Can adoption of climate resilient management practices achieve carbon neutrality in traditional green revolution states of Punjab and Haryana?

Decreasing greenhouse gas (GHG) emissions and enhancing soil carbon (C) sequestration in cropland are necessary to achieve carbon neutrality at national scale. The major objective of this study is to quantify the GHG mitigation potential of adopted climate resilient (CR) practices in CR villages using Ex-ACT tool developed by Food and Agriculture Organization (FAO). Intensively cultivated area of Punjab and Haryana was selected for carrying out this study. In both the states, villages were selected by considering the climate for past 30 years. In the selected villages, a set of CR practices were implemented in annuals, perennials, irrigated rice, fertilizer use, land use change and livestock and quantified the GHG mitigation potential in these villages for next twenty years. The tool predicted that the CR practices adopted were successful in enhancing the overall sink (carbon balance) in all the study villages. The villages of Punjab had recorded higher mitigation potential as compared to the villages of Haryana. The overall sink potential in these villages ranged from -354 to -38309 Mg CO2-eq. The change in sink potential varied from 3.16 to 112% with lowest in Radauri and highest in Badhauchhi kalan village. The sink potential got doubled in Badhauchhi kalan village due to stopping rice straw burning and increase in area under perennials by 25%. The source potential varied from 6.33 to -7.44% across the study villages. Even with the implementation of NICRA, there was increase in source by 5.58 and 6.33% in Killi Nihal Singh Wala and Radauri due to irrigated rice, land use change and livestock. Majorly, rice straw burning was seen in most of the study villages, yet, with proper residue management and adoption of CR practices (mainly intermittent flooding) in rice cultivation resulted in emissions reduction up to 5-26% with enhanced productivity up to 15-18%, which can be considered for scaling up. Fertilizer management reduced the emissions by average of 13% across the study villages. Farm gate emission intensity per ton of milk and rice recorded highest emission intensity compared to annuals and perennials suggesting strict implementation of CR practices in rice cultivation and livestock sector. Implementation and scaling up of CR practices could potentially reduce the emissions and make the village C negative in intensive rice-wheat production system.

4-Hydroxy-2-pyridones: Discovery and evaluation of a novel class of antibacterial agents targeting DNA synthesis.

The continued emergence of bacteria resistant to current standard of care antibiotics presents a rapidly growing threat to public health. New chemical entities (NCEs) to treat these serious infections are desperately needed. Herein we report the discovery, synthesis, SAR and in vivo efficacy of a novel series of 4-hydroxy-2-pyridones exhibiting activity against Gram-negative pathogens. Compound 1c, derived from the N-debenzylation of 1b, preferentially inhibits bacterial DNA synthesis as determined by standard macromolecular synthesis assays. The structural features of the 4-hydroxy-2-pyridone scaffold required for antibacterial activity were explored and compound 6q, identified through further optimization of the series, had an MIC90 value of 8 µg/mL against a panel of highly resistant strains of E. coli. In a murine septicemia model, compound 6q exhibited a PD50 of 8 mg/kg in mice infected with a lethal dose of E. coli. This novel series of 4-hydroxy-2-pyridones serves as an excellent starting point for the identification of NCEs treating Gram-negative infections.

Soil bacterial community structure and functioning in a long-term conservation agriculture experiment under semi-arid rainfed production system.

