NU-6027 Inhibits Growth of Mycobacterium tuberculosis by Targeting Protein Kinase D and Protein Kinase G.

Tuberculosis (TB) is a global health concern, and this situation has further worsened due to the emergence of drug-resistant strains and the failure of BCG vaccine to impart protection. There is an imperative need to develop highly sensitive, specific diagnostic tools, novel therapeutics, and vaccines for the eradication of TB. In the present study, a chemical screen of a pharmacologically active compound library was performed to identify antimycobacterial compounds. The phenotypic screen identified a few novel small-molecule inhibitors, including NU-6027, a known CDK-2 inhibitor. We demonstrate that NU-6027 inhibits Mycobacterium bovis BCG growth in vitro and also displayed cross-reactivity with Mycobacterium tuberculosis protein kinase D (PknD) and protein kinase G (PknG). Comparative structural and sequence analysis along with docking simulation suggest that the unique binding site stereochemistry of PknG and PknD accommodates NU-6027 more favorably than other M. tuberculosis Ser/Thr protein kinases. Further, we also show that NU-6027 treatment induces the expression of proapoptotic genes in macrophages. Finally, we demonstrate that NU-6027 inhibits M. tuberculosis growth in both macrophage and mouse tissues. Taken together, these results indicate that NU-6027 can be optimized further for the development of antimycobacterial agents.

Intestinal transport of TRH analogs through PepT1: the role of in silico and in vitro modeling.

The present study involves molecular docking, molecular dynamics (MD) simulation studies, and Caco-2 cell monolayer permeability assay to investigate the effect of structural modifications on PepT1-mediated transport of thyrotropin releasing hormone (TRH) analogs. Molecular docking of four TRH analogs was performed using a homology model of human PepT1 followed by subsequent MD simulation studies. Caco-2 cell monolayer permeability studies of four TRH analogs were performed at apical to basolateral and basolateral to apical directions. Inhibition experiments were carried out using Gly-Sar, a typical PepT1 substrate, to confirm the PepT1-mediated transport mechanism of TRH analogs. Papp of the four analogs follows the order: NP-1894 < NP-2378 < NP-1896 < NP-1895. Higher absorptive transport was observed in the case of TRH analogs, indicating the possibility of a carrier-mediated transport mechanism. Further, the significant inhibition of the uptake of Gly-Sar by TRH analogs confirmed the PepT1-mediated transport mechanism. Glide docking scores of all the four analogues were in good agreement with their transport rates, suggesting the role of substrate binding affinity in the PepT1-mediated transport of TRH analogs. MD simulation studies revealed that the polar interactions with amino acid residues present in the active site are primarily responsible for substrate binding, and a downward trend was observed with the increase in bulkiness at the N-histidyl moiety of TRH analogs.

Effect of counterions on physicochemical properties of prazosin salts.

This study evaluated the effect of counterions on the physicochemical properties of prazosin salts. Salt forms of prazosin, namely, mesylate, besylate, tosylate, camsylate, oxalate, and maleate, were prepared and compared with the marketed anhydrous and polyhydrate forms of prazosin hydrochloride. Physicochemical characterization was performed in the order of crystallinity, hygroscopicity, solubility, and stability to select the optimal salt(s). Permeability study in Caco-2 cell lines and in vivo bioavailability study in rat model were investigated to ascertain their biopharmaceutical advantage. All salt forms were crystalline, nonhygroscopic (except the anhydrous hydrochloride salt), and had solubility in the range of 0.2 to 1.6 mg/ml. All salts were physically and chemically stable at 40°C/75% relative humidity, but degraded in UV-visible light, except the anhydrous hydrochloride salt. Prazosin mesylate was selected as the optimal salt, as it possessed higher solubility, permeability, and bioavailability, compared to the commercial hydrochloride salts. Hydrochloride salt is reported to have poor bioavailability that is partially attributed to its low solubility and extensive common-ion effect in the gastric region. Factors like hydrophilicity of the counterion, hydration state of the salt, and melting point of the salt contribute to the physicochemical properties of the salts. This study has implications in the selection of an optimal salt form for prazosin, which is suitable for further development.

Beyond the polysaccharide and glycoconjugate vaccines for Streptococcus pneumoniae: Does protein/peptide nanovaccines hold promises?

