Design, Synthesis, Biological Evaluation And Molecular Modeling Studies Of Novel Multifunctional Neuroprotective Drugs For The Treatment Of Parkinson\u27s Disease: An Effort Towards The Improvement Of In Vivo Efficacy And Modulation Of Alpha Synuclein Aggregation Property Of The Neuroprotective Parent

DESIGN, SYNTHESIS, BIOLOGICAL EVALUATION AND MOLECULAR MODELING STUDIES OF NOVEL MULTIFUNCTIONAL NEUROPROTECTIVE DRUGS FOR THE TREATMENT OF PARKINSON\u27S DISEASE: AN EFFORT TOWARDS THE IMPROVEMENT OF IN VIVO EFFICACY AND MODULATION OF ALPHA SYNUCLEIN AGGREGATION PROPERTY OF THE NEUROPROTECTIVE PARENT MOLECULE (D-264) by GYAN PRAKASH MODI May 2014 Advisor: Dr. Aloke K. Dutta Major: Pharmaceutical Sciences Degree: Doctor of Philosophy Parkinson\u27s disease (PD) is a progressive age-related neurodegenerative disorder of the central nervous system that is characterized by gradual loss of dopaminergic neurons in the substantia nigra region of the brain. The research from the past two decades in PD area has provided more insights into the basic pathogenetic factors of PD such as roles of oxidative stress, aggregation of α-synuclein (ASN) proteins in the form soluble toxic aggregates and fibrils, increased concentration of iron in the PD brain. Levodopa (L-DOPA) became available in 1960 for the treatment of PD and is still being considered as one of the main stream therapy. However, prolog use of L-DOPA gives rise to on and off episode along with motor fluctuations and eventual oxidation of dopamine (DA) derived from L-DOPA further facilitates neurodegeneration. It is increasingly evident that drugs aiming a single target may be inadequate for the treatment of complex diseases such as PD, which is multifactorial in nature. Thus, it is hypothesized that multifunctional drugs having multiple pharmacological activities addressing multiple pathogenic factors of PD will be effective as disease modifying agent for the treatment of this disease. Our aim in the first study was to enhance brain penetration of one of our lead molecule D-264. Our current structure activity relationship study is focused on introduction of methoxy and hydroxyl group at various positions on the accessory binding biphenyl ring of this hybrid molecule. The introduction of hydroxyl group or combination of hydroxyl/methoxy group at a suitable position could further potentiate its antioxidant and neuroprotection property. Among all synthesized compounds in the first series, compound D-433 and D-533 exhibited the highest selectivity for the D3 over D2 receptor in both binding and functional assays. Lead compounds D-433 and D-533 also exhibited potent free radical quenching property, possibly indicating antioxidant activity. The lead compounds were tested in two PD animal models. Both the compounds exhibited higher blood brain barrier crossing ability compared to parent compounds D-264. Furthermore, in MTT assay lead compounds are able to protect MN9D cells from the exposure to neurotoxin MPP+ and 6-OHDA in a dose dependent manner. Compounds D-519 and D-520 were selected as lead molecules from the second series and they exhibited nanomolar to sub nanomolar range affinity at D2/D3 receptors in the receptor binding assay and [35S]GTPγS binding assay. It was concluded from this in vivo study that both D-519 and D-520 was able to efficiently cross blood brain barrier and exhibited high in vivo agonist efficacy. D-519 and D-520 can potentially chelate with Fe(III). Furthermore, D-520 is able to reverse the ASN aggregates induced toxicity at a significant level in PC-12 cells. Finally, three dimensional quantitative structure activity relationship (3DQSAR) studies CoMFA and CoMSIA were performed. Two alignment methods (atom base and flexible) and two charge calculation methods (Gasteinger-Huckel and MOPAC) were used. The presence of carbonyl group attached to piperazine ring and hydrophobic biphenyl ring was found to be one of the most important factors responsible for the D3 selectivity over D2

Mycobacterium tuberculosis IMPDH in Complexes with Substrates, Products and Antitubercular Compounds

Tuberculosis (TB) remains a worldwide problem and the need for new drugs is increasingly more urgent with the emergence of multidrug- and extensively-drug resistant TB. Inosine 5’-monophosphate dehydrogenase 2 (IMPDH2) from Mycobacterium tuberculosis (Mtb) is an attractive drug target. The enzyme catalyzes the conversion of inosine 5’-monophosphate into xanthosine 5’-monophosphate with the concomitant reduction of NAD+ to NADH. This reaction controls flux into the guanine nucleotide pool. We report seventeen selective IMPDH inhibitors with antitubercular activity. The crystal structures of a deletion mutant of MtbIMPDH2 in the apo form and in complex with the product XMP and substrate NAD+ are determined. We also report the structures of complexes with IMP and three structurally distinct inhibitors, including two with antitubercular activity. These structures will greatly facilitate the development of MtbIMPDH2-targeted antibiotics

