A high-throughput screening campaign to identify inhibitors of DXP reductoisomerase (IspC) and MEP cytidylyltransferase (IspD).

The rise of antibacterial resistance among human pathogens represents a problem that could change the landscape of healthcare unless new antibiotics are developed. The methyl erythritol phosphate (MEP) pathway represents an attractive series of targets for novel antibiotic design, considering each enzyme of the pathway is both essential and has no human homologs. Here we describe a pilot scale high-throughput screening (HTS) campaign against the first and second committed steps in the pathway, catalyzed by DXP reductoisomerase (IspC) and MEP cytidylyltransferase (IspD), using compounds present in the commercially available LOPAC1280 library as well as in an in-house natural product extract library. Hit compounds were characterized to deduce their mechanism of inhibition; most function through aggregation. The HTS workflow outlined here is useful for quickly screening a chemical library, while effectively identifying false positive compounds associated with assay constraints and aggregation.

The effect of chain length and unsaturation on Mtb Dxr inhibition and antitubercular killing activity of FR900098 analogs.

Inhibition of the nonmevalonate pathway (NMP) of isoprene biosynthesis has been examined as a source of new antibiotics with novel mechanisms of action. Dxr is the best studied of the NMP enzymes and several reports have described potent Dxr inhibitors. Many of these compounds are structurally related to natural products fosmidomycin and FR900098, each bearing retrohydroxamate and phosphonate groups. We synthesized a series of compounds with two to five methylene units separating these groups to examine what linker length was optimal and tested for inhibition against Mtb Dxr. We synthesized ethyl and pivaloyl esters of these compounds to increase lipophilicity and improve inhibition of Mtb growth. Our results show that propyl or propenyl linker chains are optimal. Propenyl analog 22 has an IC50 of 1.07 µM against Mtb Dxr. The pivaloyl ester of 22, compound 26, has an MIC of 9.4 µg/mL, representing a significant improvement in antitubercular potency in this class of compounds.

Retinal pigment epithelium (RPE) exosomes contain signaling phosphoproteins affected by oxidative stress.

Age-related macular degeneration (AMD) is a leading cause of vision loss and blindness among the elderly population in the industrialized world. One of the typical features of this pathology is the gradual death of retinal pigment epithelial (RPE) cells, which are essential for maintaining photoreceptor functions and survival. The etiology is multifactorial, and oxidative stress is clearly one of the key factors involved in disease pathogenesis (Plafker, Adv. Exp. Med. Biol. 664 (2010) 447-56; Qin, Drug Dev. Res. 68 (2007) 213-225). Recent work has revealed the presence of phosphorylated signaling proteins in the vitreous humour of patients affected by AMD or other retinal diseases. While the location of these signaling proteins is typically the cell membrane or intracellular compartments, vitreous samples were proven to be cell-free (Davuluri et al., Arch. Ophthalmol. 127 (2009) 613-21). To gain a better understanding of how these proteins can be shed into the vitreous, we used reverse phase protein arrays (RPMA) to analyze the protein and phosphoprotein content of exosomes shed by cultured ARPE-19 cells under oxidative stress conditions. Seventy two proteins were shown to be released by ARPE-19 cells and compartmentalized within exosomes. Forty one of them were selectively detected in their post-translationally modified form (i.e., phosphorylated or cleaved) for the first time in exosomes. Sets of these proteins were linked together reflecting activation of pathway units within exosomes. A subset of (phospho)proteins were altered in exosomes secreted by ARPE-19 cells subjected to oxidative stress, compared to that secreted by control/non stressed cells. Stress-altered exosome proteins were found to be involved in pathways regulating apoptosis/survival (i.e, Bak, Smac/Diablo, PDK1 (S241), Akt (T308), Src (Y416), Elk1 (S383), ERK 1/2 (T202/Y204)) and cell metabolism (i.e., AMPKα1 (S485), acetyl-CoA carboxylase (S79), LDHA). Exosomes may thus represent the conduit through which membrane and intracellular signaling proteins are released into the vitreous. Changes in their (phospho)protein content upon stress conditions suggest their possible role in mediating cell-cell signaling during physio-pathological events; furthermore, exosomes may represent a potential source of biomarkers.