Streptococcus pneumoniae having almost 98 serotypes and being common cause of acute otitis media, pneumonia, bacteremia, meningitis etc., which results in high mortality and morbidity globally. Although vaccines like PCV-13 and PPV-23 are available, some problems like serotype replacement and poor immunogenicity in children, old age and immunocompromised people has been observed. To overcome these drawbacks protein/peptide-based vaccine can be a good strategy as these provides wide serotype coverage. However, immunogenicity of protein subunit vaccines is lower, that issue can be solved by using adjuvants. Recently nanoparticles as an adjuvant for vaccine delivery being used, which has provided not only good immunogenicity but also improved delivery and efficiency of protein-based vaccines. In this review we have discussed the latest advancement of nanoparticles-based protein/peptide vaccine delivery for Streptococcus pneumoniae.

Mechanistic insights into PEPT1-mediated transport of a novel antiepileptic, NP-647.

The present study, in general, is aimed to uncover the properties of the transport mechanism or mechanisms responsible for the uptake of NP-647 into Caco-2 cells and, in particular, to understand whether it is a substrate for the intestinal oligopeptide transporter, PEPT1 (SLC15A1). NP-647 showed a carrier-mediated, saturable transport with Michaelis-Menten parameters K(m) = 1.2 mM and V(max) = 2.2 µM/min. The effect of pH, sodium ion (Na(+)), glycylsarcosine and amoxicillin (substrates of PEPT1), and sodium azide (Na(+)/K(+)-ATPase inhibitor) on the flux rate of NP-647 was determined. Molecular docking and molecular dynamics simulation studies were carried out to investigate molecular interactions of NP-647 with transporter using homology model of human PEPT1. The permeability coefficient (P(appCaco-2)) of NP-647 (32.5 × 10(-6) cm/s) was found to be four times higher than that of TRH. Results indicate that NP-647 is transported into Caco-2 cells by means of a carrier-mediated, proton-dependent mechanism that is inhibited by Gly-Sar and amoxicillin. In turn, NP-647 also inhibits the uptake of Gly-Sar into Caco-2 cells and, together, this evidence suggests that PEPT1 is involved in the process. Docking and molecular dynamics simulation studies indicate high affinity of NP-647 toward PEPT1 binding site as compared to TRH. High permeability of NP-647 over TRH is attributed to its increased hydrophobicity which increases its affinity toward PEPT1 by interacting with the hydrophobic pocket of the transporter through hydrophobic forces.

Antiepileptic potential and behavioral profile of L-pGlu-(2-propyl)-L-His-L-ProNH2, a newer thyrotropin-releasing hormone analog.

Thyrotropin-releasing hormone (TRH) and its analogs have a number of neurobiological functions and therapeutic uses in disorders of the central nervous system. In this study, the newly synthesized TRH analogs were evaluated for central nervous system activity in pentobarbital-induced sleeping in mice. The most potent TRH analog (L-pGlu-(2-propyl)-L-His-L-ProNH(2) coded as NP-647) was evaluated for its antiepileptic potential in various seizure models in mice in comparison with TRH. Intravenous pretreatment with NP-647 (10 and 20 micromol/kg body wt) significantly delayed the onset and reduced the frequency of convulsions in the pentylenetetrazole model, but not in the maximum electroshock seizure model. Also, it was found to be protective against picrotoxin- and kainic acid-induced seizures. However, NP-647 did not significantly affect theophylline-induced seizures. Further study of the effect of NP-647 on locomotor activity and a functional observational battery revealed that it did not significantly exhibit any undesirable effects as compared with vehicle and TRH. NP-647 did not significantly affect cerebral blood flow, whereas the native peptide TRH markedly increased cerebral blood flow. Furthermore, NP-647 exerted antiepileptic activity without significantly altering plasma thyroid-stimulating hormone levels and mean arterial blood pressure. This suggests that NP-647 is more selective for central nervous system activity and devoid of hormonal and cerebrovascular system effects. In contrast, TRH exhibited cardiac and endocrine effects as marked by significant elevation in mean arterial blood pressure and plasma thyroid-stimulating hormone levels. This study demonstrates that NP-647 has potential antiepileptic activity devoid of undesirable effects and, thus, can be exploited for the prevention and treatment of epilepsy.

NP-647, a novel TRH analogue: investigating physicochemical parameters critical for its oral and parenteral delivery.