Soil microbial communities are important drivers of biogeochemical cycling of nutrients, organic matter decomposition, soil organic carbon, and Greenhouse gas emissions (GHGs: CO2, N2O, and CH4) and are influenced by crop and soil management practices. The knowledge on the impact of conservation agriculture (CA) on soil bacterial diversity, nutrient availability, and GHG emissions in semi-arid regions under rainfed conditions is vital to develop sustainable agricultural practices, but such information has not been systemically documented. Hence, studies were conducted for 10 years in rainfed pigeonpea (Cajanus cajan L.)-castor bean (Ricinus communis L.) cropping system under semi-arid conditions to assess the effects of tillage and crop residue levels on the soil bacterial diversity, enzyme activity (Dehydrogenase, urease, acid phosphatase, and alkaline phosphatase), GHG emissions, and soil available nutrients (Nitrogen, phosphorus, and potassium). Sequencing of soil DNA through Illumina HiSeq-based 16S rRNA amplicon sequencing technology has revealed that bacterial community responded to both tillage and residue levels. The relative abundance of Actinobacteria in terms of Operational Taxonomic Unit (OTUs) at phyla, class as well as genera level was higher in CA (NTR1: No Tillage + 10 cm anchored residue and NTR2 NT + 30 cm anchored residue) over CT (conventional tillage without crop residues). CA resulted in higher enzyme activities (dehydrogenase, urease, acid phosphatase, and alkaline phosphatase) and reduction in GHG emissions over CT. CA recorded 34% higher and 3% lower OC, as compared to CT, and CTR1, respectively. CA recorded 10, 34, and 26% higher available nitrogen, phosphorus, and potassium over CT and CTR1, respectively. NTR1 recorded 25 and 38% lower N2O emissions as compared to CTR1 and CTR2, respectively. Whereas only NT recorded 12% higher N2O emissions as compared to CT. Overall, the results of the study indicate that CA improves the relative abundance of soil bacterial communities, nutrient availability, and enzyme activities, and may help to contribute to the mitigation of climate change, and sustainability in rainfed areas.

A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore.

As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.

Stereoselective synthesis of C-substituted morpholine derivatives using reductive etherification reaction: total synthesis of chelonin C.

A general strategy is developed for the stereoselective synthesis of C-substituted morpholine derivatives using intramolecular reductive etherification reaction. The method is extended to the first stereoselective total synthesis of (±)-chelonin C.

Synthesis and structure-activity studies of novel homomorpholine oxazolidinone antibacterial agents.

A novel series of oxazolidinones were synthesized in which the morpholine C-ring of linezolid was replaced with homomorpholine. In addition to investigating the effect of a homomorpholine C-ring on antibacterial activity, the effect of des-, mono-, di-, and tri-fluoro substitution on the phenyl B-ring was investigated as well. Various C-5 functional groups were also examined, including acetamides and triazoles and carboxamides.

New oxazolidinones.

Due to the emergence of resistance to known antibiotics to various organisms, for example, Staphylococcus, Streptococcus, Enterococci, and Pseudomonas there is a renewed interest in the discovery of new antibacterials. Oxazolidinones, totally synthetic class of novel antibacterials, possess activity against drug-resistant Gram-positive pathogens, especially MRSA. Linezolid, the first approved drug from this class, has shown a great promise in saving lives of many patients by acting against drug-resistant Gram-positive organisms. However, its use is somewhat limited because of its myelotoxicity when used long term (>21 days). Various research groups are active in this area either to improve myelotoxicity profile of linezolid or to expand the spectrum of activity of linezolid. In spite of active research in this area, the discovery of an oxazolidinone possessing improved myelotoxicity compared to linezolid, linezolid-like efficacy, and PK remains challenging.

Discovery and Optimization of Indolyl-Containing 4-Hydroxy-2-Pyridone Type II DNA Topoisomerase Inhibitors Active against Multidrug Resistant Gram-negative Bacteria.

There exists an urgent medical need to identify new chemical entities (NCEs) targeting multidrug resistant (MDR) bacterial infections, particularly those caused by Gram-negative pathogens. 4-Hydroxy-2-pyridones represent a novel class of nonfluoroquinolone inhibitors of bacterial type II topoisomerases active against MDR Gram-negative bacteria. Herein, we report on the discovery and structure-activity relationships of a series of fused indolyl-containing 4-hydroxy-2-pyridones with improved in vitro antibacterial activity against fluoroquinolone resistant strains. Compounds 6o and 6v are representative of this class, targeting both bacterial DNA gyrase and topoisomerase IV (Topo IV). In an abbreviated susceptibility screen, compounds 6o and 6v showed improved MIC90 values against Escherichia coli (0.5-1 µg/mL) and Acinetobacter baumannii (8-16 µg/mL) compared to the precursor compounds. In a murine septicemia model, both compounds showed complete protection in mice infected with a lethal dose of E. coli.