Synthetic Fosmidomycin analogues with altered chelating moieties do not inhibit 1-deoxy-d-xylulose 5-phosphate Reductoisomerase or Plasmodium falciparum growth in vitro.

Fourteen new fosmidomycin analogues with altered metal chelating groups were prepared and evaluated for inhibition of E. coli Dxr, M. tuberculosis Dxr and the growth of P. falciparum K1 in human erythrocytes. None of the synthesized compounds showed activity against either enzyme or the Plasmodia. This study further underlines the importance of the hydroxamate functionality and illustrates that identifying effective alternative bidentate ligands for this target enzyme is challenging.

Inhibition of DXR in the MEP pathway with lipophilic N-alkoxyaryl FR900098 analogs.

In Mycobacterium tuberculosis (Mtb) and Plasmodium falciparum (Pf), the methylerythritol phosphate (MEP) pathway is responsible for isoprene synthesis. This pathway and its products are vital to bacterial/parasitic metabolism and survival, and represent an attractive set of drug targets due to their essentiality in these pathogens but absence in humans. The second step in the MEP pathway is the conversion of 1-deoxy-d-xylulose-5-phosphate (DXP) to MEP and is catalyzed by 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR). Natural products fosmidomycin and FR900098 inhibit DXR, but are too polar to reach the desired target inside some cells, such as Mtb. Synthesized FR900098 analogs with lipophilic substitution in the position α to the phosphorous atom showed promise, resulting in increased activity against Mtb and Pf. Here, an α substitution, consisting of a 3,4-dichlorophenyl substituent, in combination with various O-linked alkylaryl substituents on the hydroxamate moiety is utilized in the synthesis of a novel series of FR900098 analogs. The purpose of the O-linked alkylaryl substituents is to further enhance DXR inhibition by extending the structure into the adjacent NADPH binding pocket, blocking the binding of both DXP and NADPH. Of the initial O-linked alkylaryl substituted analogs, compound 6e showed most potent activity against Pf parasites at 3.60 µM. Additional compounds varying the phenyl ring of 6e were synthesized. The most potent phosphonic acids, 6l and 6n, display nM activity against PfDXR and low µM activity against Pf parasites. Prodrugs of these compounds were less effective against Pf parasites but showed modest activity against Mtb cells. Data from this series of compounds suggests that this combination of substituents can be advantageous in designing a new generation of antimicrobials.

LC-QToF-Based Metabolomics Identifies Aberrant Tissue Metabolites Associated with a Higher-Fat Diet and Their 'Reversion to Healthy' with Dietary Probiotic Supplementation.

Over 33% of Americans are labeled as obese, leading the World Health Organization to designate obesity as a major public health problem. One consequence of obesity is the development of metabolic syndrome, a condition which has been correlated to an increased risk for developing cardiovascular disease and Type 2 diabetes. Prolonged ingestion of a higher-fat diet, one cause of obesity, results in alterations to the gut microbiome. These alterations are implicated to have a profound role in the evolution and progression of obesity-linked diseases. Probiotics are associated with positive health effects such as limiting pathogen colonization, aiding in digestion, and vitamin synthesis. Using Ossabaw pigs as a model for obesity, and in conjunction with our previous research, we performed an in-depth, nontargeted, metabolomic analysis on select organs to elucidate the effects of dietary supplementation with the probiotic Lacticaseibacillus paracasei. We focused our analysis on the effects of probiotic supplementation on a higher-fat (obesogenic) diet and a nutritionally balanced diet. Notably, our findings reveal that the brain cortex is highly sensitive to dietary influencers, and with probiotic supplementation, several aberrant metabolites associated with a higher-fat diet revert to healthy levels, thus demonstrating the potential for a probiotic intervention for obesity-linked disease.

