Evolution of a 4-Benzyloxy-benzylamino Chemotype to Provide Efficacious, Potent, and Isoform Selective PPARα Agonists as Leads for Retinal Disorders.

Peroxisome proliferator-activated receptor alpha (PPARα) is expressed in retinal Müller cells, endothelial cells, and in retinal pigment epithelium; agonism of PPARα with genetic or pharmacological tools ameliorates inflammation, vascular leakage, neurodegeneration, and neovascularization associated with retinal diseases in animal models. As such, PPARα is a promising drug target for diabetic retinopathy and age-related macular degeneration. Herein, we report proof-of-concept in vivo efficacy in an streptozotocin-induced vascular leakage model (rat) and preliminary pharmacokinetic assessment of a first-generation lead 4a (A91). Additionally, we present the design, synthesis, and evaluation of second-generation analogues, which led to the discovery of 4u and related compounds that reach cellular potencies <50 nM and exhibit >2,700-fold selectivity for PPARα over other PPAR isoforms. These studies identify a pipeline of candidates positioned for detailed PK/PD and pre-clinical evaluation.

Modified 2,4-diaminopyrimidine-based dihydrofolate reductase inhibitors as potential drug scaffolds against Bacillus anthracis.

The current Letter describes the synthesis and biological evaluation of dihydrophthalazine-appended 2,4-diaminopyrimidine (DAP) inhibitors (1) oxidized at the methylene bridge linking the DAP ring to the central aromatic ring and (2) modified at the central ring ether groups. Structures 4a-b incorporating an oxidized methylene bridge showed a decrease in activity, while slightly larger alkyl groups (CH2CH3 vs CH3) on the central ring oxygen atoms (R(2) and R(3)) had a minimal impact on the inhibition. Comparison of the potency data for previously reported RAB1 and BN-53 with the most potent of the new derivatives (19 b and 20a-b) showed similar values for inhibition of cellular growth and direct enzymatic inhibition (MICs 0.5-2 µg/mL). Compounds 29-34 with larger ester and ether groups containing substituted aromatic rings at R(3) exhibited slightly reduced activity (MICs 2-16 µg/mL). One explanation for this attenuated activity could be encroachment of the extended R(3) into the neighboring NADPH co-factor. These results indicate that modest additions to the central ring oxygen atoms are well tolerated, while larger modifications have the potential to act as dual-site inhibitors of dihydrofolate reductase (DHFR).

Synthesis and biological evaluation of 2,4-diaminopyrimidine-based antifolate drugs against Bacillus anthracis.

Due to the innate ability of bacteria to develop resistance to available antibiotics, there is a critical need to develop new agents to treat more resilient strains. As a continuation of our research in this area, we have synthesized a series of racemic 2,4-diaminopyrimidine-based drug candidates, and evaluated them against Bacillus anthracis. The structures are comprised of a 2,4-diaminopyrimidine ring, a 3,4-dimethoxybenzyl ring, and an N-acryloyl-substituted 1,2-dihydrophthalazine ring. Various changes were made at the C1 stereocenter of the dihydrophthalazine moiety in the structure, and the biological activity was assessed by measurement of the MIC and K(i) values to identify the most potent drug candidate.

Structure-activity relationship for enantiomers of potent inhibitors of B. anthracis dihydrofolate reductase.

BACKGROUND: Bacterial resistance to antibiotic therapies is increasing and new treatment options are badly needed. There is an overlap between these resistant bacteria and organisms classified as likely bioterror weapons. For example, Bacillus anthracis is innately resistant to the anti-folate trimethoprim due to sequence changes found in the dihydrofolate reductase enzyme. Development of new inhibitors provides an opportunity to enhance the current arsenal of anti-folate antibiotics while also expanding the coverage of the anti-folate class. METHODS: We have characterized inhibitors of B. anthracis dihydrofolate reductase by measuring the K(i) and MIC values and calculating the energetics of binding. This series contains a core diaminopyrimidine ring, a central dimethoxybenzyl ring, and a dihydrophthalazine moiety. We have altered the chemical groups extended from a chiral center on the dihydropyridazine ring of the phthalazine moiety. The interactions for the most potent compounds were visualized by X-ray structure determination. RESULTS: We find that the potency of individual enantiomers is divergent with clear preference for the S-enantiomer, while maintaining a high conservation of contacts within the binding site. The preference for enantiomers seems to be predicated largely by differential interactions with protein residues Leu29, Gln30 and Arg53. CONCLUSIONS: These studies have clarified the activity of modifications and of individual enantiomers, and highlighted the role of the less-active R-enantiomer in effectively diluting the more active S-enantiomer in racemic solutions. This directly contributes to the development of new antimicrobials, combating trimethoprim resistance, and treatment options for potential bioterrorism agents.

