Structures of human steroidogenic cytochrome P450 17A1 with substrates.

The human cytochrome P450 17A1 (CYP17A1) enzyme operates at a key juncture of human steroidogenesis, controlling the levels of mineralocorticoids influencing blood pressure, glucocorticoids involved in immune and stress responses, and androgens and estrogens involved in development and homeostasis of reproductive tissues. Understanding CYP17A1 multifunctional biochemistry is thus integral to treating prostate and breast cancer, subfertility, blood pressure, and other diseases. CYP17A1 structures with all four physiologically relevant steroid substrates suggest answers to four fundamental aspects of CYP17A1 function. First, all substrates bind in a similar overall orientation, rising ∼60° with respect to the heme. Second, both hydroxylase substrates pregnenolone and progesterone hydrogen bond to Asn(202) in orientations consistent with production of 17α-hydroxy major metabolites, but functional and structural evidence for an A105L mutation suggests that a minor conformation may yield the minor 16α-hydroxyprogesterone metabolite. Third, substrate specificity of the subsequent 17,20-lyase reaction may be explained by variation in substrate height above the heme. Although 17α-hydroxyprogesterone is only observed farther from the catalytic iron, 17α-hydroxypregnenolone is also observed closer to the heme. In conjunction with spectroscopic evidence, this suggests that only 17α-hydroxypregnenolone approaches and interacts with the proximal oxygen of the catalytic iron-peroxy intermediate, yielding efficient production of dehydroepiandrosterone as the key intermediate in human testosterone and estrogen synthesis. Fourth, differential positioning of 17α-hydroxypregnenolone offers a mechanism whereby allosteric binding of cytochrome b5 might selectively enhance the lyase reaction. In aggregate, these structures provide a structural basis for understanding multiple key reactions at the heart of human steroidogenesis.

Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways.

A specific small-molecule inhibitor of p97 would provide an important tool to investigate diverse functions of this essential ATPase associated with diverse cellular activities (AAA) ATPase and to evaluate its potential to be a therapeutic target in human disease. We carried out a high-throughput screen to identify inhibitors of p97 ATPase activity. Dual-reporter cell lines that simultaneously express p97-dependent and p97-independent proteasome substrates were used to stratify inhibitors that emerged from the screen. N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ) was identified as a selective, potent, reversible, and ATP-competitive p97 inhibitor. DBeQ blocks multiple processes that have been shown by RNAi to depend on p97, including degradation of ubiquitin fusion degradation and endoplasmic reticulum-associated degradation pathway reporters, as well as autophagosome maturation. DBeQ also potently inhibits cancer cell growth and is more rapid than a proteasome inhibitor at mobilizing the executioner caspases-3 and -7. Our results provide a rationale for targeting p97 in cancer therapy.

Overcoming product inhibition in catalysis of the intramolecular Schmidt reaction.

A method for carrying out the intramolecular Schmidt reaction of alkyl azides and ketones using a substoichiometric amount of catalyst is reported. Following extensive screening, the use of the strong hydrogen-bond-donating solvent hexafluoro-2-propanol was found to be consistent with low catalyst loadings, which ranged from 2.5 mol % for favorable substrates to 25 mol % for more difficult cases. Reaction optimization, broad substrate scope, and preliminary mechanistic studies of this improved version of the reaction are described.

Exploring chemical diversity via a modular reaction pairing strategy.

The efficient synthesis of an 80-member library of unique benzoxathiazocine 1,1-dioxides by a microwave-assisted, intermolecular nucleophilic aromatic substitution (S(N)Ar) diversification pathway is reported. Eight benzofused sultam cores were generated by means of a sulfonylation/S(N)Ar/Mitsunobu reaction pairing protocol, and subsequently diversified by intermolecular S(N)Ar with ten chiral, non-racemic amine/amino alcohol building blocks. Computational analyses were employed to explore and evaluate the chemical diversity of the library.

