A Dual-Mechanism Antibiotic Kills Gram-Negative Bacteria and Avoids Drug Resistance.

The rise of antibiotic resistance and declining discovery of new antibiotics has created a global health crisis. Of particular concern, no new antibiotic classes have been approved for treating Gram-negative pathogens in decades. Here, we characterize a compound, SCH-79797, that kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism of action (MoA) with undetectably low resistance frequencies. To characterize its MoA, we combined quantitative imaging, proteomic, genetic, metabolomic, and cell-based assays. This pipeline demonstrates that SCH-79797 has two independent cellular targets, folate metabolism and bacterial membrane integrity, and outperforms combination treatments in killing methicillin-resistant Staphylococcus aureus (MRSA) persisters. Building on the molecular core of SCH-79797, we developed a derivative, Irresistin-16, with increased potency and showed its efficacy against Neisseria gonorrhoeae in a mouse vaginal infection model. This promising antibiotic lead suggests that combining multiple MoAs onto a single chemical scaffold may be an underappreciated approach to targeting challenging bacterial pathogens.

Elucidation of Chalkophomycin Biosynthesis Reveals N-Hydroxypyrrole-Forming Enzymes.

Reactive functional groups, such as N-nitrosamines, impart unique bioactivities to the natural products in which they are found. Recent work has illuminated enzymatic N-nitrosation reactions in microbial natural product biosynthesis, motivating interest in discovering additional metabolites constructed using such reactivity. Here, we use a genome mining approach to identify over 400 cryptic biosynthetic gene clusters (BGCs) encoding homologues of the N-nitrosating biosynthetic enzyme SznF, including the BGC for chalkophomycin, a CuII-binding metabolite that contains a C-type diazeniumdiolate and N-hydroxypyrrole. Characterizing chalkophomycin biosynthetic enzymes reveals previously unknown enzymes responsible for N-hydroxypyrrole biosynthesis, including the first prolyl-N-hydroxylase, and a key step in the assembly of the diazeniumdiolate-containing amino acid graminine. Discovery of this pathway enriches our understanding of the biosynthetic logic employed in constructing unusual heteroatom-heteroatom bond-containing functional groups, enabling future efforts in natural product discovery and biocatalysis.

Binding Mode and Molecular Mechanism of the Two-Component Histidine Kinase Bos1 of Botrytis cinerea to Fludioxonil and Iprodione.

Gray mold caused by Botrytis cinerea is among the 10 most serious fungal diseases worldwide. Fludioxonil is widely used to prevent and control gray mold due to its low toxicity and high efficiency; however, resistance caused by long-term use has become increasingly prominent. Therefore, exploring the resistance mechanism of fungicides provides a theoretical basis for delaying the occurrence of diseases and controlling gray mold. In this study, fludioxonil-resistant strains were obtained through indoor drug domestication, and the mutation sites were determined by sequencing. Strains obtained by site-directed mutagenesis were subjected to biological analysis, and the binding modes of fludioxonil and iprodione to Botrytis cinerea Bos1 BcBos1 were predicted by molecular docking. The results showed that F127S, I365S/N, F127S + I365N, and I376M mutations on the Bos1 protein led to a decrease in the binding energy between the drug and BcBos1. The A1259T mutation did not lead to a decrease in the binding energy, which was not the cause of drug resistance. The biological fitness of the fludioxonil- and point mutation-resistant strains decreased, and their growth rate, sporulation rate, and pathogenicity decreased significantly. The glycerol content of the sensitive strains was significantly lower than that of the resistant strains and increased significantly after treatment with 0.1 µg/ml of fludioxonil, whereas that of the resistant strains decreased. The osmotic sensitivity of the resistant strains was significantly lower than that of the sensitive strains. Positive cross-resistance was observed between fludioxonil and iprodione. These results will help to understand the resistance mechanism of fludioxonil in Botrytis cinerea more deeply.

Bioinspired micro/nanomotor with visible light energy-dependent forward, reverse, reciprocating, and spinning schooling motion.