The Evolving Landscape of Exosomes in Neurodegenerative Diseases : Exosomes Characteristics and a Promising Role in Early Diagnosis

Neurodegenerative diseases (ND) remains to be one of the biggest burdens on healthcare systems and serves as a leading cause of disability and death. Alzheimer’s disease (AD) is among the most common of such disorders, followed by Parkinson’s disease (PD). The basic molecular details of disease initiation and pathology are still under research. Only recently, the role of exosomes has been linked to the initiation and progression of these neurodegenerative diseases. Exosomes are small bilipid layer enclosed extracellular vesicles, which were once considered as a cellular waste and functionless. These nano-vesicles of 30–150 nm in diameter carry specific proteins, lipids, functional mRNAs, and high amounts of non-coding RNAs (miRNAs, lncRNAs, and circRNAs). As the exosomes content is known to vary as per their originating and recipient cells, these vesicles can be utilized as a diagnostic biomarker for early disease detection. Here we review exosomes, their biogenesis, composition, and role in neurodegenerative diseases. We have also provided details for their characterization through an array of available techniques. Their updated role in neurodegenerative disease pathology is also discussed. Finally, we have shed light on a novel field of salivary exosomes as a potential candidate for early diagnosis in neurodegenerative diseases and compared the biomarkers of salivary exosomes with other blood/cerebrospinal fluid (CSF) based exosomes within these neurological ailmentsValiderad;2021;Nivå 2;2021-01-13 (johcin)</p

Inhibition of Inosine-5′-monophosphate Dehydrogenase from <i>Bacillus anthracis</i>: Mechanism Revealed by Pre-Steady-State Kinetics

Inosine-5′-monophosphate dehydrogenase (IMPDH) catalyzes the conversion of inosine 5′-monophosphate (IMP) to xanthosine 5′-monophosphate (XMP). The enzyme is an emerging target for antimicrobial therapy. The small molecule inhibitor <b>A110</b> has been identified as a potent and selective inhibitor of IMPDHs from a variety of pathogenic microorganisms. A recent X-ray crystallographic study reported that the inhibitor binds to the NAD⁺ cofactor site and forms a ternary complex with IMP. Here we report a pre-steady-state stopped-flow kinetic investigation of IMPDH from <i>Bacillus anthracis</i> designed to assess the kinetic significance of the crystallographic results. Stopped-flow kinetic experiments defined nine microscopic rate constants and two equilibrium constants that characterize both the catalytic cycle and details of the inhibition mechanism. In combination with steady-state initial rate studies, the results show that the inhibitor binds with high affinity (<i>K</i>_d ≈ 50 nM) predominantly to the covalent intermediate on the reaction pathway. Only a weak binding interaction (<i>K</i>_d ≈ 1 μM) is observed between the inhibitor and E·IMP. Thus, the E·IMP·<b>A110</b> ternary complex, observed by X-ray crystallography, is largely kinetically irrelevant

Spectroscopic insight into breast cancer: profiling small extracellular vesicles lipids via infrared spectroscopy for diagnostic precision

Breast cancer, a leading cause of female mortality due to delayed detection owing to asymptomatic nature and limited early diagnostic tools, was investigated using a multi-modal approach. Plasma-derived small EVs from breast cancer patients (BrCa, n = 74) and healthy controls (HC, n = 30) were analyzed. Small EVs (n = 104), isolated through chemical precipitation, underwent characterization via transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Validation involved antibody-based tests (TSG101, CD9, CD81, CD63). Infrared spectra of small EVs were obtained, revealing significant differences in lipid acyl chains, particularly in the C–H stretching of CH3. The study focused on the lipid region (3050–2900 cm−1), identifying peaks (3015 cm−1, 2960 cm−1, 2929 cm−1) as distinctive lipid characteristics. Spectroscopic lipid-to-lipid ratios [(I3015/I2929), (I2960/I2929)] emerged as prominent breast cancer markers. Exploration of protein, nucleic acid, and carbohydrate ratios indicated variations in alpha helices, asymmetric C–H stretching vibrations, and C–O stretching at 1033 cm−1. Principal component analysis (PCA) successfully differentiated BrCa and HC small EVs, and heatmap analysis and receiver operating characteristic (ROC) curve evaluations underscored the discriminatory power of lipid ratios. Notably, (I2960/I2929) exhibited 100% sensitivity and specificity, highlighting its potential as a robust BrCa sEV marker for breast cancer detection.Full text license: CC BY 4.0;Funder: Lulea University of Technology; Department of Biotechnology, India (DBT/2021-22/THSTI/1601);</p

