Structure-guided functional studies of plasmid-encoded dihydrofolate reductases reveal a common mechanism of trimethoprim resistance in Gram-negative pathogens.

Two plasmid-encoded dihydrofolate reductase (DHFR) isoforms, DfrA1 and DfrA5, that give rise to high levels of resistance in Gram-negative bacteria were structurally and biochemically characterized to reveal the mechanism of TMP resistance and to support phylogenic groupings for drug development against antibiotic resistant pathogens. Preliminary screening of novel antifolates revealed related chemotypes that showed high levels of inhibitory potency against Escherichia coli chromosomal DHFR (EcDHFR), DfrA1, and DfrA5. Kinetics and biophysical analysis, coupled with crystal structures of trimethoprim bound to EcDHFR, DfrA1 and DfrA5, and two propargyl-linked antifolates (PLA) complexed with EcDHFR, DfrA1 and DfrA5, were determined to define structural features of the substrate binding pocket and guide synthesis of pan-DHFR inhibitors.

Toward Broad Spectrum Dihydrofolate Reductase Inhibitors Targeting Trimethoprim Resistant Enzymes Identified in Clinical Isolates of Methicillin Resistant Staphylococcus aureus.

The spread of plasmid borne resistance enzymes in clinical Staphylococcus aureus isolates is rendering trimethoprim and iclaprim, both inhibitors of dihydrofolate reductase (DHFR), ineffective. Continued exploitation of these targets will require compounds that can broadly inhibit these resistance-conferring isoforms. Using a structure-based approach, we have developed a novel class of ionized nonclassical antifolates (INCAs) that capture the molecular interactions that have been exclusive to classical antifolates. These modifications allow for a greatly expanded spectrum of activity across these pathogenic DHFR isoforms, while maintaining the ability to penetrate the bacterial cell wall. Using biochemical, structural, and computational methods, we are able to optimize these inhibitors to the conserved active sites of the endogenous and trimethoprim resistant DHFR enzymes. Here, we report a series of INCA compounds that exhibit low nanomolar enzymatic activity and potent cellular activity with human selectivity against a panel of clinically relevant TMP resistant (TMPR) and methicillin resistant Staphylococcus aureus (MRSA) isolates.

Drugging the Folate Pathway in Mycobacterium tuberculosis: The Role of Multi-targeting Agents.

The folate biosynthetic pathway offers many druggable targets that have yet to be exploited in tuberculosis therapy. Herein, we have identified a series of small molecules that interrupt Mycobacterium tuberculosis (Mtb) folate metabolism by dual targeting of dihydrofolate reductase (DHFR), a key enzyme in the folate pathway, and its functional analog, Rv2671. We have also compared the antifolate activity of these compounds with that of para-aminosalicylic acid (PAS). We found that the bioactive metabolite of PAS, in addition to previously reported activity against DHFR, inhibits flavin-dependent thymidylate synthase in Mtb, suggesting a multi-targeted mechanism of action for this drug. Finally, we have shown that antifolate treatment in Mtb decreases the production of mycolic acids, most likely due to perturbation of the activated methyl cycle. We conclude that multi-targeting of the folate pathway in Mtb is associated with highly potent anti-mycobacterial activity.

Harnessing Free Energy From Nature For Efficient Operation of Compressed Air Energy Storage System and Unlocking the Potential of Renewable Power Generation.

Energy storage technologies have gained considerable momentum in the recent years owing to the rising tide of renewables. The deployment of energy storage is a trend set to continue into 2018 and beyond. In the near future, compressed air energy storage (CAES) will serve as an integral component of several energy intensive sectors. However, the major drawback in promoting CAES system in both large and small scale is owing to its minimum turn around efficiency. In the present work the major drawbacks associated with various existing configurations of CAES system are analysed. Interesting results of Isothermal CAES system are obtained through the present analysis to generate additional output energy compared to the supplied input by harnessing the free energy from the natural water bodies/ocean to enhance the overall turnaround efficiency of the system. The optimum operational characteristics of charging and discharging cycles are also addressed. In the present energy scenario, increasing the percentage of renewable energy (RE) share in the power generation is quite challenging since RE based power generation is intermittent in nature. The integration of energy storage technologies with RE source is imperative as it mitigates the intermittency of available energy. However, the development of efficient energy storage systems is one of the prime challenges in the promotion of renewable energy in a large scale. Among the various storage systems, electrochemical battery storage and pumped hydro storage (PHS) have attracted the commercial market. However, the shorter cycle life makes the battery storage more expensive and the PHS systems involves certain geographical and site constraints. Beyond the said storage systems, compressed air energy storage system which is one of the technically proven system has not been targeted the commercial market owing to its lower turnaround efficiency. Hence, the motivation behind the present research is towards developing efficient CAES configuration with higher turnaround efficiency thereby attaining economic feasibility and sustainability.

Synthesis and Pharmacokinetic Evaluation of Siderophore Biosynthesis Inhibitors for Mycobacterium tuberculosis.

