The solution structure of the bee venom neurotoxin apamin has been determined with a distance geometry program using distance constraints derived from NMR. Twenty embedded structures were generated and refined by using the program DSPACE. After error minimization using both conjugate gradient and dynamics algorithms, six structures had very low residual error. Comparisons of these show that the backbone of the peptide is quite well-defined with the largest rms difference between backbone atoms in these structures of 1.34 A. The side chains have far fewer constraints and show greater variability in their positions. The structure derived here is generally consistent with the qualitative model previously described, with most differences occurring in the loop between the beta-turn (residues 2-5) and the C-terminal alpha-helix (residues 9-17). Comparisons are made with previously derived models from NMR data and other methods.

Peptides have been synthesized that have hybrid sequences, partially derived from the bee venom peptide apamin and partially from the S peptide of ribonuclease A. The hybrid peptides were demonstrated by NMR spectroscopy to fold, forming the same disulfides and basic three-dimensional structure as native apamin, containing a f-turn and an a-helix.These hybrids were active in complementing S protein, reactivating nuclease activity. In addition, the hybrid peptide was effective in inducing antibodies that cross-react with the RNase, without conjugation to a carrier protein. The stability of the folded structure of this peptide suggests that it should be possible to elicit antibodies that will react not only with a specific sequence, but also with a specific secondary structure. Hybrid sequence peptides also provide opportunities to study separately nucleation and propagation steps in formation of secondary structure. We show that in S peptide the a-helix does not end abruptly but rather terminates gradually over four or five residues. In general, these hybrid sequence peptides, which fold predictably because of disulfide bond formation, can provide opportunities for examining structure-function relationships for many biologically active sequences.It is well known that in disulfide cross-linked proteins the relative positions of cysteines are strongly conserved. Among families of proteins containing similar disulfides, there tends to be a strong conservation of the threedimensional structure even when the overall sequence homology is rather low (1). Thus, it is clear that a wide variety of amino acid sequences can be accommodated within the fold defined by the cystine bridges. We have taken advantage of this adaptability to form structured, hybrid sequence peptides. In these hybrids the folding is largely controlled by formation of disulfides involving cysteines with relative positions taken from a natural peptide. However, many of the noncysteine residues can be replaced with a sequence from another protein. In the cases discussed here, the disulfides were derived from apamin (2-4), and the noncysteine amino acids from the helical region of apamin were replaced with those from the S peptide of RNase A (5). The S peptide, consisting of the first 20 amino acids from RNase A, has been extensively characterized and has been shown to have a helix-forming propensity (6-8) (existing as up to =40wo helix under optimal conditions). In addition, there was shown to be a helix stop signal present (9, 10) near the 13th or 14th residue. This signal persists even in trifluoroethanol, where the overall helical fraction is significantly increased (11, 12). The S peptide binds (with an association constant of 106) to S protein, the large fragment of RNase A from which it was cleaved, reactivating nuclease activity (3). In the apamin-S peptide hybrids, we show that the disulfides stabilize the helical segment at the beginning of the S peptide completely, giving us the opportunity to investigate the propagation ofthe helix af...

Modern small molecule drug design requires the optimization of not only the binding characteristics of the molecule but also its physicochemical properties for ADMET performance. A key physical property is lipophilicity and medicinal chemists need rapid access to high quality data in order to drive their decision making. Traditionally lipophilicity (log D) measurements are performed with a shake flask method and UV determination. This method suffers from low sensitivity and is not easily converted to a high throughput format. Over the past decade, several groups have taken different approaches to improve this assay, including replacing the shake flask method with one that utilizes reverse phase HPLC. Here we describe a new microscale shake flask method that utilizes UPLC-MS/MS to achieve increased throughput, sensitivity and accuracy. Approaches for assessing data quality are also described. This platform technology only requires micrograms of compound and is routinely used by most small molecule drug discovery project teams at Genentech.

Acetaminophen-induced liver toxicity is the most frequent precipitating cause of acute liver failure and liver transplant, but contemporary medical practice has mainly focused on patient management after a liver injury has been induced. An integrative genetic, transcriptional, and two-dimensional NMR-based metabolomic analysis performed using multiple inbred mouse strains, along with knowledge-based filtering of these data, identified betaine-homocysteine methyltransferase 2 (Bhmt2) as a diet-dependent genetic factor that affected susceptibility to acetaminophen-induced liver toxicity in mice. Through an effect on methionine and glutathione biosynthesis, Bhmt2 could utilize its substrate (S-methylmethionine [SMM]) to confer protection against acetaminophen-induced injury in vivo. Since SMM is only synthesized in plants, Bhmt2 exerts its beneficial effect in a diet-dependent manner. Identification of Bhmt2 and the affected biosynthetic pathway demonstrates how a novel method of integrative genomic analysis in mice can provide a unique and clinically applicable approach to a major public health problem.

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