Synthesis, characterization and antioxidant activity of angiotensin converting enzyme inhibitors

Angiotensin converting enzyme (ACE) catalyzes the conversion of angiotensin I (Ang I) to angiotensin II (Ang II). ACE also cleaves the terminal dipeptide of vasodilating hormone bradykinin (a nonapeptide) to inactivate this hormone. Therefore, inhibition of ACE is generally used as one of the methods for the treatment of hypertension. `Oxidative stress' is another disease state caused by an imbalance in the production of oxidants and antioxidants. A number of studies suggest that hypertension and oxidative stress are interdependent. Therefore, ACE inhibitors having antioxidant property are considered beneficial for the treatment of hypertension. As selenium compounds are known to exhibit better antioxidant behavior than their sulfur analogues, we have synthesized a number of selenium analogues of captopril, an ACE inhibitor used as an antihypertensive drug. The selenium analogues of captopril not only inhibit ACE activity but also effectively scavenge peroxynitrite, a strong oxidant found in vivo.

Studies on molecular evolution and structural features of double-headed inhibitors of ?-amylase and trypsin in plants

Plant seeds usually have high concentrations of proteinase and amylase inhibitors. These inhibitors exhibit a wide range of specificity, stability and oligomeric structure. In this communication, we report analysis of sequences that show statistically significant similarity to the double-headed alpha-amylase/trypsin inhibitor of ragi (Eleusine coracana). Our aim is to understand their evolutionary and structural features. The 14 sequences of this family that are available in the SWISSPROT database form three evolutionarily distinct branches. The branches relate to enzyme specificities and also probably to the oligomeric state of the proteins and not to the botanical class of the plant from which the enzymes are derived. This suggests that the enzyme specificities of the inhibitors evolved before the divergence of commercially cultivated cereals. The inhibitor sequences have three regions that display periodicity in hydrophobicity. It is likely that this feature reflects extended secondary structure in these segments. One of the most variable regions of the polypeptide corresponds to a loop, which is most probably exposed in the native structure of the inhibitors and is responsible for the inhibitory property.

GyrI: a counter-defensive strategy against proteinaceous inhibitors of DNA gyrase

DNA gyrase is the target of two plasmid-encoded toxins CcdB and microcin B17, which ensure plasmid maintenance. These proteins stabilize gyrase-DNA covalent complexes leading to double-strand breaks in the genome. In contrast, the physiological role of chromosomally encoded inhibitor of DNA gyrase (Gyrl) in Escherichia coli is unclear and its mechanism of inhibition has not been established. We demonstrate that the mode of inhibition of GyrI is distinct from all other gyrase inhibitors. It inhibits DNA gyrase prior to, or at the step of, binding of DNA by the enzyme. Gyrl reduces intrinsic as well as toxin-stabilized gyrase-DNA covalent complexes. Furthermore, Gyri reduces microcin B17-mediated double-strand breaks in vivo, imparting protection to the cells against the toxin, substantiating the in vitro results. Thus, Gyrl is an antidote to DNA gyrase-specific proteinaceous poisons encoded by plasmid addiction systems.

Angiotensin converting enzyme inhibitors in the treatment of hypertension

Angiotensin converting enzyme (ACE) catalyses the conversion of angiotensin I (Ang I) to angiotensin II (Ang II). The ACE activity directly related to hypertension as Ang II is the blood pressure regulating hormone. Therefore, ACE inhibitors are a major class of antihypertensive drugs. Captopril, chemical name, was the first orally active ACE inhibitory antihypertensive drug, discovered in 1977. Since then, a number of such drugs have been synthesized. Enzyme-inhibitor bound crystal structural studies reveal a great deal of understanding about the interactions of the inhibitors at the active site of ACE. This can be helpful in the rational design of ACE inhibitors. With the advancement of the combination therapy, it is known that ACE inhibitors having antioxidant activity can be beneficial for the treatment of hypertension. This study describes the development of ACE inhibitors in the treatment of hypertension. Importance of ACE inhibitors having antioxidant activity is also described.