Human cytochrome P450 2E1 structures with fatty acid analogs reveal a previously unobserved binding mode.

Human microsomal cytochrome P450 (CYP) 2E1 is widely known for its ability to oxidize >70 different, mostly compact, low molecular weight drugs and other xenobiotic compounds. In addition CYP2E1 oxidizes much larger C9-C20 fatty acids that can serve as endogenous signaling molecules. Previously structures of CYP2E1 with small molecules revealed a small, compact CYP2E1 active site, which would be insufficient to accommodate medium and long chain fatty acids without conformational changes in the protein. In the current work we have determined how CYP2E1 can accommodate a series of fatty acid analogs by cocrystallizing CYP2E1 with omega-imidazolyl-octanoic fatty acid, omega-imidazolyl-decanoic fatty acid, and omega-imidazolyl-dodecanoic fatty acid. In each structure direct coordination of the imidazole nitrogen to the heme iron mimics the position required for native fatty acid substrates to yield the omega-1 hydroxylated metabolites that predominate experimentally. In each case rotation of a single Phe(298) side chain merges the active site with an adjacent void, significantly altering the active site size and topology to accommodate fatty acids. The binding of these fatty acid ligands is directly opposite the channel to the protein surface and the binding observed for fatty acids in the bacterial cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium. Instead of the BM3-like binding mode in the CYP2E1 channel, these structures reveal interactions between the fatty acid carboxylates and several residues in the F, G, and B' helices at successive distances from the active site.

Effect of C-2 substitution on the stability of non-traditional cephalosporins in mouse plasma.

A systematic study of the stability of a set of cephalosporins in mouse plasma reveals that cephalosporins lacking an acidic moiety at C-2 may be vulnerable to ß-lactam cleavage in mouse plasma.

p-dimethylaminocinnamaldehyde derivatization for colorimetric detection and HPLC-UV/vis-MS/MS identification of indoles.

Cytochrome P450 2A13 (CYP2A13) is a lung specific enzyme known to activate the potent tobacco procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) into two carcinogenic metabolites. CYP2A13 has been crystallized and X-ray diffraction experiments illuminated the structure of this enzyme, but with an unknown ligand present in the enzyme active site. This unknown ligand was suspected to be indole but a selective method had to be developed to differentiate among indole and its metabolites in the protein sample. We successfully modified a microbiological colorimetric assay to spectrophotometrically differentiate between indole and a number of possible indole metabolites in nanomolar concentrations by derivatization with p-dimethylaminocinnamaldehyde (DMACA). Further differentiation of indoles was made by mass spectrometry (HPLC-UV/vis-MS/MS) utilizing the chromophore generated in the DMACA conjugation as a UV signature for HPLC detection. The ligand in the crystallized protein was identified as unsubstituted indole, which facilitated refinement of two alternate conformations in the CYP2A13 crystal structure active site.

Evaluating p97 Inhibitor Analogues for Potency against p97-p37 and p97-Npl4-Ufd1 Complexes.

We previously found that the p97 cofactor, p47, significantly decreased the potency of some ATP-competitive p97 inhibitors such as ML240 [2-(2-amino-1H-benzo[d]imidazol-1-yl)-N-benzyl-8-methoxyquinazolin-4-amine] and ML241 [2-(2H-benzo[b][1,4]oxazin-4(3H)-yl)-N-benzyl-5,6,7,8 tetrahydroquinazolin-4-amine]. In this study, we aimed to evaluate inhibitor potencies against two additional p97 cofactor complexes, p97-p37 and p97-Npl4-Ufd1. We focused on these two cofactor complexes, because the protein sequence of p37 is 50 % identical to that of p47, and the Npl4-Ufd1 heterodimer (NU) is the most-studied p97 cofactor complex. We screened 200 p97 inhibitor analogues for their ability to inhibit the ATPase activity of p97 alone and of p97-p37 and p97-NU complexes. In contrast to the effect of p47, p37 and NU did not significantly change the potencies of most of the compounds. These results highlight differences among p97 cofactors in influencing p97 conformation and effects of inhibitors on p97 complexes, as compared to p97 alone. Continued efforts are needed to advance the development of complex-specific p97 inhibitors.