In nature, microorganisms could sense the intensity of the incident visible light and exhibit bidirectional (positive or negative) phototaxis. However, it is still challenging to achieve the similar biomimetic phototaxis for the artificial micro/nanomotor (MNM) counterparts with the size from a few nanometers to a few micrometers. In this work, we report a fuel-free carbon nitride (C3N4)/polypyrrole nanoparticle (PPyNP)-based smart MNM operating in water, whose behavior resembles that of the phototactic microorganism. The MNM moves toward the visible light source under low illumination and away from it under high irradiation, which relies on the competitive interplay between the light-induced self-diffusiophoresis and self-thermophoresis mechanisms concurrently integrated into the MNM. Interestingly, the competition between these two mechanisms leads to a collective bidirectional phototaxis of an ensemble of MNMs under uniform illuminations and a spinning schooling behavior under a nonuniform light, both of which can be finely controllable by visible light energy. Our results provide important insights into the design of the artificial counterpart of the phototactic microorganism with sophisticated motion behaviors for diverse applications.

Structural basis of the protochromic green/red photocycle of the chromatic acclimation sensor RcaE.

Cyanobacteriochromes (CBCRs) are bilin-binding photosensors of the phytochrome superfamily that show remarkable spectral diversity. The green/red CBCR subfamily is important for regulating chromatic acclimation of photosynthetic antenna in cyanobacteria and is applied for optogenetic control of gene expression in synthetic biology. It is suggested that the absorption change of this subfamily is caused by the bilin C15-Z/C15-E photoisomerization and a subsequent change in the bilin protonation state. However, structural information and direct evidence of the bilin protonation state are lacking. Here, we report a high-resolution (1.63Å) crystal structure of the bilin-binding domain of the chromatic acclimation sensor RcaE in the red-absorbing photoproduct state. The bilin is buried within a "bucket" consisting of hydrophobic residues, in which the bilin configuration/conformation is C5-Z,syn/C10-Z,syn/C15-E,syn with the A- through C-rings coplanar and the D-ring tilted. Three pyrrole nitrogens of the A- through C-rings are covered in the α-face with a hydrophobic lid of Leu249 influencing the bilin pKa, whereas they are directly hydrogen bonded in the ß-face with the carboxyl group of Glu217. Glu217 is further connected to a cluster of waters forming a hole in the bucket, which are in exchange with solvent waters in molecular dynamics simulation. We propose that the "leaky bucket" structure functions as a proton exit/influx pathway upon photoconversion. NMR analysis demonstrated that the four pyrrole nitrogen atoms are indeed fully protonated in the red-absorbing state, but one of them, most likely the B-ring nitrogen, is deprotonated in the green-absorbing state. These findings deepen our understanding of the diverse spectral tuning mechanisms present in CBCRs.

Riboflavin improves grain yield, 2-acetyl-1-pyrroline accumulation, and antioxidative properties of fragrant rice.

BACKGROUND: Riboflavin, a vital water-soluble vitamin with antioxidative activity, plays a critical role in maintaining overall bodily health and defense responses. However, its impact on fragrant rice yield and aroma remains unexplored. RESULTS: In a 2022 pot experiment with Meixiangzhan and Yuxiangyouzhan fragrant rice cultivars, we applied riboflavin foliar treatments at concentrations of 0 (CK), 10 (R10), 20 (R20), and 40 (R40) mg L-1 during the initial heading stage. Riboflavin increased rice yield, 2-acetyl-1-pyrroline (2-AP) content, and antioxidative properties. It boosted 2-AP level by 13.1-50.1% for Meixiangzhan and 22.3-35.3% for Yuxiangyouzhan, with the highest levels in R20 and R10 treatments. This increase is significantly correlated with elevated levels of proline, pyrroline-5-carboxylic acid, pyrroline, and methylglyoxal, as well as heightened enzyme activities, including those of proline dehydrogenase, ornithine aminotransferase, and pyrroline-5-carboxylic acid synthetase (P5CS). The R20 treatment resulted in the highest yield due to an improved seed-setting rate. Importantly, a positive correlation emerged between 2-AP content and yield, both significantly linked to superoxide dismutase, proline, hydrogen peroxide, P5CS, catalase, and pyrroline. CONCLUSION: Riboflavin maintained enzyme activities, regulated substance synthesis pathways, and increased 2-AP and yield, especially in the R20 treatment. These insights advance fragrant rice production theory by uncovering riboflavin's role in the development of fragrant rice. © 2023 Society of Chemical Industry.