Antibacterial oxazolidinones possessing a novel C-5 side chain. (5R)-trans-3-[3-Fluoro-4- (1-oxotetrahydrothiopyran-4-yl)phenyl]-2- oxooxazolidine-5-carboxylic acid amide (PF-00422602), a new lead compound.

Oxazolidinones possessing a C-5 carboxamide functionality (reverse amides) represent a new series of compounds that block bacterial protein synthesis. These reverse amides also exhibited less potency against monoamine oxidase (MAO) enzymes and thus possess less potential for the side effects associated with MAO inhibition. The title compound (14) showed reduced in vivo myelotoxicity compared to linezolid in a 14-day safety study in rats, potent in vivo efficacy in murine systemic infection models, and excellent pharmacokinetic properties.

New hydroxyethylamine HIV protease inhibitors that suppress viral replication.

The synthesis of analogues of AcSerLeuAsn[Phe-HEA-Pro]IleValOMe (1, JG-365; where HEA stands for the hydroxyethylamine unit 2), a tight-binding inhibitor of HIVP, are reported. Systematic modification of the P3 and P3' regions of the inhibitors has led to smaller HIVP inhibitors that inhibit viral replication in HIV-infected and SIV-infected cell cultures. Six aliphatic and/or aromatic derivatives were prepared by replacing residues in the P3 regions of BocLeuAsn[Phe-HEA-Pro]IleValOMe. Aromatic side chains at P3 gave better inhibitors than aliphatic side chains. The better inhibitors in this series contained a beta-naphthylalanine or a biphenyl unit at P3. A second series of HIVP inhibitors were obtained by converting the P3 group into acyl groups. CbzAsn[Phe-HEA-Pro]IlePheOMe and Qua-Asn-[Phe-HEA-Pro]-Ile-Phe-OMe (where Qua = quinolin-2-ylcarbonyl) are potent HIVP inhibitors with Ki values equal to 1.0 and 0.1 nM, respectively. The inhibition constants were determined by using the continuous fluorometric assay developed by Toth and Marshall. The activities of the protease inhibitors for inhibition of SIV replication were determined in vitro using CEM x 174 cells. Inhibition of HIV infection was determined essentially as reported by Pauwels and co-workers. The anti-HIV assay was carried out in culture using CEM cells (a CD4+ lymphocyte line) infected with virus strain HTLV-IIIb with a multiplicity of infection of 0.1. Several analogues inhibited the cytopathic effect at concentrations of 0.1-0.8 microgram/mL. These results establish that good inhibitors of HIV protease that inhibit viral replication in infected lymphocytes in in vitro cell assays can be obtained from JG-365 when the AcSerLeu unit is replaced by aromatic acyl derivatives.

Nonpeptidic potent HIV-1 protease inhibitors: (4-hydroxy-6-phenyl-2-oxo-2H- pyran-3-yl)thiomethanes that span P1-P2' subsites in a unique mode of active site binding.

Using molecular modeling and the information derived from the X-ray crystal structure of HIV-1 protease (HIV PR) complexed with the pyran-2-one 1, a series of (4-hydroxy-6-phenyl-2-oxo-2H-pyran-3-yl)thiomethanes was designed and analyzed as novel, nonpeptidic inhibitors of HIV PR. Structure-activity studies led to the discovery of inhibitor 19 having (RS)-1-(cyclopentylthio)-3-methylbutyl functionalization at the C-3 position, which exhibited a Kc of 33 nM. A X-ray crystallographic structure of 19 bound to HIV PR showed that structural water-301 (inhibitor-flap-bridging water) was displaced by the inhibitor. Interestingly, the enol moiety of the pyran-2-one formed a hydrogen bond directly with Asp125 and with Asp25 via a bridging water molecule, thus illustrating a unique mode of active site binding by an HIV PR inhibitor. The pendant cyclopentyl and isobutyl groups of 19 occupied the S1' and S2' binding sites, respectively, whereas the 6-phenyl group occupied a region in between the S1 and S3 pockets of HIV PR. Selected compounds were tested for antiviral activity on H9 cells infected with HIV-1IIIb. A correlation between enzymatic activity and antiviral activity was not found in this series. The best antiviral compound in this series, 18, contained (RS)-3-[cyclopentyl(cyclopentylthio)methyl] functionalization at the C-3 position of the pyran-2-one ring and exhibited a CIC50 of 14 microM and TC50 of 70 microM. These studies demonstrate that potent enzyme inhibition can be achieved by inhibitors that span only three subsites.