Inhibition of bacterial dihydrofolate reductase by 6-alkyl-2,4-diaminopyrimidines.

(±)-6-Alkyl-2,4-diaminopyrimidine-based inhibitors of bacterial dihydrofolate reductase (DHFR) have been prepared and evaluated for biological potency against Bacillus anthracis and Staphylococcus aureus. Biological studies revealed attenuated activity relative to earlier structures lacking substitution at C6 of the diaminopyrimidine moiety, though minimum inhibitory concentration (MIC) values are in the 0.125-8 µg mL(-1) range for both organisms. This effect was rationalized from three- dimensional X-ray structure studies that indicate the presence of a side pocket containing two water molecules adjacent to the main binding pocket. Because of the hydrophobic nature of the substitutions at C6, the main interactions are with protein residues Leu 20 and Leu 28. These interactions lead to a minor conformational change in the protein, which opens the pocket containing these water molecules such that it becomes continuous with the main binding pocket. These water molecules are reported to play a critical role in the catalytic reaction, highlighting a new area for inhibitor expansion within the limited architectural variation at the catalytic site of bacterial DHFR.

High-throughput screening of a diversity collection using biodefense category A and B priority pathogens.

One of the objectives of the National Institutes of Allergy and Infectious Diseases (NIAID) Biodefense Program is to identify or develop broad-spectrum antimicrobials for use against bioterrorism pathogens and emerging infectious agents. As a part of that program, our institution has screened the 10 000-compound MyriaScreen Diversity Collection of high-purity druglike compounds against three NIAID category A and one category B priority pathogens in an effort to identify potential compound classes for further drug development. The effective use of a Clinical and Laboratory Standards Institute-based high-throughput screening (HTS) 96-well-based format allowed for the identification of 49 compounds that had in vitro activity against all four pathogens with minimum inhibitory concentration values of ≤16 µg/mL. Adaptation of the HTS process was necessary to conduct the work in higher-level containment, in this case, biosafety level 3. Examination of chemical scaffolds shared by some of the 49 compounds and assessment of available chemical databases indicates that several may represent broad-spectrum antimicrobials whose activity is based on novel mechanisms of action.

Synthesis and biological activity of substituted 2,4-diaminopyrimidines that inhibit Bacillus anthracis.

A series of substituted 2,4-diaminopyrimidines 1 has been prepared and evaluated for activity against Bacillus anthracis using previously reported (±)-3-{5-[(2,4-diamino-5-pyrimidinyl)methyl]-2,3-dimethoxyphenyl}-1-(1-propyl-2(1H)-phthalazinyl)-2-propen-1-one (1a), with a minimum inhibitory concentration (MIC) value of 1-3 µg/mL, as the standard. In the current work, the corresponding isobutenyl (1e) and phenyl (1h) derivatives displayed the most significant activity in terms of the lowest MICs with values of 0.5 µg/mL and 0.375-1.5 µg/mL, respectively. It is likely that the S isomers of 1 will bind the substrate-binding pocket of dihydrofolate reductase (DHFR) as in B. anthracis was found for (S)-1a. The final step in the convergent synthesis of target systems 1 from (±)-1-(1-substituted-2(1H)-phthalazinyl)-2-propen-1-ones 6 with 2,4-diamino-5-(5-iodo-3,4-dimethoxybenzyl)pyrimidine (13) was accomplished via a novel Heck coupling reaction under sealed-tube conditions.

Crystal structure of Bacillus anthracis dihydrofolate reductase with the dihydrophthalazine-based trimethoprim derivative RAB1 provides a structural explanation of potency and selectivity.

Bacillus anthracis possesses an innate resistance to the antibiotic trimethoprim due to poor binding to dihydrofolate reductase (DHFR); currently, there are no commercial antibacterials that target this enzyme in B. anthracis. We have previously reported a series of dihydrophthalazine-based trimethoprim derivatives that are inhibitors for this target. In the present work, we have synthesized one compound (RAB1) displaying favorable 50% inhibitory concentration (54 nM) and MIC (< or =12.8 microg/ml) values. RAB1 was cocrystallized with the B. anthracis DHFR in the space group P2(1)2(1)2(1), and X-ray diffraction data were collected to a 2.3-A resolution. Binding of RAB1 causes a conformational change of the side chain of Arg58 and Met37 to accommodate the dihydrophthalazine moiety. Unlike the natural substrate or trimethoprim, the dihydrophthalazine group provides a large hydrophobic anchor that embeds within the DHFR active site and accounts for its selective inhibitory activity against B. anthracis.