Benzothiophene Carboxamide Derivatives as Inhibitors of Plasmodium falciparum Enoyl-ACP Reductase

Benzothiophene derivatives like benzothiophene sulphonamides, biphenyls, or carboxyls have been synthesized and have found wide pharmacological usage. Here we report, bromo-benzothiophene carboxamide derivatives as potent, slow tight binding inhibitors of Plasmodium enoyl-acyl carrier protein (ACP) reductase (PfENR). 3-Bromo-N-(4-fluorobenzyl)-benzo[b]thiophene-2-carboxamide (compound 6) is the most potent inhibitor with an IC(50) of 115 nM for purified PfENR. The inhibition constant (K(i)) of compound 6 was 18 nM with respect to the cofactor and 91 nM with respect to crotonoyl-CoA. These inhibitors showed competitive kinetics with cofactor and uncompetitive kinetics with the substrate. Thus, these compounds hold promise for the development of potent antimalarials. (C) 2011 IUBMB IUBMB Life, 63(12): 1101-1110, 2011

Antioxidant activity of peptide-based angiotensin converting enzyme inhibitors

Angiotensin converting enzyme (ACE) inhibitors are important for the treatment of hypertension as they can decrease the formation of vasopressor hormone angiotensin II (Ang II) and elevate the levels of vasodilating hormone bradykinin. It is observed that bradykinin contains a Ser-Pro-Phe motif near the site of hydrolysis. The selenium analogues of captopril represent a novel class of ACE inhibitors as they also exhibit significant antioxidant activity. In this study, several di- and tripeptides containing selenocysteine and cysteine residues at the N-terminal were synthesized. Hydrolysis of angiotensin I (Ang I) to Ang II by ACE was studied in the presence of these peptides. It is observed that the introduction of L-Phe to Sec-Pro and Cys-Pro peptides significantly increases the ACE inhibitory activity. On the other hand, the introduction of L-Val or L-Ala decreases the inhibitory potency of the parent compounds. The presence of an L-Pro moiety in captopril analogues appears to be important for ACE inhibition as the replacement of L-Pro by L-piperidine 2-carboxylic acid decreases the ACE inhibition. The synthetic peptides were also tested for their ability to scavenge peroxynitrite (PN) and to exhibit glutathione peroxidase (GPx)-like activity. All the selenium-containing peptides exhibited good PN-scavenging and GPx activities.

Fourier transform infrared microspectroscopy identifies protein propionylation in histone deacetylase inhibitor treated glioma cells

Histone deacetylase inhibitors (HDIs) have attracted considerable attention as potential drug molecules in tumour biology. In order to optimise chemotherapy, it is important to understand the mechanisms of regulation of histone deacetylase (HDAC) enzymes and modifications brought by various HDIs. In the present study, we have employed Fourier transform infrared microspectroscopy (FT-IRMS) to evaluate modifications in cellular macromolecules subsequent to treatment with various HDIs. In addition to CH3 (methyl) stretching bands at 2872 and 2960 cm1, which arises due to acetylation, we also found major changes in bands at 2851 and 2922 cm1, which originates from stretching vibrations of CH2 (methylene) groups, in valproic acid treated cells. We further demonstrate that the changes in CH2 stretching are concentration-dependent and also induced by several other HDIs. Recently, HDIs have been shown to induce propionylation besides acetylation [1]. Since propionylation involves CH2 groups, we hypothesized that CH2 vibrational frequency changes seen in HDI treated cells could arise due to propionylation. As verification, pre-treatment of cells with propionyl CoA synthetase inhibitor resulted in loss of CH2 vibrational changes in histones, purified from valproic acid treated cells. This was further proved by western blot using propionyl-lysine specific antibody. Thus we demonstrate for the first time that propionylation could be monitored by studying CH2 stretching using IR spectroscopy and further provide a platform for monitoring HDI induced multiple changes in cells. (C) 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Binding of Gemini Bisbenzimidazole Drugs with Human Telomeric G-Quadruplex Dimers: Effect of the Spacer in the Design of Potent Telomerase Inhibitors