NP-647 (L-pGlu-(2-propyl)-L-His-L-ProNH(2)) is a novel thyrotropin releasing hormone (TRH) analogue, with potential antiepileptic activity. In the present study, the physicochemical parameters of NP-647, including its solid state properties, dissociation constant, partition coefficient, solubility (intrinsic solubility and pH-solubility profile) and stability (gastrointestinal enzymatic stability, pH-stability profile and temperature stability) were investigated for their criticality for oral and parenteral delivery. NP-647 was characterized as amorphous material having glass transition temperature of 66.73 °C at 50% RH. It was found very hygroscopic with deliquescent in nature. pK(a) of the compound, as determined using potentiometric titration, was found to be 7.2 ± 0.02 (basic). Intrinsic solubility and pH-solubility behavior were determined using dissolution titration template method. NP-647 has intrinsic solubility of 2.4 ± 0.01 mg mL(-1). Partition/distribution studies indicate that NP-647 has a low log P (-1.07 ± 0.06) and log D(7.4) (-1.20 ± 0.02), characteristic of hydrophilic molecule. It was found most stable in tartrate buffer of pH of 5.0. Arrhenius plot of NP-647 suggest its half life of ∼ 3.2 years and shelf life of ∼ 6 months. These studies conclude that amorphous nature of NP-647 with deliquescent property will be critical in its solid oral dosage formulation and need to be investigated further.

Protective effects of L-pGlu-(2-propyl)-L-His-L-ProNH2, a newer thyrotropin releasing hormone analog in in vitro and in vivo models of cerebral ischemia.

In the present study, the newly synthesized TRH analog (L-pGlu-(2-propyl)-L-His-l-ProNH(2); NP-647) was evaluated for its effects in in vitro (oxygen glucose deprivation (OGD)-, glutamate- and H(2)O(2)-induced injury in PC-12 cells) and in vivo (transient global ischemia) models of cerebral ischemic injury. PC-12 cells were subjected to oxygen and glucose deprivation for 6h. Exposure of NP-647 was given before and during OGD. In glutamate and H(2)O(2) induced injury, exposure of NP-647 was given 1, 6 and 24h prior to exposure of glutamate and H(2)O(2) exposure. NP-647, per se found to be non-toxic in 1-100µM concentrations. NP-647 showed protection against OGD at the 1 and 10µM. The concentration-dependent protection was observed in H(2)O(2)- and glutamate-induced cellular injury. In in vivo studies, NP-647 treatment showed protection of hippocampal (CA1) neuronal damage in transient global ischemia in mice and subsequent improvement in memory retention was observed using passive avoidance retention test. Moreover, administration of NP-647 resulted in decrease in inflammatory cytokines TNF-α and IL-6 as well as lipid peroxidation. These results suggest potential of NP-647 in the treatment of cerebral ischemia and its neuroprotective effect may be attributed to reduction of excitotoxicity, oxidative stress and inflammation.

Neuropharmacological profile of L-pGlu-(1-benzyl)-L-His-L-ProNH2, a newer thyrotropin-releasing hormone analog: effects on seizure models, sodium current, cerebral blood flow and behavioral parameters.

In the present study, L-pGlu-(1-benzyl)-L-His-L-ProNH(2) (NP-355), a newer CNS active thyrotropin-releasing hormone (TRH) analog was evaluated for its antiepileptic potential. NP-355 (5, 10 and 20 micromol/kg; i.v.) pretreatment significantly delayed onset and reduced the frequency of convulsions in pentylenetetrazole-induced seizures. NP-355 was also found to be protective against picrotoxin- and kainic acid-induced seizures. Maximum electroshock-induced seizures were not protected even at 20 micromol/kg in mice. Effects of NP-355 on functional observation battery did not exhibit any undesirable effects. Moreover, the antiepileptic activity produced by NP-355 was observed without significantly altering mean arterial blood pressure. NP-355 significantly increases the CBF to 17+/-3% as compared to saline (6+/-2%). NP-355 (100, 300 and 1000 microM) produces a concentration-dependent depression (16%, 63% and 77%, respectively) of the peak sodium current. NP-355 did not alter neurobehavioral parameters. This study demonstrates that NP-355 has potential antiepileptic activity and devoid of undesirable effects.