Two peptides, ProTx-I and ProTx-II, from the venom of the tarantula Thrixopelma pruriens, have been isolated and characterized. These peptides were purified on the basis of their ability to reversibly inhibit the tetrodotoxin-resistant Na channel, Na(V) 1.8, and are shown to belong to the inhibitory cystine knot (ICK) family of peptide toxins interacting with voltage-gated ion channels. The family has several hallmarks: cystine bridge connectivity, mechanism of channel inhibition, and promiscuity across channels within and across channel families. The cystine bridge connectivity of ProTx-II is very similar to that of other members of this family, i.e., C(2) to C(16), C(9) to C(21), and C(15) to C(25). These peptides are the first high-affinity ligands for tetrodotoxin-resistant peripheral nerve Na(V) channels, but also inhibit other Na(V) channels (IC(50)'s < 100 nM). ProTx-I and ProTx-II shift the voltage dependence of activation of Na(V) 1.5 to more positive voltages, similar to other gating-modifier ICK family members. ProTx-I also shifts the voltage dependence of activation of Ca(V) 3.1 (alpha(1G), T-type, IC(50) = 50 nM) without affecting the voltage dependence of inactivation. To enable further structural and functional studies, synthetic ProTx-II was made; it adopts the same structure and has the same functional properties as the native peptide. Synthetic ProTx-I was also made and exhibits the same potency as the native peptide. Synthetic ProTx-I, but not ProTx-II, also inhibits K(V) 2.1 channels with 10-fold less potency than its potency on Na(V) channels. These peptides represent novel tools for exploring the gating mechanisms of several Na(V) and Ca(V) channels.
IMP-1 metallo--lactamase (class B) is a plasmid-borne zinc metalloenzyme that efficiently hydrolyzes -lactam antibiotics, including carbapenems, rendering them ineffective. Because IMP-1 has been found in several clinically important carbapenem-resistant pathogens, there is a need for inhibitors of this enzyme that could protect broad spectrum antibiotics such as imipenem from hydrolysis and thus extend their utility. We have identified a series of 2,3-(S,S)-disubstituted succinic acids that are potent inhibitors of IMP-1. Determination of high resolution crystal structures and molecular modeling of succinic acid inhibitor complexes with IMP-1 has allowed an understanding of the potency, stereochemistry, and structure-activity relationships of these inhibitors.Carbapenems such as imipenem (Scheme 1) have proven useful for the treatment of a variety of Gram-negative and Gram-positive infections (1, 2). Carbapenems and other -lactam antibiotics covalently modify penicillin-binding proteins (PBPs) 1 involved in the peptidoglycan biosynthetic pathway of cell wall assembly in bacteria (3, 4). Resistance to carbapenems can arise because of acquisition of low affinity PBPs (3) (e.g. PBP2a of Staphylococcus aureus), altered membrane permeability (5), and expression of class A, B, and D -lactamases (6 -10). Class B -lactamases (metallo--lactamases or MBLs) can hydrolyze a wide variety of substrates of the -lactam class, including carbapenems, penicillins, and cephalosporins, rendering them ineffective as antibiotics.The IMP-1 gene encoding an MBL has been identified on a plasmid and in Japan has transferred among clinical isolates such as Pseudomonas aeruginosa (11,12), Klebsiella pneumoniae, Serratia marcescens, and other members of the Enterobacteriaceae (13,14). In addition, carbapenem-resistant clinical isolates expressing MBLs related to IMP-1 have been identified recently in Singapore (15), Italy (16), and Hong Kong (10). Such reports of plasmid-borne imipenem resistance highlight the need for inhibitors of IMP-1 that can restore the activity of carbapenems in resistant bacteria.Several classes of MBL inhibitors have been reported (for reviews, see Refs. 17 and 18)) including phenazines (19), trifluoromethyl alcohol and ketone derivatives of L-and D-alanine (20), thioesters (18, 21-23), thiols (24 -28), biphenyl tetrazoles (29, 30), and amino acid-derived hydroxamates (31). Biphenyl tetrazoles have been shown to reverse imipenem resistance in a clinical isolate of Bacteroides fragilis (29), and thioesters have been shown to reverse resistance to the carbapenem L-742,728 in a laboratory strain of Escherichia coli expressing IMP-1 (32). A 1-methylcarbapenem substituted at C-2 with a benzothienylthio moiety has been reported to be a potent IMP-1 inhibitor that can reverse resistance to imipenem in an IMP-1-producing strain of Serratia marcescens (33). Although the inhibitors described above have been reported to have good activity against a specific MBL, only certain thiols (e.g. SB 264218) exhibit broad spe...
One approach to estimating the "chemical tractability" of a candidate protein target where we know the atomic resolution structure is to examine the physical properties of potential binding sites. A number of other workers have addressed this issue. We characterize ~290,000 "pockets" from ~42,000 protein crystal structures in terms of a three parameter "pocket space": volume, buriedness, and hydrophobicity. A metric DLID (drug-like density) measures how likely a pocket is to bind a drug-like molecule. This is calculated from the count of other pockets in its local neighborhood in pocket space that contain drug-like cocrystallized ligands and the count of total pockets in the neighborhood. Surprisingly, despite being defined locally, a global trend in DLID can be predicted by a simple linear regression on log(volume), buriedness, and hydrophobicity. Two levels of simplification are necessary to relate the DLID of individual pockets to "targets": taking the best DLID per Protein Data Bank (PDB) entry (because any given crystal structure can have many pockets), and taking the median DLID over all PDB entries for the same target (because different crystal structures of the same protein can vary because of artifacts and real conformational changes). We can show that median DLIDs for targets that are detectably homologous in sequence are reasonably similar and that median DLIDs correlate with the "druggability" estimate of Cheng et al. (Nature Biotechnology 2007, 25, 71-75).
The capsaicin receptor (TRPV1) is a nonselective cation channel that is activated in nociceptors by several painful stimuli, and hence TRPV1 antagonists could represent a novel class of analgesic compounds. Resiniferatoxin (RTX), a potent agonist of TRPV1, and iodoresiniferatoxin (I-RTX), a potent antagonist of TRPV1, both bind with higher affinity to the rat TRPV1 (rTRPV1) than the human (hTRPV1) isoform. To identify the structural features responsible for this difference in affinity, [(3)H]RTX binding to chimeras between hTRPV1 and rTRPV1 was characterized. The "sensor" region within the transmembrane domain (S1-S4) was found to determine [(3)H]RTX binding affinity. All 16 different residues in this region were systematically substituted in hTRPV1 with rTRPV1 residues. A single mutation in the S4 membrane domain of hTRPV1, L547M, caused a 30-fold increase in [(3)H]RTX affinity whereas the inverse mutation in rTRPV1, M547L, caused a 30-fold decrease in affinity for [(3)H]RTX, and several other agonists and antagonists were similarly affected by these mutations. TRPV1 channels with mutations at position 547 were expressed in oocytes, and the relative response to RTX followed a pattern similar to that seen with [(3)H]RTX binding. These data suggest a model where Met-547 in the S4 domain of TRPV1 forms a binding pocket with Tyr-511 in the S3 domain. This model places RTX near the sensor domain thought to move during the gating process and should help to guide further work designed to understand the gating mechanisms of TRPV1 channels based on comparisons between the agonist RTX and the related competitive antagonist I-RTX.
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