MbtA catalyzes the first committed biosynthetic step of the mycobactins, which are important virulence factors associated with iron acquisition in Mycobacterium tuberculosis. MbtA is a validated therapeutic target for antitubercular drug development. 5'-O-[N-(Salicyl)sulfamoyl]adenosine (1) is a bisubstrate inhibitor of MbtA and exhibits exceptionally potent biochemical and antitubercular activity. However, 1 suffers from suboptimal drug disposition properties resulting in a short half-life (t(1/2)), low exposure (AUC), and low bioavailability (F). Four strategies were pursued to address these liabilities including the synthesis of prodrugs, increasing the pK(a) of the acyl-sulfonyl moiety, modulation of the lipophilicity, and strategic introduction of fluorine into 1. Complete pharmacokinetic (PK) analysis of all compounds was performed. The most successful modifications involved fluorination of the nucleoside that provided substantial improvements in t(1/2) and AUC. Increasing the pK(a) of the acyl-sulfonyl linker yielded incremental enhancements, while modulation of the lipophilicity and prodrug approaches led to substantially poorer PK parameters.

Investigation and conformational analysis of fluorinated nucleoside antibiotics targeting siderophore biosynthesis.

Antibiotic resistance represents one of the greatest threats to public health. The adenylation inhibitor 5'-O-[N-(salicyl)sulfamoyl]adenosine (SAL-AMS) is the archetype for a new class of nucleoside antibiotics that target iron acquisition in pathogenic microorganisms and is especially effective against Mycobacterium tuberculosis, the causative agent of tuberculosis. Strategic incorporation of fluorine at the 2' and 3' positions of the nucleoside was performed by direct fluorination to enhance activity and improve drug disposition properties. The resulting SAL-AMS analogues were comprehensively assessed for biochemical potency, whole-cell antitubercular activity, and in vivo pharmacokinetic parameters. Conformational analysis suggested a strong preference of fluorinated sugar rings for either a 2'-endo, 3'-exo (South), or a 3'-endo,2'-exo (North) conformation. The structure-activity relationships revealed a strong conformational bias for the C3'-endo conformation to maintain potent biochemical and whole-cell activity, whereas improved pharmacokinetic properties were associated with the C2'-endo conformation.

Propargyl-linked antifolates are dual inhibitors of Candida albicans and Candida glabrata.

Species of Candida, primarily C. albicans and with increasing prevalence, C. glabrata, are responsible for the majority of fungal bloodstream infections that cause morbidity, especially among immune compromised patients. While the development of new antifungal agents that target the essential enzyme, dihydrofolate reductase (DHFR), in both Candida species would be ideal, previous attempts have resulted in antifolates that exhibit inconsistencies between enzyme inhibition and antifungal properties. In this article, we describe the evaluation of pairs of propargyl-linked antifolates that possess similar physicochemical properties but different shapes. All of these compounds are effective at inhibiting the fungal enzymes and the growth of C. glabrata; however, the inhibition of the growth of C. albicans is shape-dependent with extended para-linked compounds proving more effective than compact, meta-linked compounds. Using crystal structures of DHFR from C. albicans and C. glabrata bound to lead compounds, 13 new para-linked compounds designed to inhibit both species were synthesized. Eight of these compounds potently inhibit the growth of both fungal species with three compounds displaying dual MIC values less than 1 µg/mL. Analysis of the active compounds shows that shape and distribution of polar functionality is critical in achieving dual antifungal activity.

Structural analysis of the active sites of dihydrofolate reductase from two species of Candida uncovers ligand-induced conformational changes shared among species.

A novel strategy for targeting the pathogenic organisms Candida albicans and Candida glabrata focuses on the development of potent and selective antifolates effective against dihydrofolate reductase. Crystal structure analysis suggested that an essential loop at the active site (Thr 58-Phe 66) differs from the analogous residues in the human enzyme, potentially providing a mechanism for achieving selectivity. In order to probe the role of this loop, we employed chemical synthesis, crystal structure determination and molecular dynamics simulations. The results of these analyses show that the loop residues undergo ligand-induced conformational changes that are similar among the fungal and human species.

Viridin analogs derived from steroidal building blocks.

Naturally occurring furanosteroids such as viridin and wortmannin have long been known as potent inhibitors of the lipid kinase PI-3K. We have been interested in directly accessing analogs of these complex natural products from abundant steroid feedstock materials. In this communication, we describe the synthesis of viridin/wortmannin hybrid molecules from readily available building blocks that function as PI-3K inhibitors and maintain their electrophilic properties. The compounds also show anti-proliferative effects against a breast cancer line.

Acetylenic linkers in lead compounds: a study of the stability of the propargyl-linked antifolates.

