Determining the Predominant Conformations of Mortiamides A-D in Solution Using NMR Data and Molecular Modeling Tools.

Macrocyclic peptidomimetics have been seriously contributing to our arsenal of drugs to combat diseases. The search for nature's discoveries led us to mortiamides A-D (found in a novel fungus from Northern Canada), which is a family of cyclic peptides that clearly have demonstrated impressive pharmaceutical potential. This prompted us to learn more about their solution-state properties as these are central for binding to target molecules. Here, we secured and isolated mortiamide D, and then acquired high-resolution nuclear magnetic resonance (NMR) data to learn more about its structure and dynamics attributes. Sets of two-dimensional NMR experiments provided atomic-level (through-bond and through-space) data to confirm the primary structure, and NMR-driven molecular dynamics (MD) simulations suggested that more than one predominant three-dimensional (3D) structure exist in solution. Further steps of MD simulations are consistent with the finding that the backbones of mortiamides A-C also have at least two prominent macrocyclic shapes, but the side-chain structures and dynamics differed significantly. Knowledge of these solution properties can be exploited for drug design and discovery.

Ligand bioactive conformation plays a critical role in the design of drugs that target the hepatitis C virus NS3 protease.

A ligand-focused strategy employed NMR, X-ray, modeling, and medicinal chemistry to expose the critical role that bioactive conformation played in the design of a variety of drugs that target the HCV protease. The bioactive conformation (bound states) were determined for key inhibitors identified along our drug discovery pathway from the hit to clinical compounds. All adopt similar bioactive conformations for the common core derived from the hit peptide DDIVPC. A carefully designed SAR analysis, based on the advanced inhibitor 1 in which the P1 to P3 side chains and the N-terminal Boc were sequentially truncated, revealed a correlation between affinity and the relative predominance of the bioactive conformation in the free state. Interestingly, synergistic conformation effects on potency were also noted. Comparisons with clinical and recently marketed drugs from the pharmaceutical industry showed that all have the same core and similar bioactive conformations. This suggested that the variety of appendages discovered for these compounds also properly satisfy the bioactive conformation requirements and allowed for a large variety of HCV protease drug candidates to be designed.

Monitoring drug self-aggregation and potential for promiscuity in off-target in vitro pharmacology screens by a practical NMR strategy.

A simple NMR assay was applied to monitor the tendency of compounds to self-aggregate in aqueous media. The observation of unusual spectral trends as a function of compound concentration appears to be signatory of the formation of self-assemblies. (1)H NMR resonances of aggregating compounds were sensitive to the presence of a range of molecular assemblies in solution including large molecular-size entities, smaller multimers, and mixtures of assembled species. The direct observation of aggregates via unusual NMR spectra also correlated with promiscuous behavior of molecules in off-target in vitro pharmacology assays. This empirical assay can have utility for predicting compound promiscuity and should complement predictive methods that principally rely on the computing of descriptors such as lipophilicity (cLogP) and topological surface area (TPSA). This assay should serve as a practical tool for medicinal chemists to monitor compound attributes in aqueous solution and various pharmacologically relevant media, as demonstrated herein.

N- versus O-alkylation: utilizing NMR methods to establish reliable primary structure determinations for drug discovery.

A classic synthetic issue that remains unresolved is the reaction that involves the control of N- versus O-alkylation of ambident anions. This common chemical transformation is important for medicinal chemists, who require predictable and reliable protocols for the rapid synthesis of inhibitors. The uncertainty of whether the product(s) are N- and/or O-alkylated is common and can be costly if undetermined. Herein, we report an NMR-based strategy that focuses on distinguishing inhibitors and intermediates that are N- or O-alkylated. The NMR strategy involves three independent and complementary methods. However, any combination of two of the methods can be reliable if the third were compromised due to resonance overlap or other issues. The timely nature of these methods (HSQC/HMQC, HMBC. ROESY, and (13)C shift predictions) allows for contemporaneous determination of regioselective alkylation as needed during the optimization of synthetic routes.

Compound aggregation in drug discovery: implementing a practical NMR assay for medicinal chemists.

