Real-world application of PBPK in drug discovery.

The utility of PBPK models in support of drug development has been well documented. During the discovery stage, PBPK has increasingly been applied for early risk assessment, prediction of human dose, toxicokinetic dose projection and early formulation assessment. Previous review articles have proposed model building and application strategies for PBPK-based first in human predictions with comprehensive descriptions of the individual components of PBPK models. This includes the generation of decision trees, based on comprehensive literature reviews, to guide the application of PBPK in the discovery setting. The goal of this mini review is to provide additional guidance on the real-world application of PBPK, in support of the discovery stage of drug development. In this mini review, our goal is to provide guidance on the typical steps involved in the development and application of a PBPK model during drug discovery to assist in decision making. We have illustrated our recommended approach through description of case examples, where PBPK has been successfully applied to aid in human PK projection, candidate selection and prediction of drug interaction liability for parent and metabolite. Through these case studies, we have highlighted fundamental issues, including pre-verification in preclinical species, the application of empirical scalars in the prediction of in vivo clearance from in vitro systems, in silico prediction of permeability and the exploration of aqueous and biorelevant solubility data to predict dissolution. In addition, current knowledge gaps have been highlighted and future directions proposed. Significance Statement Through description of three case studies, we have highlighted the fundamental principles of PBPK application during drug discovery. These include pre-verification of the model in preclinical species, application of empirical scalars where necessary in the prediction of clearance, in silico prediction of permeability, and the exploration of aqueous and biorelevant solubility data to predict dissolution. In addition, current knowledge gaps have been highlighted and future directions proposed.

Identification and characterization of the T cell receptor (TCR) repertoire of the cynomolgus macaque (Macaca Fascicularis).

BACKGROUND: Cynomolgus macaque (Macaca fascicularis) is an attractive animal model for the study of human disease and is extensively used in biomedical research. Cynomolgus macaques share behavioral, physiological, and genomic traits with humans and recapitulate human disease manifestations not observed in other animal species. To improve the use of the cynomolgus macaque model to investigate immune responses, we defined and characterized the T cell receptor (TCR) repertoire. RESULT: We identified and analyzed the alpha (TRA), beta (TRB), gamma (TRG), and delta (TRD) TCR loci of the cynomolgus macaque. The expressed repertoire was determined using 22 unique lung samples from Mycobacterium tuberculosis infected cynomolgus macaques by single cell RNA sequencing. Expressed TCR alpha (TRAV) and beta (TRBV) variable region genes were enriched and identified using gene specific primers, which allowed their functional status to be determined. Analysis of the primers used for cynomolgus macaque TCR variable region gene enrichment showed they could also be used to amplify rhesus macaque (M. mulatta) variable region genes. CONCLUSION: The genomic organization of the cynomolgus macaque has great similarity with the rhesus macaque and they shared > 90% sequence similarity with the human TCR repertoire. The identification of the TCR repertoire facilitates analysis of T cell immunity in cynomolgus macaques.

Development, validation and application of physiologically based biopharmaceutics model to justify the change in dissolution specifications for DRL ABC extended release tablets.

OBJECTIVE: The generic drug product DRL ABC is an Extended Release (ER) Tablet manufactured by Dr. Reddy's Laboratories Limited and have multi point dissolution as part of release specification. A proposal is being made to revise the dissolution specification and the aim of present work was to evaluate if this would still provide bioequivalent product. METHODS: PBBM was developed for DRL ABC using literature reported pharmacokinetic (PK) data. The intravenous PK data and in vitro metabolic rate constants were utilized for developing PBPK model first, followed by that in conjugation with mechanistic ACATTM model, a PBBM is developed for per-oral immediate release formulations. The validated model was applied to predict clinical bioequivalence (BE) study data for the Reference (Innovator ER Tablet) and Test product. For Reference and Test product, in vivo dissolution profiles were mechanistically deconvoluted from plasma concentration (Cp)-time profiles. Further, mechanistic in vitro-in vivo relationship (IVIVR) applied to in vitro release profiles of two hypothetical Test product batches (one with single point low dissolution profile (SPLP) and other with overall low dissolution profile (LP)) in order to calculate their in vivo releases and population simulation was performed with 40 virtual subjects. RESULTS: Results from the cross-over virtual trials showed BE between the Reference and various Test product batches (SPLP and LP), with maximum Cp (Cmax) and area under the Cp-time curve (AUC0-inf) well within 80-125% range. CONCLUSION: PBBM in conjugation with IVIVR and virtual BE was successfully applied for justifying changes in dissolution specification of DRL ABC.