Novel Cephalosporins Selectively Active on Nonreplicating Mycobacterium tuberculosis.

We report two series of novel cephalosporins that are bactericidal to Mycobacterium tuberculosis alone of the pathogens tested, which only kill M. tuberculosis when its replication is halted by conditions resembling those believed to pertain in the host, and whose bactericidal activity is not dependent upon or enhanced by clavulanate, a ß-lactamase inhibitor. The two classes of cephalosporins bear an ester or alternatively an oxadiazole isostere at C-2 of the cephalosporin ring system, a position that is almost exclusively a carboxylic acid in clinically used agents in the class. Representatives of the series kill M. tuberculosis within macrophages without toxicity to the macrophages or other mammalian cells.

3-Substituted Biquinolinium Inhibitors of AraC Family Transcriptional Activator VirF from S. flexneri Obtained Through In Situ Chemical Ionization of 3,4-Disubstituted Dihydroquinolines.

During a structure-activity relationship optimization campaign to develop an inhibitor of AraC family transcriptional activators, we discovered an unexpected transformation of a previously reported inhibitor that occurs under the assay conditions. Once placed in the assay media, the 3, 4-disubstituted dihydroquinoline core of the active analogue rapidly undergoes a decomposition reaction to a quaternary 3-substituted biquinolinium. Further examination established an SAR for this chemotype while also demonstrating its resilience to irreversible binding of biologically relevant nucleophiles.

Lifting the mask: identification of new small molecule inhibitors of uropathogenic Escherichia coli group 2 capsule biogenesis.

Uropathogenic Escherichia coli (UPEC) is the leading cause of community-acquired urinary tract infections (UTIs), with over 100 million UTIs occurring annually throughout the world. Increasing antimicrobial resistance among UPEC limits ambulatory care options, delays effective treatment, and may increase overall morbidity and mortality from complications such as urosepsis. The polysaccharide capsules of UPEC are an attractive target a therapeutic, based on their importance in defense against the host immune responses; however, the large number of antigenic types has limited their incorporation into vaccine development. The objective of this study was to identify small-molecule inhibitors of UPEC capsule biogenesis. A large-scale screening effort entailing 338,740 compounds was conducted in a cell-based, phenotypic screen for inhibition of capsule biogenesis in UPEC. The primary and concentration-response assays yielded 29 putative inhibitors of capsule biogenesis, of which 6 were selected for further studies. Secondary confirmatory assays identified two highly active agents, named DU003 and DU011, with 50% inhibitory concentrations of 1.0 µM and 0.69 µM, respectively. Confirmatory assays for capsular antigen and biochemical measurement of capsular sugars verified the inhibitory action of both compounds and demonstrated minimal toxicity and off-target effects. Serum sensitivity assays demonstrated that both compounds produced significant bacterial death upon exposure to active human serum. DU011 administration in mice provided near complete protection against a lethal systemic infection with the prototypic UPEC K1 isolate UTI89. This work has provided a conceptually new class of molecules to combat UPEC infection, and future studies will establish the molecular basis for their action along with efficacy in UTI and other UPEC infections.

Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) of Human Malaria via High-Throughput Screening.