Synthesis of 1-(4-(dimethylamino)phenyl)-3,4-diphenyl-1H-pyrrole-2,5-dione analogues and their anti-inflammatory activities in lipopolysaccharide-induced BV2 cells.

A series of thalidomide analogues, where the fused benzene ring in the phthalimide moiety was converted into two separated diphenyl rings in maleimide moiety and N-aminoglutarimide moiety was replaced by substituted phenyl moiety, were synthesized and evaluated for their NO inhibitory activities on BV2 cells stimulated with lipopolysaccharide (LPS). Among the synthesized compounds, the dimethylaminophenyl analogue 1s (IC50 = 7.1 µM) showed significantly higher inhibitory activity than the glutarimide analogue 1a (IC50 > 50 µM) and suppressed NO production dose-dependently without cytotoxicity. In addition, 1s inhibited the production of pro-inflammatory cytokines and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) by blocking nuclear factor-kappa B (NF-κB) and p38 MAPK pathways. These results demonstrated that 1s showed good anti-inflammatory activity and could become a leading compound for the treatment of neuroinflammatory diseases.

Resolving the Hydride Transfer Pathway in Oxidative Conversion of Proline to Pyrrole.

Thiotemplated pyrrole is a prevailing intermediate in the synthesis of numerous natural products in which the pyrrole is tethered to a carrier protein (CP). Biosynthesis of the pyrrole requires oxidation of an l-proline side chain. Herein, we investigate the biocatalytic mechanism of proline-to-pyrrole synthesis by molecular dynamics simulations, quantum mechanics/molecular mechanics simulations, and electronic structure calculations using the recently reported (Thapa, H. R., et al. Biochemistry 2019, 58, 918) structure of a type II nonribosomal protein synthetase (NRPS) Bmp3-Bmp1 (Oxidase-CP) complex. The substrate (l-proline) is attached to the Bmp1(CP), and the catalytic site is located inside the flavin-dependent oxidase (Bmp3). We show that the FAD isoalloxazine ring is stabilized in the catalytic site of Bmp3 by strong hydrogen bonding with Asn123, Ile125, Ser126, and Thr158. After the initial deprotonation followed by an enamine-imine tautomerization, oxidation of the C2-C3 or C2-N1 bond, through a hydride transfer (from either C3 or N1), is required for the pyrrole synthesis. Computational results indicate that the hydride transfer is more likely to occur from C3 than N1. Additionally, we demonstrate the elasticity in the oxidase active site through enzymatic synthesis of proline derivatives.

Pentachloropseudilin Treatment Impairs Host Cell Invasion by Trypanosoma cruzi.

Pentachloropseudilin (PClP) is a reversible and allosteric inhibitor of type 1 myosin. Here, we addressed the impact of PClP treatment of Trypanosoma cruzi and mammalian host cell on the parasite migration, cell adhesion and invasion. We observed that PClP was not toxic to either T. cruzi or host cell. Moreover, treatment of T. cruzi with PClP inhabited parasite motility, host cell adhesion and invasion. Treatment of host cell with PClP also impaired parasite invasion probably by decreasing lysosome migration to the entry site of the parasite. Therefore, PClP treatment impaired fundamental processes necessary for a successful T. cruzi infection.

Elucidating the structure and cytochrome P450-mediated mechanism for novel metabolites of GDC-0575 in rats.

1. GDC-0575 is an ATP-competitive small-molecule inhibitor of ChK1 that is being developed by Genentech for the treatment of various human malignancies.2. In a radiolabeled mass balance study of GDC-0575 in rats, two novel metabolites, named M12 (-71 Da,) and M17 (+288 Da), were detected as abundant circulating metabolites.3. Subsequent mass spectrometry and nuclear magnetic resonance analysis showed that M12 was a cyclized metabolite of GDC-0575, whereas M17 was its heterodimer to the parent. We further determined that M12 was mainly generated by cytochrome P450 (Cyp) 2d2.4. We proposed the potential mechanism was initiated by the oxidation on the pyrrole ring and subsequent cyclisation of the free primary amine onto C-3 of the pyrrole ring. This was followed by expulsion of cyclopropylcarboxamide and a loss of water to form intermediate I, which can be further oxidised to form M12, or dimerise with another molecule of GDC-0575 as nucleophile to form M17.5. To verify this hypothesis, we attempted to trap the intermediate I with glutathione (GSH) trapping assay and the GSH conjugate on the pyrrole ring was identified. This suggests the oxidation on the pyrrole led to reactive metabolite formation and supported this proposed mechanism.