In vitro efficacy of new antifolates against trimethoprim-resistant Bacillus anthracis.

Bacillus anthracis is innately resistant to trimethoprim (TMP), a synthetic antifolate that selectively inhibits several bacterial dihydrofolate reductases (DHFRs) but not human DHFR. Previously, we were able to confirm that TMP resistance in B. anthracis (MIC > 2,048 microg/ml) is due to the lack of selectivity of TMP for the B. anthracis DHFR (E. W. Barrow, P. C. Bourne, and W. W. Barrow, Antimicrob. Agents Chemother. 48:4643-4649, 2004). In this investigation, 24 2,4-diaminopyrimidine derivatives, representing a class of compounds with dihydrophthalazine side chains, were screened for their in vitro effects on B. anthracis Sterne and their selectivities for the B. anthracis DHFR. MICs were obtained by a colorimetric (Alamar blue) broth microdilution assay. Purified human recombinant DHFR (rDHFR) and B. anthracis rDHFR were used in a validated enzyme assay to determine the 50% inhibitory concentrations (IC(50)s) and the selectivity ratios of the derivatives. The MICs ranged from 12.8 to 128 microg/ml for all but nine compounds, for which the MICs were > or =128 microg/ml. The IC(50) values for B. anthracis rDHFR ranged from 46 to 600 nM, whereas the IC(50) values for human rDHFR were >16,000 nM. This is the first report on the in vitro inhibitory actions of this class of antifolates against TMP-resistant B. anthracis isolates. The selective inhibition of B. anthracis rDHFR and the in vitro activity against B. anthracis demonstrate that members of this class of compounds have the potential to be developed into clinically important therapeutic choices for the treatment of infections caused by TMP-resistant bacteria, such as B. anthracis.

Genetic basis for sulfonamide resistance in Bacillus anthracis.

Natural resistance of field strains of Bacillus anthracis to drugs from the sulfonamide class of antimicrobials that act by inhibiting dihydropteroate synthase (DHPS) has been reported. Though the structure of B. anthracis DHPS has been determined, its connection to the apparent intrinsic sulfonamide resistance of the bacterium has not been established. The aim of this study was to determine if a connection exists between DHPS and the observed sulfonamide resistance of B. anthracis. Microdilution broth assays verified that B. anthracis Sterne is highly resistant to a variety of sulfonamides with minimum inhibitory concentrations (MICs) exceeding 1250 microg/ml. A putative gene encoding DHPS (folP) was amplified from B. anthracis Sterne chromosomal DNA by polymerase chain reaction (PCR) and cloned. Sequence comparisons showed 100% identity with DHPSs from published genome sequences for various strains of B. anthracis. Additionally, expression of folP in B. anthracis Sterne was confirmed. Functionality of the B. anthracis DHPS was confirmed by complementation of an Escherichia coli folP deletion mutant as well as a standard enzyme assay. Concomitant transfer of high level sulfonamide resistance to this mutant along with increased sulfonamide IC(50)values for purified B. anthracis DHPS links DHPS to sulfonamide resistance in B. anthracis. These findings lay the groundwork that will aid future development of antimicrobics that target DHPS to treat anthrax infections.

Functional cloning of Bacillus anthracis dihydrofolate reductase and confirmation of natural resistance to trimethoprim.

Bacillus anthracis is reported to be naturally resistant to trimethoprim (TMP), a drug that inhibits dihydrofolate reductase (DHFR), a key enzyme in the folate pathway. A microdilution broth assay established that the MIC of TMP for B. anthracis Sterne is >2,048 but < or =4,096 microg/ml. A putative DHFR sequence was amplified from B. anthracis Sterne genomic DNA. The PCR product was cloned into the Invitrogen pCRT7/CT-TOPO vector, followed by transformation into Escherichia coli TOP10F' chemically competent cells. Plasmid DNA from a clone showing the correct construct with a thrombin cleavage site attached downstream from the terminus of the cloned PCR product was transformed into E. coli BL21 Star (DE3)pLysS competent cells for expression of the six-histidine-tagged fusion protein and purification on a His-Bind resin column. Functionality of the purified Sterne recombinant DHFR (Sterne rDHFR) was confirmed in an established enzyme assay. The 50% inhibitory concentrations of TMP and methotrexate for the Sterne rDHFR were found to be 77,233 and 12.2 nM, respectively. TMP resistance was observed with E. coli BL21 Star (DE3)pLysS competent cells transformed with the Sterne DHFR gene. Alignment of the amino acid sequence of the Sterne DHFR gene revealed 100% homology with various virulent strains of B. anthracis. These results confirm the natural resistance of B. anthracis to TMP and clarify that the resistance is correlated to a lack of selectivity for the chromosomally encoded gene product. These findings will assist in the development of narrow-spectrum antimicrobial agents for treatment of anthrax.

