limitations of these stereoelectronic effects in carbohydrate systems.

Docking Studies on DNA Intercalators

DNA is an important target for the treatment of multiple pathologies, most notably cancer. In particular, DNA intercalators have often been used as anticancer drugs. However, despite their relevance to drug discovery, only a few systematic computational studies were performed on DNA-intercalator complexes. In this work we have analyzed ligand binding sites preferences in 63 high resolution DNA-intercalator complexes available in the PDB and found that ligands bind preferentially between G and C and between the C and A base pairs (70% and 11%, respectively). Next, we examined the ability of AUTODOCK to accurately dock ligands into preformed intercalation sites. Following the optimization of the docking protocol, AUTODOCK was able to generate conformations with RMSD values <2.00 Å with respect to crystal structures in ∼80% of the cases while focusing on the preformed binding site (small grid box) or on the entire DNA structure (large grid box). In addition, a top ranked conformation with an RMSD < 2.00 Å was identified in 75% and 60% of the cases using small and large docking boxes, respectively. Moreover, under the large docking box setting AUTODOCK was able to successfully distinguish between the intercalation site and the minor groove site. However, in all cases the crystal structures and poses tightly clustered around it had a lower score than the best scoring poses suggesting a potential scoring problem with AUTODOCK. A close examination of all cases where the top ranked pose had an RMSD value >2.00 Å suggests that AUTODOCK may overemphasize the hydrogen bonding term. A decision tree was built to identify ligands which are likely to be accurately docked based on their characteristics. This analysis revealed that AUTODOCK performs best for intercalators characterized by a large number of aromatic rings, low flexibility, high molecular weight, and a small number of hydrogen bond acceptors. Finally, for canonical B-DNA structures (where preformed sites are unavailable), we demonstrated that intercalation sites could be formed by inserting an anthracene moiety between the (anticipated) site-flanking base pairs and by relaxing the structure using either energy minimization or preferably molecular dynamics simulations. Such sites were suitable for the docking of different intercalators by AUTODOCK.

Conformational Changes Relevant to Channel Activity and Folding within the first Nucleotide Binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator

Background: The CFTR chloride channel undergoes conformational changes during its gating cycle. Results: H620Q mutation associated with increased channel Po, and the corrector/potentiator CFFT-001 both lead to similar conformational shifts in NBD1. Conclusion: There is an intrinsic conformational equilibrium within NBD1 that is correlated with channel activity. Significance: Conformational fluctuations within NBD1 are fundamental to CFTR regulation. Deletion of Phe-508 (F508del) in the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to defects in folding and channel gating. NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between β-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. CFFT-001 appears to interact with β-strands S3/S9/S10, consistent with docking simulations. Decreases in Tm from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. We hypothesize that, in full-length CFTR, shifting the conformational equilibrium to reduce H8/H9 interactions with the uniquely conserved strands S9/S10 facilitates release of the regulatory region from the NBD dimerization interface to promote dimerization and thereby increase channel open probability. These studies enabled by our NMR assignments for F508del NBD1 provide a window into the conformational fluctuations within CFTR that may regulate function and contribute to folding energetics.

New supramolecular host systems. 2. 1,3,5,7-tetraoxadecalin, 1,2-dimethoxyethane and the Gauche effect reappraised. Theory vs. experiment.

Abstract The three 1,3,5,7-tetraoxadecalin diastereomers (1, 2 & 3), core moieties of new host and macromolecular systems, were calculated “

G Protein-Coupled Receptors: Target-Based In Silico Screening

In silico (or virtual) screening has become a common practice in current computer-aided drug design efforts. However, application to hit discovery in the G Protein-Coupled Receptors (GPCRs) arena was until recently hampered by the paucity of crystal structures available for this important class of pharmaceutical targets, forcing practitioners in the field to rely on GPCR models derived either ab initio or through homology modeling approaches. In this work we describe the EPIX in silico screening workflow which consists of the following stages: (1) Target modeling; (2) Preparation of screening library; (3) Docking; (4) Binding mode selection; (5) Scoring; (6) Consensus scoring and (7) Selection of virtual hits. This workflow was applied to the virtual screening of 13 GPCRs (5 biogenic amine receptors, 5 peptide receptors, 1 lipid receptor, 1 purinergic receptor and 1 cannabinoid receptor). Hit rates vary between 4% and 21% with higher hit rates usually obtained for biogenic amines and lower hits rates for peptide receptors. These data are analyzed in the context of the available experimental information (i.e., mutational data), the model (i.e., binding site location, and type of interactions) and the screening library. Specific examples are discussed in more detail as well as the future directions and challenges of this approach to in silico screening.

