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Establishing robust structure-activity relationships (SARs) is key to successful drug discovery campaigns, yet it often remains elusive due to screening and hit validation artifacts (false positives and false negatives), which frequently result in unproductive downstream expenditures of time and resources. To address this issue, we developed an integrative biophysics-driven strategy that expedites hit-to-lead discovery, mitigates false positives/negatives and common hit validation errors, and provides a robust approach to obtaining accurate binding and affinity measurements. The advantage of this method is that it vastly improves the clarity and reproducibility for affinity-driven SAR by monitoring and eliminating confounding factors. We demonstrate the ease at which high-quality micromolar binders can be generated from the initial millimolar fragment screening hits against an "undruggable" protein target, HRas.

Small molecules can self-assemble in aqueous solution into a wide range of nanoentity types and sizes (dimers, n -mers, micelles, colloids, etc.), each having their own unique properties. This has important consequences in the context of drug discovery including issues related to nonspecific binding, off-target effects, and false positives and negatives. Here, we demonstrate the use of the spin-spin relaxation Carr-Purcell-Meiboom-Gill NMR experiment, which is sensitive to molecular tumbling rates and can expose larger aggregate species that have slower rotational correlations. The strategy easily distinguishes lone-tumbling molecules versus nanoentities of various sizes. The technique is highly sensitive to chemical exchange between single-molecule and aggregate states and can therefore be used as a reporter when direct measurement of aggregates is not possible by NMR. Interestingly, we found differences in solution behavior for compounds within structurally related series, demonstrating structure-nanoentity relationships. This practical experiment is a valuable tool to support drug discovery efforts.

Antimicrobial peptides form part of the first line of defense against pathogens for many organisms. Current treatments for fungal infections are limited by drug toxicity and pathogen resistance. Cm-p5 (SRSELIVHQRLF), a peptide derived from the marine mollusk Cenchritis muricatus peptide Cm-p1, has a significantly increased fungistatic activity against pathogenic Candida albicans (minimal inhibitory concentration, 10 µg/ml; EC50, 1.146 µg/ml) while exhibiting low toxic effects against a cultured mammalian cell line. Cm-p5 as characterized by circular dichroism and nuclear magnetic resonance revealed an α-helical structure in membrane-mimetic conditions and a tendency to random coil folding in aqueous solutions. Additional studies modeling Cm-p5 binding to a phosphatidylserine bilayer in silico and isothermal titration calorimetry using lipid monophases demonstrated that Cm-p5 has a high affinity for the phospholipids of fungal membranes (phosphatidylserine and phosphatidylethanolamine), only moderate interactions with a mammalian membrane phospholipid, low interaction with ergosterol, and no interaction with chitin. Adhesion of Cm-p5 to living C. albicans cells was confirmed by fluorescence microscopy with FITC-labeled peptide. In a systemic candidiasis model in mice, intraperitoneal administration of Cm-p5 was unable to control the fungal kidney burden, although its low amphiphaticity could be modified to generate new derivatives with improved fungicidal activity and stability., (© FASEB.)

Most tumors exhibit increased glucose metabolism to lactate, however, the extent to which glucose-derived metabolic fluxes are used for alternative processes is poorly understood. Using a metabolomics approach with isotope labeling, we found that in some cancer cells a relatively large amount of glycolytic carbon is diverted into serine and glycine metabolism through phosphoglycerate dehydrogenase (PHGDH). An analysis of human cancers showed that PHGDH is recurrently amplified in a genomic region of focal copy number gain most commonly found in melanoma. Decreasing PHGDH expression impaired proliferation in amplified cell lines. Increased expression was also associated with breast cancer subtypes, and ectopic expression of PHGDH in mammary epithelial cells disrupted acinar morphogenesis and induced other phenotypic alterations that may predispose cells to transformation. Our findings show that the diversion of glycolytic flux into a specific alternate pathway can be selected during tumor development and may contribute to the pathogenesis of human cancer.

Heteronuclear direct-detection experiments, which utilize the slower relaxation properties of low gamma nuclei, such as (13)C have recently been proposed for sequence-specific assignment and structural analyses of large, unstructured, and/or paramagnetic proteins. Here we present two novel (15)N direct-detection experiments. The CAN experiment sequentially connects amide (15)N resonances using (13)C(alpha) chemical shift matching, and the CON experiment connects the preceding (13)C' nuclei. When starting from the same carbon polarization, the intensities of nitrogen signals detected in the CAN or CON experiments would be expected four times lower than those of carbon resonances observed in the corresponding (13)C-detecting experiment, NCA-DIPAP or NCO-IPAP (Bermel et al. 2006b; Takeuchi et al. 2008). However, the disadvantage due to the lower gamma is counteracted by the slower (15)N transverse relaxation during detection, the possibility for more efficient decoupling in both dimensions, and relaxation optimized properties of the pulse sequences. As a result, the median S/N in the (15)N observe CAN experiment is 16% higher than in the (13)C observe NCA-DIPAP experiment. In addition, significantly higher sensitivity was observed for those residues that are hard to detect in the NCA-DIPAP experiment, such as Gly, Ser and residues with high-field C(alpha) resonances. Both CAN and CON experiments are able to detect Pro resonances that would not be observed in conventional proton-detected experiments. In addition, those experiments are free from problems of incomplete deuterium-to-proton back exchange in amide positions of perdeuterated proteins expressed in D(2)O. Thus, these features and the superior resolution of (15)N-detected experiments provide an attractive alternative for main chain assignments. The experiments are demonstrated with the small model protein GB1 at conditions simulating a 150 kDa protein, and the 52 kDa glutathione S-transferase dimer, GST.

We have previously established that ATP binds to mammalian metallothionein-2 (MT). The interaction between ATP and MT and the associated conformational change of the protein affect the sulfhydryl reactivity and zinc transfer potential of MT [Jiang, L.-J., Maret, W., and Vallee, B. L. (1998) The ATP-metallothionein complex. Proc. Natl. Acad. Sci. U.S.A. 95, 9146-9149]. NMR spectroscopic investigations have now provided further evidence for the interaction. (35)Cl NMR spectroscopy has further identified chloride as an additional biological MT ligand, which can interfere with the interaction of ATP with MT. (1)H NMR/TOCSY spectra demonstrate that ATP binding affects the N- and C-terminal amino acids of the MT molecule. Scanning tunneling microscopy recorded images of single MT molecules in buffered solutions. Moreover, this technique demonstrates that the otherwise nearly linear MT molecule bends by about 20 degrees at its central hinge region between the domains in the presence of ATP. These results may bear on the development of mild obesity in MT null mice and the role of MT in the regulation of energy balance. The interaction suggests a mechanism for the cellular translocation, retention, and reactivity of the ATP*MT complex in the mitochondrial intermembrane space. Both MT and ATP are localized there, and MT and thionein alternately bind and release zinc, thereby affecting mitochondrial respiration.