The target of this study, the PfM18 aspartyl aminopeptidase (PfM18AAP), is the only AAP present in the genome of the malaria parasite Plasmodium falciparum. PfM18AAP is a metallo-exopeptidase that exclusively cleaves N-terminal acidic amino acids glutamate and aspartate. It is expressed in parasite cytoplasm and may function in concert with other aminopeptidases in protein degradation, of, for example, hemoglobin. Previous antisense knockdown experiments identified a lethal phenotype associated with PfM18AAP, suggesting that it is a valid target for new antimalaria therapies. To identify inhibitors of PfM18AAP function, a fluorescence enzymatic assay was developed using recombinant PfM18AAP enzyme and a fluorogenic peptide substrate (H-Glu-NHMec). This was screened against the Molecular Libraries Probe Production Centers Network collection of ~292,000 compounds (the Molecular Libraries Small Molecule Repository). A cathepsin L1 (CTSL1) enzyme-based assay was developed and used as a counter screen to identify compounds with nonspecific activity. Enzymology and phenotypic assays were used to determine mechanism of action and efficacy of selective and potent compounds identified from high-throughput screening. Two structurally related compounds, CID 6852389 and CID 23724194, yielded micromolar potency and were inactive in CTSL1 titration experiments (IC50>59.6 µM). As measured by the K(i) assay, both compounds demonstrated micromolar noncompetitive inhibition in the PfM18AAP enzyme assay. Both CID 6852389 and CID 23724194 demonstrated potency in malaria growth assays (IC504 µM and 1.3 µM, respectively).

Solution-phase synthesis of a diverse library of benzisoxazoles utilizing the [3 + 2] cycloaddition of in situ-generated nitrile oxides and arynes.

A library of benzisoxazoles has been synthesized by the [3 + 2] cycloaddition of nitrile oxides with arynes and further diversified by acylation/sulfonylation and palladium-catalyzed coupling processes. The eight key intermediate benzisoxazoles have been prepared by the reaction of o-(trimethylsilyl)aryl triflates and chlorooximes in the presence of CsF in good to excellent yields under mild reaction conditions. These building blocks have been used as the key components of a diverse set of 3,5,6-trisubstituted benzisoxazoles.

Synthesis of an Isoindoline-Annulated, Tricyclic Sultam Library via Microwave-Assisted, Continuous-Flow Organic Synthesis (MACOS).

A microwave-assisted, continuous-flow organic synthesis (MACOS) protocol for the synthesis of an isoindoline-annulat-ed, tricyclic sultam library, utilizing a Heck-aza-Michael (HaM) strategy, is reported. This sequence involves a Heck reaction on vi-nylsulfonamides with batch microwave heating followed by a one-pot, sequential intramolecular aza-Michael cyclization/Boc-deprot-ection using MACOS. Subsequent cyclization with either 1,1'-carbonyldiimidazole or chloromethyl pivalate using MACOS provided an array of tricyclic sultams. This efficient three-step protocol requires only a few hours to produce the target sultams starting from simple starting materials. Using this strategy, a 38-member library of isoindoline-annulated sultams was generated in good to excellent overall yields (53-87%).

Synthesis of a unique isoindoline/tetrahydroisoquinoline-based tricyclic sultam library utilizing a Heck-aza-Michael strategy.

The synthesis of a unique isoindoline- and tetrahydroisoquinoline (THIQ)-containing tricyclic sultam library, utilizing a Heck-aza-Michael (HaM) strategy is reported. Both isoindoline and THIQ rings are installed through a Heck reaction on a vinylsulfonamide, followed by one-pot deprotection and intramolecular aza-Michael reaction. Subsequent cyclization with either paraformaldehyde condensation or 1,1'-carbonyldiimidazole coupling generates a variety of tricyclic sultams. Overall, a 160-member library of these sultams, together with their isoindolines/THIQ and secondary sulfonamides precursors, were constructed using this strategy.

Facile (triazolyl)methylation of MACOS-derived benzofused sultams utilizing ROMP-derived OTP reagents.

A combination of MACOS scale-out and ROMP-derived oligomeric triazole phosphates (OTP(n)) have been successfully utilized for the preparation of a 106-member library of triazole containing benzothiaoxazepine-1,1-dioxides. This report demonstrates the utilization of a suite of soluble OTP(n) reagents for facile (triazolyl)methylation of 10 MACOS-derived sultam scaffolds in purification-free process for parallel synthesis of small molecule collections for HTS.