The study of anticancer agents that act via stabilization of telomeric G-quadruplex DNA (G4DNA) is important because such agents often inhibit telomerase activity. Several types of G4DNA binding ligands are known. In these studies, the target structures often involve a single G4 DNA unit formed by short DNA telomeric sequences. However, the 3'-terminal single-stranded human telomeric DNA can form higher-order structures by clustering consecutive quadruplex units (dimers or nmers). Herein, we present new synthetic gemini (twin) bisbenzimidazole ligands, in which the oligo-oxyethylene spacers join the two bisbenzimidazole units for the recognition of both monomeric and dimeric G4DNA, derived from d(T2AG3)4 and d(T2AG3) 8 human telomeric DNA, respectively. The spacer between the two bisbenzimidazoles in the geminis plays a critical role in the G4DNA stability. We report here (i) synthesis of new effective gemini anticancer agents that are selectively more toxic towards the cancer cells than the corresponding normal cells; (ii) formation and characterization of G4DNA dimers in solution as well as computational construction of the dimeric G4DNA structures. The gemini ligands direct the folding of the single-stranded DNA into an unusually stable parallel-stranded G4DNA when it was formed in presence of the ligands in KCl solution and the gemini ligands show spacer length dependent potent telomerase inhibition properties.

Infrared Spectroscopic Studies to Understand the Effect of Drugs at Molecular Level

In the recent past, there have been enormous efforts to understand effect of drugs on human body. Prior to understand the effect of drugs on human body most of the experiments are carried out on cells or model organisms. Here we present our study on the effect of chemotherapeutic drugs on cancer cells and the acetaminophen (APAP) induced hepatotoxicity in mouse model. Histone deacetylase inhibitors (HDIs) have attracted attention as potential drug molecules for the treatment of cancer. These are the chemotherapeutic drugs which have indirect mechanistic action against cancer cells via acting against histone deacetylases (HDAC). It has been known that different HDAC enzymes are over-expressed in various types of cancers for example; HDAC1 is over expressed in prostate, gastric and breast carcinomas. Therefore, in order to optimise chemotherapy, it is important to determine the efficacy of various classes of HDAC inhibitor drugs against variety of over-expressed HDAC enzymes. In the present study, FTIR microspectroscopy has been employed to predict the acetylation and propionylation brought in by HDIs. The liver plays an important role in cellular metabolism and is highly susceptible to drug toxicity. APAP which is an analgesic and antipyretic drug is extensively used for therapeutic purposes and has become the most common cause of acute liver failure (ALF). In the current study, we have focused to understand APAP induced hepatotoxicity using FTIR microspectroscopy. In the IR spectrum the bands corresponding to glycogen, ester group and were found to be suitable markers to predict liver injury at early time point (0.5hr) due to APAP both in tissue and serum in comparison to standard biochemical assays. Our studies show the potential of FTIR spectroscopy as a rapid, sensitive and non invasive detection technique for future clinical diagnosis.