Induction of Mitochondrial Cell Death and Reversal of Anticancer Drug Resistance via Nanocarriers Composed of a Triphenylphosphonium Derivative of Tocopheryl Polyethylene Glycol Succinate.

We have devised a nanocarrier using "tocopheryl polyethylene glycol succinate (TPGS) conjugated to triphenylphosphonium cation" (TPP-TPGS) for improving the efficacy of doxorubicin hydrochloride (DOX). Triphenylphosphonium cation (TPP) has affinity for an elevated transmembrane potential gradient (mitochondrial), which is usually high in cancer cells. Consequently, when tested in molecular docking and cytotoxicity assays, TPP-TPGS, owing to its structural similarity to mitochondrially directed anticancer compounds of the "tocopheryl succinate" family, interferes specifically in mitochondrial CII enzyme activity, increases intracellular oxidative stress, and induces apoptosis in breast cancer cells. DOX loaded nanocarrier (DTPP-TPGS) constructed using TPP-TPGS was positively charged, spherical in shape, sized below 100 nm, and had its drug content distributed evenly. DTPP-TPGS offers greater intracellular drug delivery due to its rapid endocytosis and subsequent endosomal escape. DTPP-TPGS also efficiently inhibits efflux transporter P glycoprotein (PgP), which, along with greater cell uptake and inherent cytotoxic activity of the construction material (TPP-TPGS), cumulatively results in 3-fold increment in anticancer activity of DOX in resistant breast cancer cells as well as greater induction of necroapoptosis and arrest in all phases of the cell cycle. DTPP-TPGS after intravenous administration in Balb/C mice with breast cancer accumulates preferentially in tumor tissue, which produces significantly greater antitumor activity when compared to DOX solution. Toxicity evaluation was also performed to confirm the safety of this formulation. Overall TPP-TPGS is a promising candidate for delivery of DOX.

Protein tyrosine kinase A modulates intracellular survival of mycobacteria through Galectin 3.

Mycobacterium tuberculosis (MTB) is a successful pathogen which increases persistence inside the host macrophage by subverting its defence mechanism. Mycobacteria regulate the pathogenesis and intracellular survival by controlling its interaction with host protein(s). Galectin 3 is a member of the ß-galactoside binding gene family which is involved in several biological functions. In the present study, we have expressed the mycobacterial protein tyrosine kinase (PtkA) in the cytosol of host macrophages through a eukaryotic promoter vector and found that it down-regulates Galectin 3. Infection by ptkA knocked-out (KO) mycobacterial strain shows increased level of Galectin 3 in the cytosol of macrophages. PtkA regulates Galectin 3 level and stimulates host macrophage through MEK-JNK-cJUN pathway and initiates early apoptosis in H37Ra infected macrophage. The ptkA KO strain showed decreased progression of apoptosis confirming Galectin 3 as anti-apoptotic molecule. The intracellular survival was also found to be impaired in the mice infected with ptkA KO mycobacteria. The hypothesis was also confirmed by looking at the intracellular survival of mycobacteria in Galectin 3 silenced macrophages. The overall findings suggest the significance of Galectin 3 and PtkA interaction in intracellular persistence of mycobacteria.

Antimicrobial and antibiofilm activity of curcumin-silver nanoparticles with improved stability and selective toxicity to bacteria over mammalian cells.