Combined Metabolomic and Quantitative RT-PCR Analyses Revealed the Synthetic Differences of 2-Acetyl-1-pyrroline in Aromatic and Non-Aromatic Vegetable Soybeans.

Aroma is an important economic trait of vegetable soybeans, which greatly influences their market value. The 2-acetyl-1-pyrroline (2AP) is considered as an important substance affecting the aroma of plants. Although the 2AP synthesis pathway has been resolved, the differences of the 2AP synthesis in the aromatic and non-aromatic vegetable soybeans are unknown. In this study, a broad targeted metabolome analysis including measurement of metabolites levels and gene expression levels was performed to reveal pathways of aroma formation in the two developmental stages of vegetable soybean grains [35 (S5) and 40 (S6) days after anthesis] of the 'Zhexian No. 8' (ZX8, non-aromatic) and ZK1754 (aromatic). The results showed that the differentially accumulated metabolites (DAMs) of the two varieties can be classified into nine main categories including flavonoids, lipids, amino acids and derivatives, saccharides and alcohols, organic acids, nucleotides and derivatives, phenolic acids, alkaloids and vitamin, which mainly contributed to their phenotypic differences. Furthermore, in combination with the 2AP synthesis pathway, the differences of amino acids and derivatives were mainly involved in the 2AP synthesis. Furthermore, 2AP precursors' analysis revealed that the accumulation of 2AP mainly occurred from 1-pyrroline-5-carboxylate (P5C), not 4-aminobutyraldehyde (GABald). The quantitative RT-PCR showed that the associated synthetic genes were 1-pyrroline-5-carboxylate dehydrogenase (P5CDH), ∆1-pyrroline-5-carboxylate synthetase (P5CS), proline dehydrogenase (PRODH) and pyrroline-5-carboxylate reductase (P5CR), which further verified the synthetic pathway of 2AP. Furthermore, the betaine aldehyde dehydrogenase 2 (GmBADH2) mutant was not only vital for the occurrence of 2AP, but also for the synthesis of 4-aminobutyric acid (GABA) in vegetable soybean. Therefore, the differences of 2AP accumulation in aromatic and non-aromatic vegetable soybeans have been revealed, and it also provides an important theoretical basis for aromatic vegetable soybean breeding.

Anticancer Approach Inspired by the Hepatotoxic Mechanism of Pyrrolizidine Alkaloids with Glycosylated Artificial Metalloenzymes.

Metabolic oxidation of pyrrolizidine alkaloids (PAs) from herbal and dietary supplements by cytochrome P450 produces dehydro-PAs (DHPs), which leads to toxicities. A highly reactive cation species generated from the active pyrrole ring of DHPs readily reacts with various cellular components, causing hepatotoxicity and cytotoxicity. Inspired by PA-induced hepatic damage, we developed a therapeutic approach based on a cyclization precursor that can be transformed into a synthetic DHP under physiological conditions through gold-catalyzed 5-endo-dig cyclization using a gold-based artificial metalloenzyme (ArM) instead of through metabolic oxidation by cytochrome P450. In cell-based assays, the synthesis of the DHP by a cancer-targeting glycosylated gold-based ArM substantially suppressed cell growth of the targeted cancer cells without causing cytotoxicity to untargeted cells, highlighting the potential of the strategy to be used therapeutically in vivo.

Nucleobase mutants of a bacterial preQ1-II riboswitch that uncouple metabolite sensing from gene regulation.