MEPicides: α,ß-unsaturated Fosmidomycin N-Acyl Analogs as Efficient Inhibitors of Plasmodium falciparum 1-Deoxy-d-xylulose-5-phosphate reductoisomerase.

Malaria, a mosquito-borne disease caused by several parasites of the Plasmodium genus, remains a huge threat to global public health. There are an estimated 0.5 million malaria deaths each year, mostly among African children. Unlike humans, Plasmodium parasites and a number of important pathogenic bacteria employ the methyl erythritol phosphate (MEP) pathway for isoprenoid synthesis. Thus, the MEP pathway represents a promising set of drug targets for antimalarial and antibacterial compounds. Here, we present new unsaturated MEPicide inhibitors of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), the second enzyme of the MEP pathway. A number of these compounds have demonstrated robust inhibition of Plasmodium falciparum DXR, potent antiparasitic activity, and low cytotoxicity against HepG2 cells. Parasites treated with active compounds are rescued by isopentenyl pyrophosphate, the product of the MEP pathway. With higher levels of DXR substrate, parasites acquire resistance to active compounds. These results further confirm the on-target inhibition of DXR in parasites by the inhibitors. Stability in mouse liver microsomes is high for the phosphonate salts, but remains a challenge for the prodrugs. Taken together, the potent activity and on-target mechanism of action of this series further validate DXR as an antimalarial drug target and the α,ß-unsaturation moiety as an important structural component.

Alterations in the porcine colon microbiota induced by the gastrointestinal nematode Trichuris suis.

Helminth parasites ensure their survival by regulating host immunity through mechanisms that dampen inflammation. These properties have recently been exploited therapeutically to treat human diseases. The biocomplexity of the intestinal lumen suggests that interactions between the parasite and the intestinal microbiota would also influence inflammation. In this study, we characterized the microbiota in the porcine proximal colon in response to Trichuris suis (whipworm) infection using 16S rRNA gene-based and whole-genome shotgun (WGS) sequencing. A 21-day T. suis infection in four pigs induced a significant change in the composition of the proximal colon microbiota compared to that of three parasite-naive pigs. Among the 15 phyla identified, the abundances of Proteobacteria and Deferribacteres were changed in infected pigs. The abundances of approximately 13% of genera were significantly altered by infection. Changes in relative abundances of Succinivibrio and Mucispirillum, for example, may relate to alterations in carbohydrate metabolism and niche disruptions in mucosal interfaces induced by parasitic infection, respectively. Of note, infection by T. suis led to a significant shift in the metabolic potential of the proximal colon microbiota, where 26% of all metabolic pathways identified were affected. Besides carbohydrate metabolism, lysine biosynthesis was repressed as well. A metabolomic analysis of volatile organic compounds (VOCs) in the luminal contents showed a relative absence in infected pigs of cofactors for carbohydrate and lysine biosynthesis, as well as an accumulation of oleic acid, suggesting altered fatty acid absorption contributing to local inflammation. Our findings should facilitate development of strategies for parasitic control in pigs and humans.

Bacteria-induced static batch fungal fermentation of the diterpenoid cyathin A(3), a small-molecule inducer of nerve growth factor.

