Using HMBC and ADEQUATE NMR data to define and differentiate long-range coupling pathways: is the Crews rule obsolete?

It is well known that as molecules become progressively more proton-deficient, structure elucidation becomes correspondingly more challenging. When the ratio of (1)H to (13)C and the sum of other heavy atoms falls below 2, an axiom that has been dubbed the "Crews rule" comes into play. The general premise of the Crews rule is that highly proton-deficient molecules may have structures that are difficult, and in some cases impossible, to elucidate using conventional suites of NMR experiments that include proton and carbon reference spectra, COSY, multiplicity-edited HSQC, and HMBC (both (1)H-(13)C and (1)H-(15)N).

Application of fragment-based NMR screening, X-ray crystallography, structure-based design, and focused chemical library design to identify novel microM leads for the development of nM BACE-1 (beta-site APP cleaving enzyme 1) inhibitors.

Fragment-based NMR screening, X-ray crystallography, structure-based design, and focused chemical library design were used to identify novel inhibitors for BACE-1. A rapid optimization of an initial NMR hit was achieved by a combination of NMR and a functional assay, resulting in the identification of an isothiourea hit with a K(d) of 15 microM for BACE-1. NMR data and the crystal structure revealed that this hit makes H-bond interactions with the two catalytic aspartates, occupies the nonprime side region of the active site of BACE-1, and extends toward the S3 subpocket (S3sp).

Unambiguous structural characterization of hydantoin reaction products using 2D HMBC NMR spectroscopy.

Data from two-dimensional (2D) NMR experiments were used to identify the reaction products resulting from the opening of pyroglutamates with isocyanates or thioisocyanates. The reaction has the potential to produce compounds that would have very similar one-dimensional proton ((1)H) or carbon-13 ((13)C) NMR spectra. Careful analysis of (1)H--(1)H COSY, (1)H--(1)H NOESY, and HMBC data, including chemical shifts and coupling constants, were used to distinguish correctly between carbamoyl-2-pyrrolidinone, hydantoin, and perhydro-1,3-diazepine-2,4-dione type structures that could result from this reaction.

Characterization of autocatalytic conversion of precursor BACE1 by heteronuclear NMR spectroscopy.

Accumulation of the cytotoxic 40- to 42-residue beta-amyloid peptide represents the primary pathological process in Alzheimer's disease (AD). BACE1 (beta-site APP cleaving enzyme 1) is responsible for the initial required step in the neuronal amyloidogenic processing of beta-amyloid precursor protein and is a major drug target for the therapeutic intervention of AD. In the present study, BACE1 is initially synthesized as an immature precursor protein containing part of the pre domain and the entire pro domain, and undergoes autocatalytic conversion to yield the well-folded mature BACE1 enzyme.

Screening of protein kinases by ATP-STD NMR spectroscopy.

ATP-STD NMR takes advantage of Mg2+ binding to ATP to adjust the ATP affinity for protein kinases permitting a wide range of Ki's to be determined for ATP competitive ligands. Substituting Mn2+ for Mg2+ creates a paramagnetic probe (MnATP) from which the proximity of non-ATP competitive ligands can be inferred. Internal standards and references are used to reduce false positives due to protein or compound degradation.

Non-peptidic small-molecule inhibitors of the single-chain hepatitis C virus NS3 protease/NS4A cofactor complex discovered by structure-based NMR screening.

NMR-based screening of a customized fragment library identified 16 small-molecule hits that bind weakly (K(D) approximately 100 microM to 10 mM) to substrate binding sites of the NS4A-bound NS3 protease of the hepatitis C virus (HCV). Analogues for five classes of NMR hits were evaluated by a combination of NMR and biochemical data yielding SAR and, in most cases, optimized hits with improved potencies (K(D) approximately K(I) approximately 40 microM to 1 mM). NMR chemical shift perturbation data were used to establish the binding location and orientation of the active site directed scaffolds in these five analogue series.

Flexible lid to the p53-binding domain of human Mdm2: implications for p53 regulation.

The stabilization of p53 against Mdm2-mediated degradation is an important event in DNA damage response. Initial models of p53 stabilization focused on posttranslational modification of p53 that would disrupt the p53-Mdm2 interaction. The N-terminal regions of both p53 and Mdm2 are modified in vivo in response to cellular stress, suggesting that modifications to Mdm2 also may affect the p53-Mdm2 interaction.

NMR-based approaches for lead discovery.

NMR methods have long been used for studying molecular interactions. In the last few years, various NMR approaches have been developed to aid lead discovery. These involve different NMR screening methods to identify initial compounds, which often bind only weakly (in the micro- to millimolar range) to the drug target.