Structure-based design and combinatorial chemistry yield low nanomolar inhibitors of cathepsin D.

BACKGROUND: The identification of potent small molecule ligands to receptors and enzymes is one of the major goals of chemical and biological research. Two powerful new tools that can be used in these efforts are combinatorial chemistry and structure-based design. Here we address how to join these methods in a design protocol that produces libraries of compounds that are directed against specific macromolecular targets. The aspartyl class of proteases, which is involved in numerous biological processes, was chosen to demonstrate this effective procedure. RESULTS: Using cathepsin D, a prototypical aspartyl protease, a number of low nanomolar inhibitors were rapidly identified. Although cathepsin D is implicated in a number of therapeutically relevant processes, potent nonpeptide inhibitors have not been reported previously. The libraries, synthesized on solid support, displayed nonpeptide functionality about the (hydroxyethyl)amine isostere. The (hydroxyethyl)amine isostere, which targets the aspartyl protease class, is a stable mimetic of the tetrahedral intermediate of amide hydrolysis. Structure-based design, using the crystal structure of cathepsin D complexed with the peptide-based natural product pepstatin, was used to select the building blocks for the library synthesis. The library yielded a 'hit rate' of 6-7% at 1 microM inhibitor concentrations, with the most potent compound having a Ki value of 73 nM. More potent, nonpeptide inhibitors (Ki = 9-15 nM) of cathepsin D were rapidly identified by synthesizing and screening a small second generation library. CONCLUSIONS: The success of these studies clearly demonstrates the power of coupling the complementary methods of combinatorial chemistry and structure-based design. We anticipate that the general approaches described here will be successful for other members of the aspartyl protease class and for many other enzyme classes.

Complications following insertion and replacement of percutaneous endoscopic gastrostomy (PEG) tubes.

Percutaneous endoscopic gastrostomy (PEG) tube insertion was introduced in 1980 as an alternative to nasogastric tubes and surgically placed gastrostomy tubes. The procedure is indicated in those patients who have an inability to sustain adequate nutrition in the presence of a functioning gastrointestinal tract. We report four deaths that arose within a ten-week period in 1998.

A case presentation of hemolytic anemia in ulcerative colitis and review of the literature.

Autoimmune hemolytic anemia associated with ulcerative colitis is a rare occurrence, and its management poses a therapeutic dilemma. Herein we present a case of ulcerative colitis involving an autoimmune hemolytic anemia and discuss the proposed mechanisms for the anemia. A review of past reported cases and their outcomes is included. In the past, a patient with autoimmune anemia unresponsive to corticosteroids was subjected first to splenectomy and then to colectomy if splenectomy failed to resolve the anemia. In view of the high failure rate for splenectomy, we recommend that ulcerative colitis patients with anemia unresponsive to corticosteroids proceed directly to total colectomy.

BUILDER v.2: improving the chemistry of a de novo design strategy.

Significant improvements have been made to the de novo drug design program BUILDER. The BUILDER strategy is to find molecule templates that bind tightly to 'hot spots' in the target receptor, and then generate bridges to join these templates. In this paper, the bridging algorithm has been further developed to improve the chemical sense and diversity of the bridges, as well as the robustness of the technique. The improved algorithm is then applied to rebuild known bridges in methotrexate and HIV protease. Finally, the entire BUILDER approach is tested by rebuilding methotrexate de novo.

Fluoroacetate-mediated toxicity of fluorinated ethanes.