Three-dimensional structures of a humanized anti-IFN-gamma Fab (HuZAF) in two crystal forms.

Three-dimensional structures were determined for two crystal forms (orthorhombic P2(1)2(1)2(1) and monoclinic C2) of the Fab from the humanized version of a murine monoclonal antibody (AF2) that possesses binding and potent neutralizing activity against human interferon gamma (IFN-gamma). This humanized antibody (HuZAF; USAN name fontolizumab) is currently in phase II clinical trials for the treatment of Crohn's disease. HuZAF exhibits binding and IFN-gamma neutralizing capacities that closely approximate those of the original antibody. It is shown that HuZAF, whose VH domain was designed using a best-sequence-fit approach, is closer structurally to its mouse precursor than is a version whose VH was constructed using a human sequence with lower homology to the original mouse sequence. This work thus offers direct structural evidence in support of the best-sequence-fit approach and adds to previous results of biological and biochemical evaluations of distinctly engineered antibodies that also favored the use of a best-sequence-fit strategy. A second crystal type appeared during attempts to crystallize the Fab-IFN-gamma complex. The antibody-antigen complex that existed in solution dissociated in the crystallization mixture. A conformationally altered but unliganded HuZAF protein crystallized in a different space group (C2), with two Fab molecules in the asymmetric unit. In this crystal lattice, no space was available for accommodating the IFN-gamma antigen. Thus, there are currently three slightly different structures of the HuZAF Fab.

Comparison of the three-dimensional structures of a human Bence-Jones dimer crystallized on Earth and aboard US Space Shuttle Mission STS-95.

Crystals of a human (Sea) Bence-Jones dimer were produced in a capillary by vapor diffusion under microgravity conditions in the 9 day US Space Shuttle Mission STS-95. In comparison to ground-based experiments, nucleation was facile and spontaneous in space. Appearance of a very large (8 x 1.6 x 1.0 mm) crystal in a short time period is a strong endorsement for the use of microgravity to produce crystals sufficiently large for neutron diffraction studies. The Sea dimer crystallized in the orthorhombic space group P2(1)2(1)2(1), with a = 48.9 A, b = 85.2 A, and c = 114.0 A. The crystals grown in microgravity exhibited significantly lower mosaicities than those of ground-based crystals and the X-ray diffraction data had a lower overall B factor. Three-dimensional structures determined by X-ray analysis at two temperatures (100 and 293 K) were indistinguishable from those obtained from ground-based crystals. However, both the crystallographic R factor and the free R factor were slightly lower in the models derived from crystals produced in microgravity. The major difference between the two crystal growth systems is a lack of convection and sedimentation in a microgravity environment. This environment resulted in the growth of much larger, higher-quality crystals of the Sea Bence-Jones protein. Structurally, heretofore unrecognized grooves on the external surfaces of the Sea and other immunoglobulin-derived fragments are regular features and may offer supplementary binding regions for super antigens and other elongated ligands in the bloodstream and perivascular tissues.

Three-dimensional structure of an immunoglobulin light-chain dimer with amyloidogenic properties.

The X-ray structure of an immunoglobulin light-chain dimer isolated from the urine as a "Bence-Jones protein" from a patient with multiple myeloma and amyloidosis (Sea) was determined at 1.94 A resolution and refined to R and R(free) factors of 0.22 and 0.25, respectively. This "amyloidogenic" protein crystallized in the orthorhombic P2(1)2(1)2(1) space group with unit-cell parameters a = 48.28, b = 83.32, c = 112.59 A as determined at 100 K. In the vital organs (heart and kidneys), the equivalent of the urinary protein produced fibrillar amyloid deposits which were fatal to the patient. Compared with the amyloidogenic Mcg light-chain dimer, the Sea protein was highly soluble in aqueous solutions and only crystallized at concentrations approaching 100 mg ml(-1). Both the Sea and Mcg proteins packed into crystals in highly ordered arrangements typical of strongly diffracting crystals of immunoglobulin fragments. Overall similarities and significant differences in the three-dimensional structures and crystalline properties are discussed for the Sea and Mcg Bence-Jones proteins, which together provide a generalized model of abnormalities present in lambda chains, facilitating a better understanding of amyloidosis of light-chain origin (AL).