Riboswitches are a class of nonprotein-coding RNAs that directly sense cellular metabolites to regulate gene expression. They are model systems for analyzing RNA-ligand interactions and are established targets for antibacterial agents. Many studies have analyzed the ligand-binding properties of riboswitches, but this work has outpaced our understanding of the underlying chemical pathways that govern riboswitch-controlled gene expression. To address this knowledge gap, we prepared 15 mutants of the preQ1-II riboswitch-a structurally and biochemically well-characterized HLout pseudoknot that recognizes the metabolite prequeuosine1 (preQ1). The mutants span the preQ1-binding pocket through the adjoining Shine-Dalgarno sequence (SDS) and include A-minor motifs, pseudoknot-insertion helix P4, U·A-U base triples, and canonical G-C pairs in the anti-SDS. As predicted-and confirmed by in vitro isothermal titration calorimetry measurements-specific mutations ablated preQ1 binding, but most aberrant binding effects were corrected by compensatory mutations. In contrast, functional analysis in live bacteria using a riboswitch-controlled GFPuv-reporter assay revealed that each mutant had a deleterious effect on gene regulation, even when compensatory changes were included. Our results indicate that effector binding can be uncoupled from gene regulation. We attribute loss of function to defects in a chemical interaction network that links effector binding to distal regions of the fold that support the gene-off RNA conformation. Our findings differentiate effector binding from biological function, which has ramifications for riboswitch characterization. Our results are considered in the context of synthetic ligands and drugs that bind tightly to riboswitches without eliciting a biological response.

Lanthanum (La) improves growth, yield formation and 2-acetyl-1-pyrroline biosynthesis in aromatic rice (Oryza sativa L.).

BACKGROUND: Lanthanum (La) is a rare earth element that can influence plant growth and development. However, the effect of La on growth, yield formation and 2-acetyl-1-pyrroline (2-AP, a key compound responsible for the aroma of rice) biosynthesis in aromatic rice (Oryza sativa L. subsp. japonica Kato) has not been reported. Therefore, the present study investigated the effects of La on growth, photosynthesis, yield formation and 2-AP biosynthesis in aromatic rice through three experiments. RESULTS: Two pot experiments and a two-year field trial were conducted with different rates of La application (20-120 LaCl3 mg kg-1 and 12 kg ha-1 LaCl3), and treatments without La application were used as controls. The results showed that the application of LaCl3 at 80 and 100 mg kg-1 and at 12 kg ha-1 greatly increased the 2-AP content (by 6.45-43.03%) in aromatic rice seedlings and mature grains compared with the control. The La treatments also increased the chlorophyll content, net photosynthetic rate and total aboveground biomass of rice seedlings. Higher antioxidant enzyme (superoxide, peroxidase, and catalase) activity was detected in the La treatments than in the control. The La treatments also increased the grain yield, grain number per panicle and seed-setting rate of aromatic rice relative to the control. Moreover, the grain proline and γ-aminobutyric acid contents and the activity of betaine aldehyde dehydrogenase significantly decreased under the La treatment. The application of La to soil enhanced the activity of proline dehydrogenase by 20.62-56.95%. CONCLUSIONS: La improved the growth, yield formation and 2-AP content of aromatic rice and enhanced 2-AP biosynthesis by increasing the conversion of proline to 2-AP and decreasing the conversion of GABald to GABA.

Chemical and Genetic Studies on the Formation of Pyrrolones During the Biosynthesis of Cytochalasans.

A key step during the biosynthesis of cytochalasans is a proposed Knoevenagel condensation to form the pyrrolone core, enabling the subsequent 4+2 cycloaddition reaction that results in the characteristic octahydroisoindolone motif of all cytochalasans. In this work, we investigate the role of the highly conserved α,ß-hydrolase enzymes PyiE and ORFZ during the biosynthesis of pyrichalasin H and the ACE1 metabolite, respectively, using gene knockout and complementation techniques. Using synthetic aldehyde models we demonstrate that the Knoevenagel condensation proceeds spontaneously but results in the 1,3-dihydro-2H-pyrrol-2-one tautomer, rather than the required 1,5-dihydro-2H-pyrrol-2-one tautomer. Taken together our results suggest that the α,ß-hydrolase enzymes are essential for first ring cyclisation, but the precise nature of the intermediates remains to be determined.

Kinetics of human pyrroline-5-carboxylate reductase in L-thioproline metabolism.