Automated synthesis of a library of triazolated 1,2,5-thiadiazepane 1,1-dioxides via a double aza-Michael strategy.

The construction of a 96-member library of triazolated 1,2,5-thiadiazepane 1,1-dioxides was performed on a Chemspeed Accelerator (SLT-100) automated parallel synthesis platform, culminating in the successful preparation of 94 out of 96 possible products. The key step, a one-pot, sequential elimination, double-aza-Michael reaction, and [3 + 2] Huisgen cycloaddition pathway has been automated and utilized in the production of two sets of triazolated sultam products.

Synthesis of amino-benzothiaoxazepine-1,1-dioxides utilizing a microwave-assisted, S(N)Ar protocol.

The development of a microwave-assisted, intermolecular S(N)Ar protocol for the synthesis of a 126-member benzothiaoxazepine-1,1-dioxide library is reported. Diversification of 12 benzothiaoxazepine-1,1-dioxides was achieved in rapid fashion utilizing a variety of 2° amines and amino alcohols to generate an 80-member library. A second 48-member library was subsequently generated via a two-step alkylation, intermolecular S(N)Ar diversification protocol.

Triazole-containing isothiazolidine 1,1-dioxide library synthesis: one-pot, multi-component protocols for small molecular probe discovery.

The construction of two libraries of triazole-containing isothiazolidine 1,1-dioxides is reported utilizing either a one-pot click/aza-Michael or click/OACC esterification protocol. One core dihydroisothiazole 1,1-dioxide scaffold was prepared rapidly on multigram scale via ring-closing metathesis (RCM) and was subjected to a one-pot multicomponent click/aza-Michael protocol with an array of amines and azides for the generation of a 180-member triazole-containing isothiazolidine 1,1-dioxide library. Alternatively, three daughter scaffolds were generated via the aza-Michael of three amino alcohols, followed by a one-pot, multicomponent click/esterification protocol utilizing a ring-opening metathesis polymerization (ROMP)-derived coupling reagent, oligomeric alkyl carbodiimide (OACC) to generate a 41-member library of triazole-containing isothiazole 1,1-dioxides.

Structures of human cytochrome P-450 2E1. Insights into the binding of inhibitors and both small molecular weight and fatty acid substrates.

Human microsomal cytochrome P-450 2E1 (CYP2E1) monooxygenates > 70 low molecular weight xenobiotic compounds, as well as much larger endogenous fatty acid signaling molecules such as arachidonic acid. In the process, CYP2E1 can generate toxic or carcinogenic compounds, as occurs with acetaminophen overdose, nitrosamines in cigarette smoke, and reactive oxygen species from uncoupled catalysis. Thus, the diverse roles that CYP2E1 has in normal physiology, toxicity, and drug metabolism are related to its ability to metabolize diverse classes of ligands, but the structural basis for this was previously unknown. Structures of human CYP2E1 have been solved to 2.2 angstroms for an indazole complex and 2.6 angstroms for a 4-methylpyrazole complex. Both inhibitors bind to the heme iron and hydrogen bond to Thr303 within the active site. Complementing its small molecular weight substrates, the hydrophobic CYP2E1 active site is the smallest yet observed for a human cytochrome P-450. The CYP2E1 active site also has two adjacent voids: one enclosed above the I helix and the other forming a channel to the protein surface. Minor repositioning of the Phe478 aromatic ring that separates the active site and access channel would allow the carboxylate of fatty acid substrates to interact with conserved 216QXXNN220 residues in the access channel while positioning the hydrocarbon terminus in the active site, consistent with experimentally observed omega-1 hydroxylation of saturated fatty acids. Thus, these structures provide insights into the ability of CYP2E1 to effectively bind and metabolize both small molecule substrates and fatty acids.