Heat Shock Protein 90 Inhibitors as Broad Spectrum Anti-Infectives

Combating stress is one of the prime requirements for any organism. For parasitic microbes, stress levels are highest during the growth inside the host. Their survival depends on their ability to acclimatize and adapt to new environmental conditions. Robust cellular machinery for stress response is, therefore, both critical and essential especially for pathogenic microorganisms. Microbes have cleverly exploited stress proteins as virulence factors for pathogenesis in their hosts. Owing to its ability to sense and respond to the stress conditions, Heat shock protein 90 (Hsp90) is one of the key stress proteins utilized by parasitic microbes. There are growing evidences for the critical role played by Hsp90 in the growth of pathogenic organisms like Candida, Giardia, Plasmodium, Trypanosoma, and others. This review, therefore, explores potential of exploiting Hsp90 as a target for the treatment of infectious diseases. This molecular chaperone has already gained attention as an effective anti-cancer drug target. As a result, a lot of research has been done at laboratory, preclinical and clinical levels for several Hsp90 inhibitors as potential anti-cancer drugs. In addition, lot of data pertaining to toxicity studies, pharmacokinetics and pharmacodynamics studies, dosage regime, drug related toxicities, dose limiting toxicities as well as adverse drug reactions are available for Hsp90 inhibitors. Therefore, repurposing/repositioning strategies are also being explored for these compounds which have gone through advanced stage clinical trials. This review presents a comprehensive summary of current status of development of Hsp90 as a drug target and its inhibitors as candidate anti-infectives. A particular emphasis is laid on the possibility of repositioning strategies coupled with pharmaceutical solutions required for fulfilling needs for ever growing pharmaceutical infectious disease market.

Assay development for identifying inhibitors of the mycobacterial FadD32 activity

FadD32, a fatty acyl-AMP ligase (FAAL32) involved in the biosynthesis of mycolic acids, major and specific lipid components of the mycobacterial cell envelope, is essential for the survival of Mycobacterium tuberculosis, the causative agent of tuberculosis. The protein catalyzes the conversion of fatty acid to acyl-adenylate (acyl-AMP) in the presence of adenosine triphosphate and is conserved in all the mycobacterial species sequenced so far, thus representing a promising target for the development of novel antituberculous drugs. Here, we describe the optimization of the protein purification procedure and the development of a high-throughput screening assay for FadD32 activity. This spectrophotometric assay measuring the release of inorganic phosphate was optimized using the Mycobacterium smegmatis FadD32 as a surrogate enzyme. We describe the use of Tm (melting temperature) shift assay, which measures the modulation of FadD32 thermal stability, as a tool for the identification of potential ligands and for validation of compounds as inhibitors. Screening of a selected library of compounds led to the identification of five novel classes of inhibitors.

Small molecule inhibitors of HCV replication from Pomegranate

Hepatitis C virus (HCV) is the causative agent of end-stage liver disease. Recent advances in the last decade in anti HCV treatment strategies have dramatically increased the viral clearance rate. However, several limitations are still associated, which warrant a great need of novel, safe and selective drugs against HCV infection. Towards this objective, we explored highly potent and selective small molecule inhibitors, the ellagitannins, from the crude extract of Pomegranate (Punica granatum) fruit peel. The pure compounds, punicalagin, punicalin, and ellagic acid isolated from the extract specifically blocked the HCV NS3/4A protease activity in vitro. Structural analysis using computational approach also showed that ligand molecules interact with the catalytic and substrate binding residues of NS3/4A protease, leading to inhibition of the enzyme activity. Further, punicalagin and punicalin significantly reduced the HCV replication in cell culture system. More importantly, these compounds are well tolerated ex vivo and `no observed adverse effect level' (NOAEL) was established upto an acute dose of 5000 mg/kg in BALB/c mice. Additionally, pharmacokinetics study showed that the compounds are bioavailable. Taken together, our study provides a proof-of-concept approach for the potential use of antiviral and non-toxic principle ellagitannins from pomegranate in prevention and control of HCV induced complications.

Targeting Mycobacterium tuberculosis nucleoid-associated protein HU with structure-based inhibitors

The nucleoid-associated protein HU plays an important role in maintenance of chromosomal architecture and in global regulation of DNA transactions in bacteria. Although HU is essential for growth in Mycobacterium tuberculosis (Mtb), there have been no reported attempts to perturb HU function with small molecules. Here we report the crystal structure of the N-terminal domain of HU from Mtb. We identify a core region within the HU-DNA interface that can be targeted using stilbene derivatives. These small molecules specifically inhibit HU-DNA binding, disrupt nucleoid architecture and reduce Mtb growth. The stilbene inhibitors induce gene expression changes in Mtb that resemble those induced by HU deficiency. Our results indicate that HU is a potential target for the development of therapies against tuberculosis.