A series of 1-(di)halo-2-fluoroethanes reported in the literature to be nontoxic or of low toxicity were found to be highly toxic by the inhalation route. Experiments were performed that showed the compounds, 1,2-difluoroethane, 1-chloro-2-fluoroethane, 1-chloro-1,2-difluoroethane, and 1-bromo-2-fluoroethane to be highly toxic to rats upon inhalation for 4 hr. All four compounds had 4-hr approximate lethal concentrations of < or = 100 ppm in rats. In contrast, 1,1-difluoroethane (commonly referred to as HFC-152a) has very low acute toxicity with a 4-hr LC50 of > 400,000 ppm in rats. Rats exposed to the selected toxic fluoroethanes showed clinical signs of fluoroacetate toxicity (lethargy, hunched posture, convulsions). 1,2-Difluoroethane, 1-chloro-2-fluoroethane, 1-chloro-1,2-difluoroethane, and 1-bromo-2-fluoroethane were shown to increase concentrations of citrate in serum and heart tissue, a hallmark of fluoroacetate intoxication. 19F NMR analysis confirmed that fluoroacetate was present in the urine of rats exposed to each toxic compound. Fluorocitrate, a condensation product of fluoroacetate and oxaloacetate, was identified in the kidney of rats exposed to 1,2-difluoroethane. There was a concentration-related elevation of serum and heart citrate in rats exposed to 0-1000 ppm 1,2-fluoroethane. Serum citrate was increased up to 5-fold and heart citrate was increased up to 11-fold over control citrate levels. Metabolism of 1,2-difluoroethane by cytochrome P450 (most likely CYP2E1) is suspected because pretreatment of rats or mice with SKF-525F, disulfiram, or dimethyl sulfoxide prevented or delayed the toxicity observed in rats not pretreated. Experimental evidence indicates that the metabolism of the toxic fluoroethanes is initiated at the carbon-hydrogen bond, with metabolism to fluoroacetate via an aldehyde or an acyl fluoride. The results of these studies show that 1-(di)halo-2-fluoroethanes are highly toxic to rats and should be considered a hazard to humans unless demonstrated otherwise.

Evidence for multiple forms of p-trifluoromethylbenzenesulfonyl-alpha-chymotrypsin.

p-Trifluoromethylbenzenesulfonyl-alpha-chymotrypsin, an analog of tosylchymotrypsin, has been prepared and shown to be stable enough to permit fluorine nuclear magnetic resonance experiments. Up to four distinct trifluoromethyl resonances can be observed for the modified protein at 94.1 MHz even when the enzyme derivative is prepared from protein which has been purified by several methods. The resonances observed appear to represent proteins which are grossly similar as regards molecular size and the ability to bind and hydrolyze substrates, but nonetheless distinctive enough in the active-site region to produce appreciable chemical-shift effects.

A hexamer peptide ligand that binds selectively to staphylococcal enterotoxin B: isolation from a solid phase combinatorial library.

By screening a solid-phase combinatorial peptide library, a short peptide ligand, YYWLHH, has been discovered that binds with high affinity and selectivity to staphylococcal enterotoxin B (SEB), but only weakly to other SEs that share sequence and structural homology with SEB. Using column affinity chromatography with an immobilized YYWLHH stationary phase, it was possible to separate SEB quantitatively from Staphylococcus aureus fermentation broth, a complex mixture of proteins, carbohydrates and other biomolecules. The immobilized peptide was also used to purify native SEB from a mixture containing denatured and hydrolyzed SEB, and showed little cross-reactivity with other SEs. To our knowledge this is the first report of a highly specific short peptide ligand for SEB. Such a ligand is a potential candidate to replace antibodies for detection, removal and purification strategies for SEB.

Finding potential DNA-binding compounds by using molecular shape.

For the first time a general shape-search docking algorithm (DOCK) has been applied to the minor and major grooves of A-, B- and Z-type DNA dodecamers and to an intercalation site in a B-DNA-type hexamer. Both experimentally and theoretically derived geometries for the various DNA fragments were used. The DOCK searches were carried out on a subset of the Cambridge Crystallographic Database, consisting of almost 10,000 molecules. One of the molecules that scored best in terms of the DOCK algorithm was CC-1065, a potent antitumor agent known to (covalently) bind the AT-rich parts of the minor groove of B-DNA. Several known DNA-binding agents also scored highly. Molecules with shapes complementary to A-, B- and Z-type DNA were indicated by DOCK. In addition, compounds were extracted from the database that might be selective for the GC-rich regions of the minor groove of B-DNA. Many of the compounds in the present study may serve as a starting point for further molecular design of novel DNA-binding ligands.