4-hydroxy-5,6-dihydropyrones. 2. Potent non-peptide inhibitors of HIV protease.

The 4-hydroxy-5,6-dihydropyrone template was utilized as a flexible scaffolding from which to build potent active site inhibitors of HIV protease. Dihydropyrone 1c (5,6-dihydro-4-hydroxy-6-phenyl-3-[(2-phenylethyl)thio]-2H-pyran-2-one) was modeled in the active site of HIV protease utilizing a similar binding mode found for the previously reported 4-hydroxybenzopyran-2-ones. Our model led us to pursue the synthesis of 6,6-disubstituted dihydropyrones with the aim of filling S1 and S2 and thereby increasing the potency of the parent dihydropyrone 1c which did not fill S2. Toward this end we attached various hydrophobic and hydrophilic side chains at the 6-position of the dihydropyrone to mimic the natural and unnatural amino acids known to be effective substrates at P2 and P2'. Parent dihydropyrone 1c (IC50 = 2100 nM) was elaborated into compounds with greater than a 100-fold increase in potency [18c, IC50 = 5 nM, 5-(3,6-dihydro-4-hydroxy-6-oxo-2-phenyl-5-[2-phenylethyl)thio] -2H-pyran-2-yl)pentanoic acid and 12c, IC50 = 51 nM, 5,6-dihydro-4-hydroxy-6-phenyl-6-(2-phenylethyl)-3- [(2-phenyl-ethyl)thio]-2H-pyran-2-one]. Optimization of the 3-position fragment to fill S1' and S2' afforded potent HIV protease inhibitor 49 [IC50 = 10 nM, 3-[(2-tert-butyl-5-methylphenyl)sulfanyl]-5,6-dihydro-4 -hydroxy-6-phenyl-6-(2-phenylethyl)-2H-pyran-2-one]. The resulting low molecular weight compounds (< 475) have one or no chiral centers and are readily synthesized.

5,6-Dihydropyran-2-ones possessing various sulfonyl functionalities: potent nonpeptidic inhibitors of HIV protease.

On the basis of previous SAR findings and molecular modeling studies, a series of compounds were synthesized which possessed various sulfonyl moieties substituted at the 4-position of the C-3 phenyl ring substituent of the dihydropyran-2-one ring system. The sulfonyl substituents were added in an attempt to fill the additional S(3)' pocket and thereby produce increasingly potent inhibitors of the target enzyme. Racemic and enantiomerically resolved varieties of selected compounds were synthesized. All analogues in the study displayed decent binding affinity to HIV protease, and several compounds were shown to possess very good antiviral efficacy and safety margins. X-ray crystallographic structures confirmed that the sulfonamide and sulfonate moieties were filling the S(3)' pocket of the enzyme. However, the additional substituent did not provide improved enzymatic inhibitory or antiviral activity as compared to the resolved unsubstituted aniline. The addition of the sulfonyl moiety substitution does not appear to provide favorable pharamacokinectic parameters. Selected inhibitors were tested for antiviral activity in clinical isolates and exhibited similar antiviral activity against all of the HIV-1 strains tested as they did against the wild-type HIV-1. In addition, the inhibitors exhibited good antiviral efficacies against HIV-1 strains that displayed resistance to the currently marketed protease inhibitors.

PD0084430: a non-nucleoside inhibitor of human cytomegalovirus replication in vitro.