Cyathin A(3), produced by the fungus Cyathus helenae, is a member of the cyathane family of diterpene natural products. While many of the cyathanes display antibacterial/antimicrobial activity or have cytotoxic activity against human cancer cell lines, their most exciting therapeutic potential is derived from their ability to induce nerve growth factor (NGF) release from glial cells, making the cyathanes attractive lead molecules for the development of neuroprotective therapeutics to prevent/treat Alzheimer's disease. To investigate if cyathin A(3) has NGF-inducing activity, we set out to obtain it using published C. helenae bench-scale fungal fermentations. However, to overcome nonproducing fermentations, we developed an alternative, bacteria-induced static batch fermentation approach to the production of cyathin A(3), as described in this report. HPLC, UV absorption spectra, and mass spectrometry identify cyathin A(3) in fungal fermentations induced by the timely addition of Escherichia coli K12 or Bacillus megabacterium. Pre-filtration of the bacterial culture abolishes cyathin A(3) induction, suggesting that bacteria-associated media changes or physical interaction between the fungus and bacteria underlie the induction mechanism. Through alteration of incubation conditions, including agitation, the timing of induction, and media composition, we optimized the fermentation to yield nearly 1 mg cyathin A(3)/ml media, a sixfold increase over previously described yields. Additionally, by comparison of fermentation profiles, we reveal that cyathin A(3) biosynthesis is regulated by carbon catabolite repression. We have used an enzyme-linked immunosorbent assay to illustrate that cyathin A(3) induces NGF release from cultured glial cells, and therefore cyathin A(3) warrants further examination in the development of neuroprotective therapeutics.

Characterization and Inhibition of 1-Deoxy-d-Xylulose 5-Phosphate Reductoisomerase: A Promising Drug Target in Acinetobacter baumannii and Klebsiella pneumoniae.

The ESKAPE pathogens comprise a group of multidrug-resistant bacteria that are the leading cause of nosocomial infections worldwide. The prevalence of antibiotic resistant strains and the relative ease by which bacteria acquire resistance genes highlight the continual need for the development of novel antibiotics against new drug targets. The methylerythritol phosphate (MEP) pathway is an attractive target for the development of new antibiotics. The MEP pathway governs the synthesis of isoprenoids, which are key lipid precursors for vital cell components such as ubiquinone and bacterial hopanoids. Additionally, the MEP pathway is entirely distinct from the corresponding mammalian pathway, the mevalonic acid (MVA) pathway, making the first committed enzyme of the MEP pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC), an attractive target for antibiotic development. To facilitate drug development against two of the ESKAPE pathogens, Acinetobacter baumannii and Klebsiella pneumoniae, we cloned, expressed, purified, and characterized IspC from these two Gram-negative bacteria. Enzyme inhibition assays using IspC from these two pathogens, and compounds fosmidomycin and FR900098, indicate IC50 values ranging from 19.5-45.5 nM. Antimicrobial susceptibility tests with these inhibitors reveal that A. baumannii is susceptible to FR900098, whereas K. pneumoniae is susceptible to both compounds. Finally, to facilitate structure-based drug design of inhibitors targeting A. baumannii IspC, we determined the 2.5 Å crystal structure of IspC from A. baumannii in complex with inhibitor FR900098, and cofactors NADPH and magnesium.

Mechanism of Action of N-Acyl and N-Alkoxy Fosmidomycin Analogs: Mono- and Bisubstrate Inhibition of IspC from Plasmodium falciparum, a Causative Agent of Malaria.

Malaria is a global health threat that requires immediate attention. Malaria is caused by the protozoan parasite Plasmodium, the most severe form of which is Plasmodium falciparum. The methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis is essential to the survival of many human pathogens, including P. falciparum, but is absent in humans, and thus shows promise as a new antimalarial drug target. The enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) catalyzes the first committed step in the MEP pathway. In addition to a divalent cation (Mg2+), the enzyme requires the substrates 1-deoxy-D-xylulose 5-phosphate (DXP) and NADPH to catalyze its reaction. We designed N-alkoxy and N-acyl fosmidomycin analogs to inhibit the activity of P. falciparum IspC in a bisubstrate manner. Enzyme assays reveal that the N-alkoxy fosmidomycin analogs have a competitive mode of inhibition relative to both the DXP- and NADPH-binding sites, confirming a bisubstrate mode of inhibition. In contrast, the N-acyl fosmidomycin analogs demonstrate competitive inhibition with respect to DXP but uncompetitive inhibition with respect to NADPH, indicating monosubstrate inhibitory activity. Our results will have a positive impact on the discovery of novel antimalarial drugs.