Propargyl-linked antifolates that target dihydrofolate reductase are potent inhibitors of several species of pathogenic bacteria and fungi. This novel class of antifolates possesses a relatively uncommon acetylenic linker designed to span a narrow passage in the enzyme active site and join two larger functional domains. Because the use of alkyne functionality in drug molecules is limited, it was important to evaluate some key physicochemical properties of these molecules and specifically to assess the overall stability of the acetylene. Herein, we report studies on four compounds from our lead series that vary specifically in the environment of the alkyne. We show that the compounds are soluble, chemically stable in water, as well as simulated gastric and intestinal fluids with half-lives of approximately 30 min after incubation with mouse liver microsomes. Their primary in vitro route of metabolism involves oxidative transformations of pendant functionality with little direct alteration of the alkyne. Identification of several major metabolites indicated the formation of N-oxides; the rate of formation of these oxides was highly influenced by branching substitutions around the propargyl linker. On the basis of the lessons of these metabolic studies, a more advanced inhibitor was designed, synthesized, and shown to have increased (t(1/2) = 65 min) metabolic stability while maintaining potent enzyme inhibition.

Prospective screening of novel antibacterial inhibitors of dihydrofolate reductase for mutational resistance.

Resistance to trimethoprim (TMP) resulting from point mutations in the enzyme drug target dihydrofolate reductase (DHFR) drives the development of new antifolate inhibitors effective against methicillin-resistant Staphylococcus aureus (MRSA). For the past several years we have used structure-based design to create propargyl-linked antifolates that are highly potent antibacterial agents. In order to focus priority on the development of lead compounds with a low propensity to induce resistance, we prospectively evaluated resistance profiles for two of these inhibitors in an MRSA strain. By selection with the lead inhibitors, we generated resistant strains that contain single point mutations F98Y and H30N associated with TMP resistance and one novel mutation, F98I, in DHFR. Encouragingly, the pyridyl propargyl-linked inhibitor selects mutants at low frequency (6.85 × 10(-10) to 1.65 × 10(-9)) and maintains a low MIC (2.5 µg/ml) and a low mutant prevention concentration (1.25 µg/ml), strongly supporting its position as a lead compound. Results from this prospective screening method inform the continued design of antifolates effective against mutations at the Phe 98 position. Furthermore, the method can be used broadly to incorporate ideas for overcoming resistance early in the development process.

Toward new therapeutics for skin and soft tissue infections: propargyl-linked antifolates are potent inhibitors of MRSA and Streptococcus pyogenes.

Hospital- and community-acquired, complicated skin and soft tissue infections, often attributed to Staphylococcus aureus and Streptococcus pyogenes, present a significant health burden that is associated with increased health care costs and mortality. As these two species are difficult to discern on diagnosis and are associated with differential profiles of drug resistance, the development of an efficacious antibacterial agent that targets both organisms is a high priority. Herein we describe a structure-based drug development effort that has produced highly potent inhibitors of dihydrofolate reductase from both species. Optimized propargyl-linked antifolates containing a key pyridyl substituent display antibacterial activity against both methicillin-resistant S. aureus and S. pyogenes at MIC values below 0.1 µg/mL and minimal cytotoxicity against mammalian cells. Further evaluation against a panel of clinical isolates shows good efficacy against a range of important phenotypes such as hospital- and community-acquired strains as well as strains resistant to vancomycin.

Z-Phe-Ala-diazomethylketone (PADK) disrupts and remodels early oligomer states of the Alzheimer disease Aß42 protein.

The oligomerization of the amyloid-ß protein (Aß) is an important event in Alzheimer disease (AD) pathology. Developing small molecules that disrupt formation of early oligomeric states of Aß and thereby reduce the effective amount of toxic oligomers is a promising therapeutic strategy for AD. Here, mass spectrometry and ion mobility spectrometry were used to investigate the effects of a small molecule, Z-Phe-Ala-diazomethylketone (PADK), on the Aß42 form of the protein. The mass spectrum of a mixture of PADK and Aß42 clearly shows that PADK binds directly to Aß42 monomers and small oligomers. Ion mobility results indicate that PADK not only inhibits the formation of Aß42 dodecamers, but also removes preformed Aß42 dodecamers from the solution. Electron microscopy images show that PADK inhibits Aß42 fibril formation in the solution. These results are consistent with a previous study that found that PADK has protective effects in an AD transgenic mouse model. The study of PADK and Aß42 provides an example of small molecule therapeutic development for AD and other amyloid diseases.

Nonpeptidic lysosomal modulators derived from z-phe-ala-diazomethylketone for treating protein accumulation diseases.

Lysosomes are involved in protein turnover and removing misfolded species, and their enzymes have the potential to offset the defect in proteolytic clearance that contributes to the age-related dementia Alzheimer's disease (AD). The weak cathepsin B and L inhibitor Z-Phe-Ala-diazomethylketone (PADK) enhances lysosomal cathepsin levels at low concentrations, thereby eliciting protective clearance of PHF-τ and Aß42 in the hippocampus and other brain regions. Here, a class of positive modulators is established with compounds decoupled from the cathepsin inhibitory properties. We utilized PADK as a departure point to develop nonpeptidic structures with the hydroxyethyl isostere. The first-in-class modulators SD1002 and SD1003 exhibit improved levels of cathepsin up-regulation but almost complete removal of cathepsin inhibitory properties as compared to PADK. Isomers of the lead compound SD1002 were synthesized, and the modulatory activity was determined to be stereoselective. In addition, the lead compound was tested in transgenic mice with results indicating protection against AD-type protein accumulation pathology.