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.

The interactions of the NS3 protease domain with inhibitors that are based on N-terminal cleavage products of peptide substrates were studied by NMR methods. Transferred nuclear Overhauser effect experiments showed that these inhibitors bind the protease in a well defined, extended conformation. Protease-induced linebroadening studies helped identify the segments of inhibitors which come into contact with the protease. A comparison of the NMR data of the free and proteasebound states suggests that these ligands undergo rigidification upon complexation. This work provides the first structure of an inhibitor when bound to NS3 protease and should be valuable for designing more potent inhibitors. Hepatitis C virus (HCV)1 infection is an important cause of chronic hepatitis, cirrhosis, hepatocellular carcinoma, and liver failure worldwide (1). Approved therapies with proven benefit for patients with chronic hepatitis C include various drug regimens of interferon-␣. These therapies have limited efficacy with a low sustained response rate and frequent side effects (1). Therefore, there is an urgent need for the development of new therapies for the treatment of HCV infections.HCV is a small enveloped virus containing a single-stranded RNA genome of positive polarity, which encodes a unique polyprotein of approximately 3000 amino acids (for reviews see Refs. 2 and 3). This polyprotein is the precursor of four structural and six nonstructural (NS) proteins (4 -10). The structural proteins are proteolytically processed by host signal peptidases, whereas two virally encoded proteases within the NS2 and NS3 regions process the remaining nonstructural proteins.The NS3 serine protease domain (20 kDa), located within the N-terminal portion of the NS3 protein, mediates the proteolysis at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions (6 -11). We and others have recently reported that N-terminal cleavage products of peptide substrates are competitive inhibitors of NS3 protease activity (12, 13), which has served as the basis for designing substrate-based inhibitors (14, 15). To date, there have been no reports in the literature on the structure of substrates or inhibitors when bound to NS3 protease, which would certainly be valuable for inhibitor design efforts. However, x-ray crystal structures have been determined for NS3 protease alone (16) and for NS3 protease in the presence of an NS4A peptide cofactor (17,18). These structures show that NS3 protease adopts a chymotrypsin/trypsin-like fold.In this report we applied NMR methods to study the structure of peptides and inhibitors, based on N-terminal cleavage products of peptide substrates when bound to the NS3 protease domain of HCV. Transferred NOESY experiments were used to determine the conformation of ligands when bound to the protease, and differential line-broadening experiments were used to identify which segments of the ligands contact the protease. EXPERIMENTAL PROCEDURESPurification of NS3 Protease-A modification of a previously published procedure (19) was used ...

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.

The conformational properties of the N-tert-butylacetyl-l-tert-butylglycyl-l-N δ,N δ-dimethylasparagyl-l-alanyl methyl ketone (MK) 1 and its terminal N-isopropylacetyl analogue 2 were investigated. Whereas these compounds are weak (mM IC50 range) inhibitors of the human cytomegalovirus (HCMV) protease, their activated carbonyl analogues are >1000-fold more potent (e.g., trifluoromethyl ketone 3, IC50 = 1.1 μM). A combination of NMR techniques demonstrated that MK 2 exists in solution as a relatively rigid and extended peptide structure and that the bulky side chains, notably the P3 tert-butyl group, greatly contribute to maintaining this solution conformation. Furthermore, transferred nuclear Overhauser effect (TRNOE) studies provided an enzyme-bound conformation of MK 2 that was found to be similar to its free solution structure and compares very well to the X-ray crystallographic structure of a related peptidyl inhibitor complexed to the enzyme. The fact that ligands such as MK 2 exist in solution in the bioactive conformation accounts, in part, for the observed inhibitory activity of activated ketone inhibitors bearing comparable peptidyl sequences. Comparison of the X-ray structures of HCMV protease apoenzyme and that of its complex with a related peptidyl α-ketoamide inhibitor allowed for a detailed analysis of the previously reported conformational change of the enzyme upon complexation of inhibitors such as 1 and 3. The above observations indicate that HCMV protease is a novel example of a serine protease that operates by an induced-fit mechanism for which complexation of peptidyl ligands results in structural changes which bring the enzyme to a catalytically active (or optimized) form. Kinetic and fluorescence studies are also consistent with an induced-fit mechanism in which a considerable proportion of the intrinsic ligand-binding energy is used to carry out the conformational reorganization of the protease. Issues related to the rational design of both mechanism- and nonmechanism-based inhibitors of HCMV protease, notably in light of the peptidyl ligand-induced optimization of its catalytic functioning, are discussed.

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.