The pharmaceutical industry has recognized that many drug-like molecules can self-aggregate in aqueous media and have physicochemical properties that skew experimental results and decisions. Herein, we introduce the use of a simple NMR strategy for detecting the formation of aggregates using dilution experiments that can be performed on equipment prevalent in most synthetic chemistry departments. We show that (1)H NMR resonances are sensitive to large molecular-size entities and to smaller multimers and mixtures of species. Practical details are provided for sample preparation and for determining the concentrations of single molecule, aggregate entities, and precipitate. The critical concentrations above which aggregation begins can be found and were corroborated by comparisons with light scattering techniques. Disaggregation can also be monitored using detergents. This NMR assay should serve as a practical and readily available tool for medicinal chemists to better characterize how their compounds behave in aqueous media and influence drug design decisions.

Importance of ligand bioactive conformation in the discovery of potent indole-diamide inhibitors of the hepatitis C virus NS5B.

Significant advances have led to receptor induced-fit and conformational selection models for describing bimolecular recognition, but a more comprehensive view must evolve to also include ligand shape and conformational changes. Here, we describe an example where a ligand's "structural hinge" influences potency by inducing an "L-shape" bioactive conformation, and due to its solvent exposure in the complex, reasonable conformation-activity-relationships can be qualitatively attributed. From a ligand design perspective, this feature was exploited by successful linker hopping to an alternate "structural hinge" that led to a new and promising chemical series which matched the ligand bioactive conformation and the pocket bioactive space. Using a combination of X-ray crystallography, NMR and modeling with support from binding-site resistance mutant studies and photoaffinity labeling experiments, we were able to derive inhibitor-polymerase complexes for various chemical series.

Dimethylthiazolidine carboxylic acid as a rigid p3 unit in inhibitors of serine proteases: application to two targets.

Serine proteases are a very large class of enzymes, many of which represent important targets for therapeutic agents against a wide variety of disease states. The similarity in active site architecture for these proteases has often allowed inhibitor design strategies for a particular target to be successfully applied to other enzymes in the class. In many cases, the presence of a bulky P3 amino acid residue in peptide-based inhibitors is central to conferring an extended peptide conformation, critical to binding of the ligands to serine protease active sites. The dimethylthiazolidine carboxylic acid 'residue' was found to be effective as a novel P3 replacement in peptidomimetic inhibitors of two distinct serine proteases, the hepatitis C NS3 protease and the human cytomegalovirus maturational protease. An array of NMR methods was used to confirm that the dimethylthiazolidine carboxylic acid unit indeed confers conformational and dynamic properties very similar to that of the rigidified parent structures.

Quantifying trifluoroacetic acid as a counterion in drug discovery by 19F NMR and capillary electrophoresis.

Drug discovery compounds are often isolated as salts of trifluoroacetate from preparative high performance liquid chromatography, which are then used for biological assays in order to assess their efficacy against the biochemical target of interest. It is, therefore, imperative to determine the TFA content in order to ascertain the correct formula weight and when required, to ensure that the TFA has been completely exchanged for another counterion in order to have superior pharmacokinetic properties and to avoid potential toxicity effects. In this paper, we present capillary electrophoresis and (19)F nuclear magnetic resonance methods for determining the TFA content of drug discovery compounds. Furthermore, these methods have been successfully applied in a high-throughput fashion, which is a key feature for general applicability in a pharmaceutical setting.

RCM of tripeptide dienes containing a chiral vinylcyclopropane moiety: impact of different Ru-based catalysts on the stereochemical integrity of the macrocyclic products.

[structures: see text] Tripeptide dienes containing an (1R,2S)-vinyl aminocyclopropylcarboxylate residue were cyclized to beta-strand scaffolds under ring-closing metathesis (RCM). Conformational factors, ligand effects, and reaction conditions were evaluated. A protocol was developed for the efficient synthesis of 15-membered ring peptides in high diastereomeric purity. These peptides are key synthetic precursors to antiviral agents that target the hepatitis C virus and represent the first class of clinically validated pharmaceutical agents that are synthesized in large scale using RCM.

The Conformation of a Peptidyl Methyl Ketone Inhibitor Bound to the Human Cytomegalovirus Protease.

A weak inhibitor means faster exchange! Since the methyl ketone MK2 is a weak noncovalent peptidyl inhibitor of the human cytomegalovirus protease, exchange between the free and enzyme-bound forms is rapid. This allows for the use of transferred NOE NMR methods and molecular modeling, which show that the bound conformation of MK2 is an extended peptide. This is confirmed by the results of an X-ray crystallographic analysis of a related enzyme-inhibitor complex.