Human hydroxymethylglutaryl-coenzyme A reductase (HMGCR) and statin sensitivity.

While statins, hydroxymethylglutaryl-coenzyme A reductase (HMGCR) inhibitors, are clinically proven to reduce plasma cholesterol levels, a wide variation in inter-individual response to statin therapy has been observed. Pharmacogenetic studies have identified multiple loci that potentially contribute towards the statin response, including the HMGCR gene. To examine, if a statin-resistant, catalytically-active isoform of the human HMGCR could be generated, we have rationally altered the protein to include additional residues in the flap domain, which has a role in statin binding. Comparative enzyme assays with purified wild-type and mutant isoforms reveal the alteration imposes a slight (38%) decrease in the K(app)(M) for the substrate, a near 2-fold increase in turnover number, and a 480% increase in the Ki for lovastatin. Thus, alterations in HMGCR could contribute towards the synergistic effects of multiple loci in the statin response.

VOC fingerprints: metabolomic signatures of biothreat agents with and without antibiotic resistance.

Category A and B biothreat agents are deemed to be of great concern by the US Centers for Disease Control and Prevention (CDC) and include the bacteria Francisella tularensis, Yersinia pestis, Burkholderia mallei, and Brucella species. Underscored by the impact of the 2020 SARS-CoV-2 outbreak, 2016 Zika pandemic, 2014 Ebola outbreak, 2001 anthrax letter attacks, and 1984 Rajneeshee Salmonella attacks, the threat of future epidemics/pandemics and/or terrorist/criminal use of pathogenic organisms warrants continued exploration and development of both classic and alternative methods of detecting biothreat agents. Volatile organic compounds (VOCs) comprise a large and highly diverse group of carbon-based molecules, generally related by their volatility at ambient temperature. Recently, the diagnostic potential of VOCs has been realized, as correlations between the microbial VOC metabolome and specific bacterial pathogens have been identified. Herein, we describe the use of microbial VOC profiles as fingerprints for the identification of biothreat-relevant microbes, and for differentiating between a kanamycin susceptible and resistant strain. Additionally, we demonstrate microbial VOC profiling using a rapid-throughput VOC metabolomics method we refer to as 'simultaneous multifiber headspace solid-phase microextraction' (simulti-hSPME). Finally, through VOC analysis, we illustrate a rapid non-invasive approach to the diagnosis of BALB/c mice infected with either F. tularensis SCHU S4 or Y. pestis CO92.

Inhibition of the Yersinia pestis Methylerythritol Phosphate Pathway of Isoprenoid Biosynthesis by α-Phenyl-Substituted Reverse Fosmidomycin Analogues.

Fosmidomycin inhibits IspC (1-deoxy-d-xylulose 5-phosphate reductoisomerase), the first committed enzyme in the methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis. The MEP pathway of isoprenoid biosynthesis is essential to the causative agent of the plague, Yersinia pestis, and is entirely distinct from the corresponding mammalian pathway. To further drug development, we established structure-activity relationships of fosmidomycin analogues by assessing a suite of 17 α-phenyl-substituted reverse derivatives of fosmidomycin against Y. pestis IspC. Several of these compounds showed increased potency over fosmidomycin with IC50 values in the nanomolar range. Additionally, we performed antimicrobial susceptibility testing with Y. pestis A1122 (YpA1122). The bacteria were susceptible to several compounds with minimal inhibitory concentration (MIC) values ranging from 128 to 512 µg/mL; a correlation between the IC50 and MIC values was observed.

Induction of nerve growth factor by phorbol 12-myristate 13-acetate is dependent upon the mitogen activated protein kinase pathway.