Novel Bivalent Ligands for D2/D3 Dopamine Receptors: Significant Cooperative Gain in D2 Affinity and Potency

This report describes development of a series of novel bivalent molecules with a pharmacophore derived from the D2/D3 agonist 5-OH-DPAT. The spacer length in the bivalent compounds had a pronounced influence on affinity for D2 receptors. A 23-fold increase of D2 affinity was observed at a spacer length of 9 or 10 (compounds <b>11d</b> and <b>14b</b>) as compared to monovalent 5-OH-DPAT (<i>K</i>_i; 2.5 and 2.0 vs 59 nM for <b>11d</b> and <b>14b</b> vs 5-OH-DPAT, respectively). The functional potency of <b>11d</b> and <b>14b</b> indicated a 24- and 94-fold increase in potency at the D2 receptor as compared to 5-OH-DPAT (EC₅₀; 1.7 and 0.44 vs 41 nM for <b>11d</b> and <b>14b</b> vs 5-OH-DPAT, respectively). These are the most potent bivalent agonists for the D2 receptor known to date. This synergism is consonant with cooperative interaction at the two orthosteric binding sites in the homodimeric receptor

<i>Mycobacterium tuberculosis</i> IMPDH in Complexes with Substrates, Products and Antitubercular Compounds

Tuberculosis (TB) remains a worldwide problem and the need for new drugs is increasingly more urgent with the emergence of multidrug- and extensively-drug resistant TB. Inosine 5’-monophosphate dehydrogenase 2 (IMPDH2) from Mycobacterium tuberculosis (Mtb) is an attractive drug target. The enzyme catalyzes the conversion of inosine 5’-monophosphate into xanthosine 5’-monophosphate with the concomitant reduction of NAD+ to NADH. This reaction controls flux into the guanine nucleotide pool. We report seventeen selective IMPDH inhibitors with antitubercular activity. The crystal structures of a deletion mutant of MtbIMPDH2 in the apo form and in complex with the product XMP and substrate NAD+ are determined. We also report the structures of complexes with IMP and three structurally distinct inhibitors, including two with antitubercular activity. These structures will greatly facilitate the development of MtbIMPDH2-targeted antibiotics

Circulating plasma miR-23b-3p as a biomarker target for idiopathic Parkinson's disease: comparison with small extracellular vesicle miRNA

Background: Parkinson's disease (PD) is an increasingly common neurodegenerative condition, which causes movement dysfunction and a broad range of non-motor symptoms. There is no molecular or biochemical diagnosis test for PD. The miRNAs are a class of small non-coding RNAs and are extensively studied owing to their altered expression in pathological states and facile harvesting and analysis techniques. Methods: A total of 48 samples (16 each of PD, aged-matched, and young controls) were recruited. The small extracellular vesicles (sEVs) were isolated and validated using Western blot, transmission electron microscope, and nanoparticle tracking analysis. Small RNA isolation, library preparation, and small RNA sequencing followed by differential expression and targeted prediction of miRNA were performed. The real-time PCR was performed with the targeted miRNA on PD, age-matched, and young healthy control of plasma and plasma-derived sEVs to demonstrate their potential as a diagnostic biomarker. Results: In RNA sequencing, we identified 14.89% upregulated (fold change 1.11 to 11.04, p &lt; 0.05) and 16.54% downregulated (fold change −1.04 to −7.28, p &lt; 0.05) miRNAs in PD and controls. Four differentially expressed miRNAs (miR-23b-3p, miR-29a-3p, miR-19b-3p, and miR-150-3p) were selected. The expression of miR-23b-3p was “upregulated” (p = 0.002) in plasma, whereas “downregulated” (p = 0.0284) in plasma-derived sEVs in PD than age-matched controls. The ROC analysis of miR-23b-3p revealed better AUC values in plasma (AUC = 0.8086, p = 0.0029) and plasma-derived sEVs (AUC = 0.7278, p = 0.0483) of PD and age-matched controls. Conclusion: We observed an opposite expression profile of miR-23b-3p in PD and age-matched healthy control in plasma and plasma-derived sEV fractions, where the expression of miR-23b-3p is increased in PD plasma while decreased in plasma-derived sEV fractions. We further observed the different miR-23b-3p expression profiles in young and age-matched healthy control.Validerad;2023;Nivå 2;2023-11-21 (hanlid);Funder: Indian Council of Medical Research (ICMR) (2020-1194); Department of Health Research (DHR) (R.11013/21/2021-GIA/HR); Ministry of Health and Family WelfareFull text license: CC BY</p