Antibiotic resistance has necessitated search for new antibacterials for combating threat of pathogenic bacteria. Though chemically synthesized silver nanoparticles are a well-known antimicrobial agent, they are toxic to human cells at higher concentrations. Hence in the present study, curcumin-silver nanoparticles (Cur-AgNPs) of size 25-35 nm, were synthesized using curcumin, a phytochemical. These nanoparticles were effective against both Gram positive and Gram-negative bacteria and were less toxic to human keratinocytes. They had very low total silver content and high stability. The antibacterial activity of Cur-AgNPs, as studied by minimum inhibitory concentration (MIC = 5 mg/L), time kill kinetics and post agent effect, was better than silver nanoparticles (AgNPs, size ≈ 35 nm, MIC = 20 mg/L). The inhibitory effect of Cur-AgNPs on biofilm formation was also ≈ 20% more than AgNPs as demonstrated by live-dead imaging and scanning electron microscopy. The cytotoxic test to skin keratinocytes (HaCaT) showed that Cur-AgNPs were toxic at a concentration of 156 mg/L which is much higher than the bacterial MIC (selective toxicity). They also showed anti-inflammatory effect on human macrophages (THP1) by reducing secretion of pro-inflammatory cytokines IL-6 and TNF-α as compared to chemically synthesized AgNPs.

Click Biotinylation of PLGA Template for Biotin Receptor Oriented Delivery of Doxorubicin Hydrochloride in 4T1 Cell-Induced Breast Cancer.

PLGA was functionalized with PEG and biotin using click chemistry to generate a biotin receptor targeted copolymer (biotinylated-PEG-PLGA) which in turn was used to fabricate ultrafine nanoparticles (BPNP) of doxorubicin hydrochloride (DOX) for effective delivery in 4T1 cell induced breast cancer. However, adequate entrapment of a hydrophilic bioactive like DOX in a hydrophobic polymer system made of PLGA is not usually possible. We therefore modified a conventional W/O/W emulsion method by utilizing NH4Cl in the external phase to constrain DOX in dissolved polymer phase by suppressing DOX's inherent aqueous solubility as per common ion effect. This resulted in over 8-fold enhancement in entrapment efficiency of DOX inside BPNP, which otherwise is highly susceptible to leakage due to its relatively high aqueous solubility. TEM and DLS established BPNP to be sized below 100 nm, storage stability studies showed that BPNP were stable for one month at 4 °C, and in vitro release suggested significant control in drug release. Extensive in vitro and in vivo studies were conducted to propound anticancer and antiproliferative activity of BPNP. Plasma and tissue distribution study supplemented by pertinent in vivo fluorescence imaging mapped the exact fate of DOX contained inside BPNP once it was administered intravenously. A comparative safety profile via acute toxicity studies in mice was also generated to out rightly establish usefulness of BPNP. Results suggest that BPNP substantially enhance anticancer activity of DOX while simultaneously mitigating its toxic potential due to altered spatial and temporal presentation of drug and consequently deserve further allometric iteration.

A combination of complexation and self-nanoemulsifying drug delivery system for enhancing oral bioavailability and anticancer efficacy of curcumin.

OBJECTIVE: Curcumin, the golden spice from Indian saffron, has shown chemoprotective action against many types of cancer including breast cancer. However, poor oral bioavailability is the major hurdle in its clinical application. In the recent years, self-nanoemulsifying drug delivery system (SNEDDS) has emerged as a promising tool to improve the oral absorption and enhancing the bioavailability of poorly water-soluble drugs. In this context, complexation with lipid carriers like phospholipid has also shown the tremendous potential to improve the solubility and therapeutic efficacy of certain drugs with poor oral bioavailability. METHODS: In the present investigation, a systematic combination of both the approaches is utilized to prepare the phospholipid complex of curcumin and facilitate its incorporation into SNEDDS. The combined use of both the approaches has been explored for the first time to enhance the oral bioavailability and in turn increase the anticancer activity of curcumin. RESULTS: As evident from the pharmacokinetic studies and in situ single pass intestinal perfusion studies in Sprague-Dawley rats, the optimized SNEDDS of curcumin-phospholipid complex has shown enhanced oral absorption and bioavailability of curcumin. The cytotoxicity study in metastatic breast carcinoma cell line has shown the enhancement of cytotoxic action by 38.7%. The primary tumor growth reduction by 58.9% as compared with the control group in 4T1 tumor-bearing BALB/c mice further supported the theory of enhancement of anticancer activity of curcumin in SNEDDS. CONCLUSION: The developed formulation can be a potential and safe carrier for the oral delivery of curcumin.