L-Thioproline (L-thiazolidine-4-carboxylate, L-T4C) is a cyclic sulfur-containing analog of L-proline found in multiple kingdoms of life. The oxidation of L-T4C leads to L-cysteine formation in bacteria, plants, mammals, and protozoa. The conversion of L-T4C to L-Cys in bacterial cell lysates has been attributed to proline dehydrogenase and L-Δ1-pyrroline-5-carboxylate (P5C) reductase (PYCR) enzymes but detailed kinetic studies have not been conducted. Here, we characterize the dehydrogenase activity of human PYCR isozymes 1 and 2 with L-T4C using NAD(P)+ as the hydride acceptor. Both PYCRs exhibit significant L-T4C dehydrogenase activity; however, PYCR2 displays nearly tenfold higher catalytic efficiency (136 M-1 s-1) than PYCR1 (13.7 M-1 s-1). Interestingly, no activity was observed with either L-Pro or the analog DL-thiazolidine-2-carboxylate, indicating that the sulfur at the 4-position is critical for PYCRs to utilize L-T4C as a substrate. Inhibition kinetics show that L-Pro is a competitive inhibitor of PYCR1 [Formula: see text] with respect to L-T4C, consistent with these ligands occupying the same binding site. We also confirm by mass spectrometry that L-T4C oxidation by PYCRs leads to cysteine product formation. Our results suggest a new enzyme function for human PYCRs in the metabolism of L-T4C.

Discovery of IDO1 inhibitors containing a decahydroquinoline, decahydro-1,6-naphthyridine, or octahydro-1H-pyrrolo[3,2-c]pyridine scaffold.

A series of IDO1 inhibitors containing a decahydroquinoline, decahydro-1,6-naphthyridine, or octahydro-1H-pyrrolo[3,2-c]pyridine scaffold were identified with good cellular and human whole blood activity against IDO1. These inhibitors contain multiple chiral centers and all diastereomers were separated. The absolute stereochemistry of each isomers were not determined. Compounds 15 and 27 stood out as leads due to their good cellular as well as human whole blood IDO1 inhibition activity, low unbound clearance, and reasonable mean residence time in rat cassette PK studies.

Pyrrole carboxamide introduction in the total synthesis of pyrrole-imidazole alkaloids.

In this review various strategies for the incorporation of the signature pyrrole carboxamide moiety in the total syntheses of pyrrole-imidazole alkaloids (PIA) are discussed. These so-called oroidin alkaloids have a broad range of biological activities and display interesting skeletal diversity and complexity. These alkaloids are sponge-derived secondary metabolites and thus far more than 200 members of the PIA family have been isolated over the past few decades. Methods range from classical amide bond forming processes to non-traditional bond formation including the de novo synthesis of the pyrrole itself.

Peculiar substrate specificity of δ1-pyrroline-5-carboxylate reductase in the obligately fermentative bacterium Zymomonas mobilis.

BACKGROUND: The enzyme that catalyzes the last step in proline synthesis, δ1-pyrroline-5-carboxylate reductase, showed in most cases a distinct preference in vitro for NADPH as the electron donor. METHODS AND RESULTS: A Zymomonas mobilis gene coding for a δ1-pyrroline-5-carboxylate reductase was cloned and heterologously expressed, and the recombinant protein was purified and characterized. The enzyme showed higher affinity to, and higher catalytic rate with NADH, with a specific activity of about 600 nkat (mg protein)-1. The molecular basis of this feature was investigated by analysis of the dinucleotide binding domain in silico. CONCLUSIONS: We postulate that the main determinants of coenzyme preference for P5C reductases are the length and the sequence of the motif A, whereas the overall sequence identity is insufficient to predict it a priori. Results are discussed in view of the obligately fermentative metabolism of this bacterium.

Neuropathic pain-alleviating activity of novel 5-HT6 receptor inverse agonists derived from 2-aryl-1H-pyrrole-3-carboxamide.

The diverse signaling pathways engaged by serotonin type 6 receptor (5-HT6R) together with its high constitutive activity suggests different types of pharmacological interventions for the treatment of CNS disorders. Non-physiological activation of mTOR kinase by constitutively active 5-HT6R under neuropathic pain conditions focused our attention on the possible repurposing of 5-HT6R inverse agonists as a strategy to treat painful symptoms associated with neuropathies of different etiologies. Herein, we report the identification of compound 33 derived from the library of 2-aryl-1H-pyrrole-3-carboxamides as a potential analgesic agent. Compound 33 behaves as a potent 5-HT6R inverse agonist at Gs, Cdk5, and mTOR signaling. Preliminary ADME/Tox studies revealed preferential distribution of 33 to the CNS and placed it in the low-risk safety space. Finally, compound 33 dose-dependently reduced tactile allodynia in spinal nerve ligation (SNL)-induced neuropathic rats.