G Protein Coupled Receptors - In Silico Drug Discovery and Design

In silico drug discovery is a complex process requiring flexibility and ingenuity in method selection and a careful validation of work protocols. GPCR in silico drug discovery poses additional challenges due to the paucity of crystallographic data. This paper starts by reviewing selected GPCR in silico screening programs reported in the literature, including both structure-based and ligand-based approaches. Particular emphasis is given to library design, binding mode selection, process validation and compound selection for biological testing. Following literature review, we provide insights into in silico methodologies and process workflows used at EPIX to drive over 20 highly successful screening and lead optimization programs performed since 2001. Applications of the various methodologies discussed are demonstrated by examples from recent programs that have not yet been published.

SeleX-CS : A New Consensus Scoring Algorithm for Hit Discovery and Lead Optimization

Identifying active compounds (hits) that bind to biological targets of pharmaceutical relevance is the cornerstone of drug design efforts. Structure based virtual screening, namely, the in silico evaluation of binding energies and geometries between a protein and its putative ligands, has emerged over the past few years as a promising approach in this field. The success of the method relies on the availability of reliable 3-dimensional (3D) structures of the target protein and its candidate ligands (the screening library), a reliable docking method that can fit the different ligands into the proteins binding site, and an accurate scoring function that can rank the resulting binding modes in accord with their binding affinities. This last requirement is arguably the most difficult to meet due to the complexity of the binding process. A potential solution to this so-called scoring problem is the usage of multiple scoring functions in an approach known as consensus scoring. Several consensus scoring methods were suggested in the literature and have generally demonstrated an improved ranking of screening libraries relative to individual scoring functions. Nevertheless, current consensus scoring strategies suffer from several shortcomings, in particular, strong dependence on the initial parameters and an incomplete treatment of inactive compounds. In this work we present a new consensus scoring algorithm (SeleX-Consensus Scoring abbreviated to SeleX-CS) specifically designed to address these limitations: (i) A subset of the initial set of the scoring functions is allowed to form the consensus score, and this subset is optimized via a Monte Carlo/Simulated Annealing procedure. (ii) Rank redundancy between the members of the screening library is removed. (iii) The method explicitly considers the presence of inactive compounds. The new algorithm was applied to the ranking of screening libraries targeting two G-protein coupled receptors (GPCR). Excellent enrichment factors were obtained in both cases: For the cannabinoid receptor 1 (CB1), SeleX-CS outperformed the best single score and afforded an enrichment factor of 41 at 1% of the screening library compared with the best single score value of 15 (GOLD_Fitness). For the chemokine receptor type 2 (CCR2) SeleX-CS afforded an enrichment factor of 72 (again at 1% of the screening library) once more outperforming any single score (enrichment factor of 20 by GSCORE). Moreover, SeleX-CS demonstrated success rates of 67% (CCR2) and 73% (CB1) when applied to ranking an external test set. In both cases, the new algorithm also afforded good derichment of inactive compounds (i.e., the ability to push inactive compounds to the bottom of the ranked library). The method was then extended to rank a lead optimization series targeting the Kv4.3 potassium ion channel, resulting in a Spearmans correlation coefficient, p = 0.63 (n = 40), between the SeleX-CS-based rank and the actual pKi values. These results suggest that SeleX-CS is a powerful method for ranking screening libraries in the lead discovery phase and also merits consideration as a lead optimization tool.

Computation of O-C-F and N-C-F systems: AB-initio calculations and a MM2 parameterization study. Theory vs. experiment☆

Abstract A parameterization scheme of Allingers MM2 force field for the anomeric effect in O-C-F and N-C-F systems is presented. The scarcity of experimental data, in particular for the N-C-F case, dictated the use of ab-initio calculations to account for the energetic and structural manifestations of the effect. The resulting modified force field was tested against available X-ray, microwave and NMR results leading to a very good agreement between calculations and experiment. In addition, ab-initio results were used to demonstrate the role of the anomeric effect in lowering barriers to N-inversion and elevating barriers for rotation around single CN bonds. The results for the fluoro compounds, when juxtaposed to other systems, provide a complete treatment of the anomeric effect for the first row elements.