Protein kinase C-δ inhibitor, Rottlerin inhibits growth and survival of mycobacteria exclusively through Shikimate kinase.

The molecular bases of disease provide exceptional prospect to translate research findings into new drugs. Nevertheless, to develop new and novel chemical entities takes huge amount of time and efforts, mainly due to the stringent processes. Therefore, drug repurposing is one of such strategies which is being used in recent times to identify new pharmacophores. The essential first step in discovery of the specific inhibitor with low toxicity is the identification and elucidation of pathways exclusive to target pathogen. One such target is the shikimate pathway, which is essential for algae, higher plants, bacteria and fungi. Since, this enzyme system is absent in higher eukaryotes and in mammals, the enzymes involved in the pathway provide an attractive target for the development of potentially selective and non toxic antimicrobial agents. Since, so far there is no specific inhibitor which is able to restrain mycobacterial shikimate pathway; we expanded the use of a known kinase inhibitor; Rottlerin, in order to predict the prototype in discovering the specific molecules against this enzyme. For the first time we have shown that Rottlerin inhibits extracellular mycobacteria by affecting Shikimate Kinase (SK) and this effect is further enhanced during the intracellular infection due to the added effect of PKC- δ down-regulation. The molecular docking of Rottlerin with both the mycobacterial SKs, corroborated the inhibition data, and revealed that the effects of SK, in slow and in fast grower mycobacteria are due to the changes in affinity of binding with the drug.

Role of enterohepatic recirculation in drug disposition: cooperation and complications.

Enterohepatic recirculation (EHC) concerns many physiological processes and notably affects pharmacokinetic parameters such as plasma half-life and AUC as well as estimates of bioavailability of drugs. Also, EHC plays a detrimental role as the compounds/drugs are allowed to recycle. An in-depth comprehension of this phenomenon and its consequences on the pharmacological effects of affected drugs is important and decisive in the design and development of new candidate drugs. EHC of a compound/drug occurs by biliary excretion and intestinal reabsorption, sometimes with hepatic conjugation and intestinal deconjugation. EHC leads to prolonged elimination half-life of the drugs, altered pharmacokinetics and pharmacodynamics. Study of the EHC of any drug is complicated due to unavailability of the apposite model, sophisticated procedures and ethical concerns. Different in vitro and in vivo methods for studies in experimental animals and humans have been devised, each having its own merits and demerits. Involvement of the different transporters in biliary excretion, intra- and inter-species, pathological and biochemical variabilities obscure the study of the phenomenon. Modeling of drugs undergoing EHC has always been intricate and exigent models have been exploited to interpret the pharmacokinetic profiles of drugs witnessing multiple peaks due to EHC. Here, we critically appraise the mechanisms of bile formation, factors affecting biliary drug elimination, methods to estimate biliary excretion of drugs, EHC, multiple peak phenomenon and its modeling.

Novel, potent, orally bioavailable and selective mycobacterial ATP synthase inhibitors that demonstrated activity against both replicating and non-replicating M. tuberculosis.