Human cytomegalovirus (HCMV) is a major opportunistic pathogen in immunocompromised individuals. Current therapies target viral DNA replication and accumulate mutations that yield cross-resistance among the approved drugs. A novel, non-nucleoside inhibitor of HCMV replication, PD0084430, was identified in a screening assay using the HCMV beta-galactosidase recombinant RC256. The EC(50) for PD0084430 by inhibition of beta-galactosidase production is 1+/-0.7 microM. This antiviral activity was confirmed by yield reduction and plaque reduction assays using HCMV strain AD169. The TC(50) of PD0084430 as measured by (4C)thymidine incorporation is approximately 30 microM and by XTT is approximately 90 microM. The TC(50) for inhibition of cellular proliferation is approximately 20 microM. Time of addition experiments displayed a similar drop in efficacy for both PD0084430 and GCV when added after the onset of viral DNA replication. The transcomplementation assay for viral DNA replication, using a transfected ori(Lyt) containing plasmid, confirmed that viral DNA synthesis was inhibited at the same concentrations that showed antiviral activity. Western blots showed no apparent block of immediate early or early gene expression. Two ganciclovir (GCV) resistant isolates of HCMV tested showed no cross-resistance to PD0084430. These data suggested a potentially promising novel compound that inhibited HCMV at or before viral DNA replication. However, in vivo testing in mice dosed either orally or intraperitoneally showed rapid glucuronidation on the -OH group. SAR studies on this backbone showed that the -OH group was essential for the antiviral activity in vitro.

Implementation of an ADME enabling selection and visualization tool for drug discovery.

The pharmaceutical industry has large investments in compound library enrichment, high throughput biological screening, and biopharmaceutical (ADME) screening. As the number of compounds submitted for in vitro ADME screens increases, data analysis, interpretation, and reporting will become rate limiting in providing ADME-structure-activity relationship information to guide the synthetic strategy for chemical series. To meet these challenges, a software tool was developed and implemented that enables scientists to explore in vitro and in silico ADME and chemistry data in a multidimensional framework. The present work integrates physicochemical and ADME data, encompassing results for Caco-2 permeability, human liver microsomal half-life, rat liver microsomal half-life, kinetic solubility, measured log P, rule of 5 descriptors (molecular weight, hydrogen bond acceptors, hydrogen bond donors, calculated log P), polar surface area, chemical stability, and CYP450 3A4 inhibition. To facilitate interpretation of this data, a semicustomized software solution using Spotfire was designed that allows for multidimensional data analysis and visualization. The solution also enables simultaneous viewing and export of chemical structures with the corresponding ADME properties, enabling a more facile analysis of ADME-structure-activity relationship. In vitro and in silico ADME data were generated for 358 compounds from a series of human immunodeficiency virus protease inhibitors, resulting in a data set of 5370 experimental values which were subsequently analyzed and visualized using the customized Spotfire application. Implementation of this analysis and visualization tool has accelerated the selection of molecules for further development based on optimum ADME characteristics, and provided medicinal chemistry with specific, data driven structural recommendations for improvements in the ADME profile.

Discovery of antibacterial biotin carboxylase inhibitors by virtual screening and fragment-based approaches.

As part of our effort to inhibit bacterial fatty acid biosynthesis through the recently validated target biotin carboxylase, we employed a unique combination of two emergent lead discovery strategies. We used both de novo fragment-based drug discovery and virtual screening, which employs 3D shape and electrostatic property similarity searching. We screened a collection of unbiased low-molecular-weight molecules and identified a structurally diverse collection of weak-binding but ligand-efficient fragments as potential building blocks for biotin carboxylase ATP-competitive inhibitors. Through iterative cycles of structure-based drug design relying on successive fragment costructures, we improved the potency of the initial hits by up to 3000-fold while maintaining their ligand-efficiency and desirable physicochemical properties. In one example, hit-expansion efforts resulted in a series of amino-oxazoles with antibacterial activity. These results successfully demonstrate that virtual screening approaches can substantially augment fragment-based screening approaches to identify novel antibacterial agents.

Synthesis and SAR of novel conformationally restricted oxazolidinones possessing gram-positive and fastidious gram-negative antibacterial activity. Part 2: Amino substitutions on heterocyclic D-ring system.

A novel series of conformationally restricted oxazolidinones was synthesized, in which the heterocyclic D ring was substituted with various amino groups. Several analogs exhibited potent activity against both gram-positive and fastidious gram-negative organisms. Certain amino-substituted analogs also exhibited improved aqueous solubility compared to the corresponding un-substituted heterocyclic D-ring analogs.