Automated site-directed drug design using molecular lattices.

Receptor-based drug design is predicated on the knowledge of the structure of a target receptor and the principles of molecule recognition. The objective is to produce a wide diversity of structures that are sterically and electrostatically complementary to a specified receptor site. Many drug-receptor interactions are controlled by a few key receptor groups. This observation leads to a design approach in which one focuses on chemical fragments that putatively interact with the key receptor groups. There then remains the difficult task of joining the fragments into molecular structures that match the spatial patterns of recognition forces in the receptor site. In this paper, we describe a new modeling program, BUILDER, that combines database searching techniques and structure generation algorithms within an interactive graphics modeling environment (MidasPlus). A novel tool for process communication (delegate) is introduced and examples of its use are given. To demonstrate the functionality of the package and its ability to produce novel structures, we examine the active site of HIV-1 protease.

Identification of novel small molecule ligands that bind to tetanus toxin.

Tetanus toxin belongs to a family of clostridial protein neurotoxins for which there are no known antidotes. Another closely related member of this family, botulinum toxin, is being used with increasing frequency by physicians to treat severe muscle disorders. Botulinum toxin has also been produced in large quantities by terrorists for use as a biological weapon. To identify small molecule ligands that might bind to the targeting domain of tetanus and botulinum toxins and to facilitate the design of inhibitors and new reagents for their detection, molecular docking calculations were used to screen a large database of compounds for their potential to bind to the C fragment of tetanus toxin. Eleven of the predicted ligands were assayed by electrospray ionization mass spectrometry (ESI-MS) for binding to the tetanus toxin C fragment, and five ligands (45%) were found to bind to the protein. One of these compounds, doxorubicin, was observed to have strong hydrophobic interactions with the C fragment. To check the ligands for their ability to compete with ganglioside binding, each was also tested using a GT1b liposome assay. Doxorubicin was the only ligand found to competitively bind the tetanus toxin C fragment with an appreciable binding constant (9.4 microM).

Dethiobiotin synthetase: the carbonylation of 7,8-diaminonanoic acid proceeds regiospecifically via the N7-carbamate.

Dethiobiotin synthetase (DTBS) catalyzes the penultimate step in biotin biosynthesis, the formation of the ureido ring of dethiobiotin from (7R,8S)-7,8-diaminononanoic acid (7,8-diaminopelargonic acid, DAPA), CO2, and ATP. Solutions of DAPA at neutral pH readily formed a mixture of the N7- and N8-carbamates in the presence of CO2. However, four lines of evidence together indicated that only the N7-carbamate of DAPA was an intermediate in the reaction catalyzed by DTBS. (1) Addition of diazomethane to mixtures of DAPA and [14C]CO2 yielded a mixture of the N7- and N8-methyl carbamate esters, consistent with carbamate formation in free solution. In the presence of excess DTBS (over DAPA), the ratio of N7:N8-methyl carbamate esters recovered was roughly doubled, suggesting that the enzyme preferentially bound the N7-DAPA-carbamate. (2) Both N7- and N8-DAPA-carbamates were observed directly by 1H and 13C NMR in solutions containing DAPA and [13C]CO2. In the presence of excess DTBS (over DAPA) only one carbamate was observed, showing that carbamate binding to the enzyme was regiospecific. 13C NMR of mixtures containing enzyme, [7-15N]DAPA, and [13C]CO2 showed that the enzyme-bound carbamate was at N7 of DAPA. In addition, pulse-chase experiments showed that the binary complex of DTBS and N7-DAPA-carbamate became kinetically committed upon addition of MgATP. (3) The N7-DAPA-carbamate mimic, 3-(1-aminoethyl)nonanedioic acid, in which the carbamate nitrogen was replaced with a methylene group, cyclized to the corresponding lactam in the presence of DTBS and ATP; ADP and P(i) were also formed.(ABSTRACT TRUNCATED AT 250 WORDS)