The mycobacterial F0F1-ATP synthase (ATPase) is a validated target for the development of tuberculosis (TB) therapeutics. Therefore, a series of eighteen novel compounds has been designed, synthesized and evaluated against Mycobacterium smegmatis ATPase. The observed ATPase inhibitory activities (IC50) of these compounds range between 0.36 and 5.45µM. The lead compound 9d [N-(7-chloro-2-methylquinolin-4-yl)-N-(3-((diethylamino)methyl)-4-hydroxyphenyl)-2,3-dichlorobenzenesulfonamide] with null cytotoxicity (CC50>300µg/mL) and excellent anti-mycobacterial activity and selectivity (mycobacterium ATPase IC50=0.51µM, mammalian ATPase IC50>100µM, and selectivity >200) exhibited a complete growth inhibition of replicating Mycobacterium tuberculosis H37Rv at 3.12µg/mL. In addition, it also exhibited bactericidal effect (approximately 2.4log10 reductions in CFU) in the hypoxic culture of non-replicating M. tuberculosis at 100µg/mL (32-fold of its MIC) as compared to positive control isoniazid [approximately 0.2log10 reduction in CFU at 5µg/mL (50-fold of its MIC)]. The pharmacokinetics of 9d after p.o. and IV administration in male Sprague-Dawley rats indicated its quick absorption, distribution and slow elimination. It exhibited a high volume of distribution (Vss, 0.41L/kg), moderate clearance (0.06L/h/kg), long half-life (4.2h) and low absolute bioavailability (1.72%). In the murine model system of chronic TB, 9d showed 2.12log10 reductions in CFU in both lung and spleen at 173µmol/kg dose as compared to the growth of untreated control group of Balb/C male mice infected with replicating M. tuberculosis H37Rv. The in vivo efficacy of 9d is at least double of the control drug ethambutol. These results suggest 9d as a promising candidate molecule for further preclinical evaluation against resistant TB strains.

Coadministration of HMG-CoA reductase inhibitors, atorvastatin and rosuvastatin, does not affect contraceptive efficacy of centchroman.

OBJECTIVE: The study aimed to investigate the effect of concomitant use of atorvastatin or rosuvastatin on the pharmacokinetic and pharmacodynamic profile of centchroman, a non-steroidal female oral contraceptive. METHODS: A rat model was used to predict pharmacokinetic drug-drug interactions between centchroman and atorvastatin or rosuvastatin. A dried blood spot sampling technique followed by liquid chromatography-tandem mass spectrometry detection was employed for analysis of the pharmacokinetic interaction study samples. Sperm-positive female rats were investigated for postcoital contraceptive activity of centchroman with or without coadministration of atorvastatin or rosuvastatin. RESULTS: Coadministration of atorvastatin or rosuvastatin may increase the systemic availability of centchroman in blood, but it does not affect the pharmacodynamic profile of centchroman. CONCLUSION: Atorvastatin or rosuvastatin may be prescribed with centchroman without compromising the contraceptive efficacy of centchroman.

Novel pre-clinical methodologies for pharmacokinetic drug-drug interaction studies: spotlight on "humanized" animal models.

Poly-therapy is common due to co-occurrence of several ailments in patients, leading to the elevated possibility of drug-drug interactions (DDI). Pharmacokinetic DDI often accounts for severe adverse drug reactions in patients resulting in withdrawal of drug from the market. Hence, the prediction of DDI is necessary at pre-clinical stage of drug development. Several human tissue and cell line-based in vitro systems are routinely used for screening metabolic and transporter pathways of investigational drugs and for predicting their clinical DDI potentials. However, ample constraints are associated with the in vitro systems and sometimes in vitro-in vivo extrapolation (IVIVE) fail to assess the risk of DDI in clinic. In vitro-in vivo correlation model in animals combined with human in vitro studies may be helpful in better prediction of clinical outcome. Native animal models vary remarkably from humans in drug metabolizing enzymes and transporters, hence, the interpretation of results from animal DDI studies is difficult. With the advent of modern molecular biology and engineering tools, novel pre-clinical animal models, namely, knockout rat/mouse, transgenic rat/mouse with humanized drug metabolizing enzymes and/or transporters and chimeric rat/mouse with humanized liver are developed. These models nearly simulate human-like drug metabolism and help to validate the in vivo relevance of the in vitro human DDI data. This review briefly discusses the application of such novel pre-clinical models for screening various type of DDI along with their advantages and limitations.

Targeting Solute Carrier Transporters (SLCs) as a Therapeutic Target in Different Cancers.

