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Drug candidates are generally discovered using biochemical screens employing an isolated target protein or by utilizing cell-based phenotypic assays. Both noncovalent and covalent hits emerge from such endeavors. Herein, we exemplify an "Inverse Drug Discovery" strategy in which organic compounds of intermediate complexity harboring weak, but activatable, electrophiles are matched with the protein(s) they react with in cells or cell lysate. An alkyne substructure in each candidate small molecule enables affinity chromatography-mass spectrometry, which produces a list of proteins that each distinct compound reacts with. A notable feature of this approach is that it is agnostic with respect to the cellular proteins targeted. To illustrate this strategy, we employed aryl fluorosulfates, an underexplored class of sulfur(VI) halides, that are generally unreactive unless activated by protein binding. Reversible aryl fluorosulfate binding, correct juxtaposition of protein side chain functional groups, and transition-state stabilization of the S(VI) exchange reaction all seem to be critical for conjugate formation. The aryl fluorosulfates studied thus far exhibit chemoselective reactivity toward Lys and, particularly, Tyr side chains, and can be used to target nonenzymes (e.g., a hormone carrier or a small-molecule carrier protein) as well as enzymes. The "Inverse Drug Discovery" strategy should be particularly attractive as a means to explore latent electrophiles not typically used in medicinal chemistry efforts, until one reacts with a protein target of exceptional interest. Structure-activity data can then be used to enhance the selectivity of conjugate formation or the covalent probe can be used as a competitor to develop noncovalent drug candidates. Here we use the "Inverse Drug Discovery" platform to identify and validate covalent ligands for 11 different human proteins. In the case of one of these proteins, we have identified and validated a small-molecule probe for the first time.
Inhibition of factor XIa (FXIa) is a novel paradigm for developing anticoagulants without major bleeding consequences. We present the discovery of sulfated pentagalloylglucoside (6) as a highly selective inhibitor of human FXIa. Biochemical screening of a focused library led to the identification of 6, a sulfated aromatic mimetic of heparin. Inhibitor 6 displayed a potency of 551 nM against FXIa, which was at least 200-fold more selective than other relevant enzymes. It also prevented activation of factor IX and prolonged human plasma and whole blood clotting. Inhibitor 6 reduced V(MAX) of FXIa hydrolysis of chromogenic substrate without affecting the K(M), suggesting an allosteric mechanism. Competitive studies showed that 6 bound in the heparin-binding site of FXIa. No allosteric small molecule has been discovered to date that exhibits equivalent potency against FXIa. Inhibitor 6 is expected to open up a major route to allosteric FXIa anticoagulants with clinical relevance.
BACKGROUND - Acute irritant contact dermatitis induced by cutaneous exposure to chemicals is a common dermatologic problem in the workplace. In severe cases, irritant contact responses can result in a caustic burn. Chemical burn induced by concentrated sodium hypochlorite (the active ingredient in bleach) has been reported infrequently in the literature, with no previously reported cases of chemical burn due to an alkyl sulfate (a common surfactant in cleaning fluids). Here we describe a chemical burn in a 16-year-old girl resulting from exposure to a solution of concentrated sodium hypochlorite and alkyl sulfate applied as a sanitizer to the interior of roller skates worn at work.
OBSERVATIONS - The diagnosis was made on the basis of the patient's exposure history, clinical appearance, and laboratory results. On physical examination, the erythematous plaque, located at the site of chemical exposure, had intact skin lines, surrounding edema, and decreased sensitivity to touch. The peripheral white blood cell count was within normal limits and bacterial and fungal cultures from the lesion were negative.
CONCLUSIONS - The irritant effect of exposure to chemicals, including those that usually are not major irritants, and the possible additive effect of simultaneous exposure to different chemicals, should be considered in the differential diagnosis of acute dermatitis of unknown etiology. Moreover, increased reporting of cases of chemical-induced acute irritant contact dermatitis will help lead to crucial early and appropriate treatment.
The Bacillus subtilis phage phi 105 repressor, a lambda repressor-like transcriptional regulatory protein, was overproduced in Escherichia coli and purified to near homogeneity in order to examine its in vitro DNA-binding properties. The active form of repressor appears to be a tetramer. DNase I protection experiments demonstrate that repressor can specifically bind to six distinct sites, all located within the phi 105 EcoRI-F immunity region (immF). Three of these sites had been identified earlier as functional operators by genetic analysis. They share a common 14-base pair, asymmetric "core" sequence, 5'-GACGGAAATACAAG-3', termed OR. The three additional sites show significant homology with OR. For an individual binding site, hydroxyl-radical footprinting reveals symmetrical repressor-DNA interactions established via one side of the helix. Dimethyl sulfate protection indicates that guanines at the conserved OR base pair positions 1, 3, and 4 may participate in sequence-specific interactions with repressor in agreement with a previously proposed recognition model. However, since the OR sequence is not symmetrical with respect to this GNCG motif, at present it remains difficult to completely understand the molecular basis of this interaction.