Several small molecules have been identified that induce glial cells to synthesize and secrete nerve growth factor (NGF), a critical neurotrophin that supports neuronal growth and survival, and as such show promise in the development of drugs for the chemoprevention of Alzheimer's disease. To map the signal transduction cascade leading to NGF synthesis and secretion, cultured human glial cells were stimulated by phorbol 12-myristate 13-acetate (PMA), an agonist of Protein Kinase C. Changes in intracellular protein phosphorylation states were evaluated by reverse phase protein microarrays (RPPA), selectively screening over 130 protein endpoints. Of these, 55 proteins showed statistically significant changes in phosphorylation state due to cellular exposure to PMA. A critical signal transduction pathway was identified, and subsequent validation by ELISA and qPCR revealed that the signaling proteins Raf, MEK, ERK, and the signal transduction factor CREB are all essential to the upregulation of NGF gene expression by PMA. Additionally, members of the RSK family of kinases appear to be involved in glial secretion (exocytosis) of the NGF protein. Furthermore, through RPPA, the effects of PMA on apoptosis signaling events and cell proliferation were differentiated from the pathway to NGF upregulation. Overall, this study reveals potential protein targets for the rational design of Alzheimer's therapeutics.

MEPicides: α,ß-Unsaturated Fosmidomycin Analogues as DXR Inhibitors against Malaria.

Severe malaria due to Plasmodium falciparum remains a significant global health threat. DXR, the second enzyme in the MEP pathway, plays an important role to synthesize building blocks for isoprenoids. This enzyme is a promising drug target for malaria due to its essentiality as well as its absence in humans. In this study, we designed and synthesized a series of α,ß-unsaturated analogues of fosmidomycin, a natural product that inhibits DXR in P. falciparum. All compounds were evaluated as inhibitors of P. falciparum. The most promising compound, 18a, displays on-target, potent inhibition against the growth of P. falciparum (IC50 = 13 nM) without significant inhibition of HepG2 cells (IC50 > 50 µM). 18a was also tested in a luciferase-based Plasmodium berghei mouse model of malaria and showed exceptional in vivo efficacy. Together, the data support MEPicide 18a as a novel, potent, and promising drug candidate for the treatment of malaria.

Structure-Activity Relationships of the MEPicides: N-Acyl and O-Linked Analogs of FR900098 as Inhibitors of Dxr from Mycobacterium tuberculosis and Yersinia pestis.

Despite continued research efforts, the threat of drug resistance from a variety of bacteria continues to plague clinical communities. Discovery and validation of novel biochemical targets will facilitate development of new drugs to combat these organisms. The methylerythritol phosphate (MEP) pathway to make isoprene units is a biosynthetic pathway essential to many bacteria. We and others have explored inhibitors of the MEP pathway as novel antibacterial agents. Mycobacterium tuberculosis, the causative agent of tuberculosis, and Yersinia pestis, resulting in the plague or "black death", both rely on the MEP pathway for isoprene production. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (Dxr) catalyzes the first committed step in the MEP pathway. We examined two series of Dxr inhibitors based on the parent structure of the retrohydroxamate natural product FR900098. The compounds contain either an extended N-acyl or O-linked alkyl/aryl group and are designed to act as bisubstrate inhibitors of the enzyme. While nearly all of the compounds inhibited both Mtb and Yp Dxr to some extent, compounds generally displayed more potent inhibition against the Yp homologue, with the best analogs displaying nanomolar IC50 values. In bacterial growth inhibition assays, the phosphonic acids generally resulted in poor antibacterial activity, likely a reflection of inadequate permeability. Accordingly, diethyl and dipivaloyloxymethyl (POM) prodrug esters of these compounds were made. While the added lipophilicity did not enhance Yersinia activity, the compounds showed significantly improved antitubercular activities. The most potent compounds have Mtb MIC values of 3-12 µg/mL. Taken together, we have uncovered two series of analogs that potently inhibit Dxr homologues from Mtb and Yp. These inhibitors of the MEP pathway, termed MEPicides, serve as leads for future analog development.