Solute carrier (SLC) transporters constitute a vast superfamily of transmembrane proteins tasked with regulating the transport of various substances such as metabolites, nutrients, ions, and drugs across cellular membranes. SLC transporters exhibit coordinated expression patterns across normal tissues, suggesting a tightly regulated regulatory network governing normal cellular functions. These transporters are crucial for the transport of various metabolites, including carbohydrates, proteins, lipids, and nucleic acids. However, during tumor development, metabolic changes drive an increased demand for energy and nutrients. Consequently, tumor cells alter the expression of SLC transporters to meet their heightened nutrient requirements. Targeting SLCs through inhibition or activation presents a promising therapeutic approach in cancer treatment. Certain SLCs also serve as intriguing chemo-sensitizing targets, as modulating their activity can potentially alter the response to chemotherapy. This review underscores the significance of various SLCs in tumor progression and underscores their potential as both direct and indirect targets for cancer therapy.

Exploring the role of the protein tyrosine kinase a (PtkA) in mycobacterial intracellular survival.

Mycobacterium tuberculosis (Mtb) continues to define new paradigms of host-pathogen interaction. There are several host proteins known which are regulated by Mtb infection. The proteins which regulate host biological processes like apoptosis, cell processes, stress proteins, metabolic enzymes, etc. are targeted by the pathogens. Mtb proteins interact directly or indirectly with host proteins and play an important role in their persistence and intracellular growth. Mtb is an intracellular pathogen. It remains dormant for years within the host without activating its immune system. Mtb Protein tyrosine kinase (PtkA) regulates host anti-apoptotic protein, metabolic enzymes, and several other proteins that are involved in stress regulation, cell proliferation, protein folding, DNA repair, etc. PtkA regulates other mycobacterial proteins and plays an important role in its growth and survival. Here we summarized the current knowledge of PtkA and reviewed its role in mycobacterial intracellular survival as it regulates several other mycobacterial proteins and host proteins. PtkA regulates PtpA secretion which is essential for mycobacterial virulence and could be used as an attractive drug target.

Synthesis and validation of click-modified NOD1/2 agonists.

NOD1 and NOD2 sense small bacterial peptidoglycan fragments, often called muropeptides, that access the cytosol. These muropeptides include iE-DAP and MDP, the minimal agonists for NOD1 and NOD2, respectively. Here, we synthesized and validated alkyne-modified muropeptides, iE-DAP-Alk and MDP-Alk, for use in click-chemistry reactions. While it has long been known that many cell types respond to extracellular exposure to muropeptides, it is unclear how these innate immune activators access their cytosolic innate immune receptors, NOD1 and NOD2. The subcellular trafficking and transport mechanisms by which muropeptides access these cytosolic innate immune receptors are a major gap in our understanding of these critical host responses. The click-chemistry-enabled agonists developed here will be particularly powerful to decipher the underlying cell biology and biochemistry of NOD1 and NOD2 innate immune sensing.

Enriching and Characterizing T Cell Repertoires from 3' Barcoded Single-Cell Whole Transcriptome Amplification Products.

Antigen-specific T cells play an essential role in immunoregulation and many diseases such as cancer. Characterizing the T cell receptor (TCR) sequences that encode T cell specificity is critical for elucidating the antigenic determinants of immunological diseases and designing therapeutic remedies. However, methods of obtaining single-cell TCR sequencing data are labor and cost intensive, typically requiring both cell sorting and full-length single-cell RNA-sequencing (scRNA-seq). New high-throughput 3' cell-barcoding scRNA-seq methods can simplify and scale this process; however, they do not routinely capture TCR sequences during library preparation and sequencing. While 5' cell-barcoding scRNA-seq methods can be used to examine TCR repertoire at single-cell resolution, doing so requires specialized reagents which cannot be applied to samples previously processed using 3' cell-barcoding methods.Here, we outline a method for sequencing TCRα and TCRß transcripts from samples already processed using 3' cell-barcoding scRNA-seq platforms, ensuring TCR recovery at a single-cell resolution. In short, a fraction of the 3' barcoded whole transcriptome amplification (WTA) product typically used to generate a massively parallel 3' scRNA-seq library is enriched for TCR transcripts using biotinylated probes and further amplified using the same universal primer sequence from WTA. Primer extension using TCR V-region primers and targeted PCR amplification using a second universal primer result in a 3' barcoded single-cell CDR3-enriched library that can be sequenced with custom sequencing primers. Coupled with 3' scRNA-seq of the same WTA, this method enables simultaneous analysis of single-cell transcriptomes and TCR sequences which can help interpret inherent heterogeneity among antigen-specific T cells and salient disease biology. The method presented here can also be adapted readily to enrich and sequence other transcripts of interest from both 3' and 5' barcoded scRNA-seq WTA libraries.

ATP synthase, an essential enzyme in growth and multiplication is modulated by protein tyrosine phosphatase in Mycobacterium tuberculosis H37Ra.

Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase (PtpA) has so far been known to control intracellular survival of mycobacteria; whereas the ATP synthase which is essential for mycobacterial growth has recently been contemplated in developing a breakthrough anti-TB drug, diarylquinoline. Since both of these enzymes have been established as validated drug targets; we report a robust and functional relationship between these two enzymes through a series of experiments using Mtb H37Ra. In the present study we report that the mycobacterial ATP synthase alpha subunit is regulated by PtpA. We generated gene knock-out for the enzyme PtpA and subjected to determine the mycobacterial replication and the proteome profile of wild type, mutant (ΔptpA) and complemented (ΔptpA:ptpA) strains of Mtb H37Ra. A substantial amount of decrease in the protein level of ATP synthase alpha subunit (AtpA) in case of mutant H37Ra was observed, while the levels of the enzyme were either increased or remained unchanged, in wild type and in the complemented strains.

Mycobacterial protein tyrosine kinase, PtkA phosphorylates PtpA at tyrosine residues and the mechanism is stalled by the novel series of inhibitors.

Phosphorylation and dephosphorylation are the key mechanisms for mycobacterial physiology and play critical roles in mycobacterial survival and in its pathogenesis. Mycobacteria evade host immune mechanism by inhibiting phagosome - lysosome fusion in which mycobacterial protein tyrosine phosphatase A (PtpA;TP) plays an indispensable role. Tyrosine kinase (PtkA;TK) activated by autophosphorylation; phosphorylates TP, which subsequently leads to increase in its phosphatase activity. The phosphorylated TP is secreted in phagosome of macrophage. In the present study, we have shown that the phosphorylation at two sites of TP; Y128 and Y129 are critical for TK-mediated phosphatase activity. The disruption of this interaction between TK and TP inhibits activation of later which further leads to the decrease in intracellular survival of mycobacteria. Furthermore, the proof of concept has been established using benzylbenzofurans and benzofuranamides, which inhibit the growth and intracellular survival of mycobacteria, associate with the functional sites of TP and contend with the TK. This binding was further restated by looking at the anchorage of protein-protein and the protein-inhibitor complexes in the homology-based structure models and by surface plasmon resonance analysis.

Novel Tetrahydroquinazolinamines as Selective Histamine 3 Receptor Antagonists for the Treatment of Obesity.

The histamine 3 receptor (H3R) is a presynaptic receptor, which modulates several neurotransmitters including histamine and various essential physiological processes, such as feeding, arousal, cognition, and pain. The H3R is considered as a drug target for the treatment of several central nervous system disorders. We have synthesized and identified a novel series of 4-aryl-6-methyl-5,6,7,8-tetrahydroquinazolinamines that act as selective H3R antagonists. Among all the synthesized compounds, in vitro and docking studies suggested that the 4-methoxy-phenyl-substituted tetrahydroquinazolinamine compound 4c has potent and selective H3R antagonist activity (IC50 < 0.04 µM). Compound 4c did not exhibit any activity on the hERG ion channel and pan-assay interference compounds liability. Pharmacokinetic studies showed that 4c crosses the blood brain barrier, and in vivo studies demonstrated that 4c induces anorexia and weight loss in obese, but not in lean mice. These data reveal the therapeutic potential of 4c as an anti-obesity candidate drug via antagonizing the H3R.