Alcohol induced alterations to the human fecal VOC metabolome.

Studies have shown that excessive alcohol consumption impacts the intestinal microbiota composition, causing disruption of homeostasis (dysbiosis). However, this observed change is not indicative of the dysbiotic intestinal microbiota function that could result in the production of injurious and toxic products. Thus, knowledge of the effects of alcohol on the intestinal microbiota function and their metabolites is warranted, in order to better understand the role of the intestinal microbiota in alcohol associated organ failure. Here, we report the results of a differential metabolomic analysis comparing volatile organic compounds (VOC) detected in the stool of alcoholics and non-alcoholic healthy controls. We performed the analysis with fecal samples collected after passage as well as with samples collected directly from the sigmoid lumen. Regardless of the approach to fecal collection, we found a stool VOC metabolomic signature in alcoholics that is different from healthy controls. The most notable metabolite alterations in the alcoholic samples include: (1) an elevation in the oxidative stress biomarker tetradecane; (2) a decrease in five fatty alcohols with anti-oxidant property; (3) a decrease in the short chain fatty acids propionate and isobutyrate, important in maintaining intestinal epithelial cell health and barrier integrity; (4) a decrease in alcohol consumption natural suppressant caryophyllene; (5) a decrease in natural product and hepatic steatosis attenuator camphene; and (6) decreased dimethyl disulfide and dimethyl trisulfide, microbial products of decomposition. Our results showed that intestinal microbiota function is altered in alcoholics which might promote alcohol associated pathologies.

Synthesis and bioactivity of ß-substituted fosmidomycin analogues targeting 1-deoxy-D-xylulose-5-phosphate reductoisomerase.

Blocking the 2-C-methyl-d-erythrithol-4-phosphate (MEP) pathway for isoprenoid biosynthesis offers interesting prospects for inhibiting Plasmodium or Mycobacterium spp. growth. Fosmidomycin (1) and its homologue FR900098 (2) potently inhibit 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this pathway. Here we introduced aryl or aralkyl substituents at the ß-position of the hydroxamate analogue of 2. While direct addition of a ß-aryl moiety resulted in poor inhibition, longer linkers between the carbon backbone and the phenyl ring were generally associated with better binding to the enzymes. X-ray structures of the parasite Dxr-inhibitor complexes show that the "longer" compounds generate a substantially different flap structure, in which a key tryptophan residue is displaced, and the aromatic group of the ligand lies between the tryptophan and the hydroxamate's methyl group. Although the most promising new Dxr inhibitors lack activity against Escherichia coli and Mycobacterium smegmatis, they proved to be highly potent inhibitors of Plasmodium falciparum in vitro growth.

Design, synthesis, and biological evaluation of biotin conjugates of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid for the isolation of the protein targets.

2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO, 1) and related compounds [for example, CDDO-Me (2) and CDDO-Im (3)] are potential anti-inflammatory, cancer chemopreventive, and chemotherapeutic agents. However, the mechanisms responsible for the multiple effects of CDDO are still unclear. Clarification of these mechanisms and particularly isolation of the protein targets are essential for the development of CDDO and its analogues as clinically useful drugs. Such knowledge would provide superior opportunities for designing new compounds with improved potency and selectivity. Therefore, to isolate protein targets using affinity chromatography with immobilized streptavidin as a carrier, we have designed and synthesized C-17 and C-23 biotin conjugates of CDDO (4, 5, and 6) on the basis of our established structure-activity relationships. For the synthesis of 6, a new important precursor, 23-hydroxy-CDDO-Me (29) was synthesized from 20 by a C-23 oxidation protocol, which involves cyclopalladation of the C-4 methyl group from a 3-one oxime. The inhibitory activity of C-23 conjugate 6 is only about 3 times less potent than the mother compound, CDDO, against the proliferation of MCF-7 breast cancer cells. Consequently, 6 may be a very promising tool for the isolation of the protein targets of CDDO.