Nonsmall-cell lung cancer treatment: current status of drug repurposing and nanoparticle-based drug delivery systems.

Drug repurposing is the strategy of drug utilization for a treatment option other than the intended indications. This strategy has witnessed increased adoption over the past decades, especially within cancer nanomedicine. Cancer nanomedicine has been facilitated through nanoparticle-based (NP-based) delivery systems which can combat nonsmall-cell lung cancer (NSCLC) via recent advances in nanotechnology and apply its benefits to existing drugs. The repurposing of drugs, coupled with NP-based drug delivery systems, presents a promising avenue for achieving effective therapeutic solutions with accelerated outcomes. This review aims to present an overview of NSCLC treatments, with a specific focus on drug repurposing. It seeks to elucidate the latest advances in clinical studies and the utilization of NP-based drug delivery systems tailored for NSCLC treatment. First, the molecular mechanisms of Food and Drug Administration (FDA)-approved drugs for NSCLC, including ROS1 tyrosine kinase inhibitors (TKI) like repotrectinib, approved in November 2023, are detailed. Further, in vitro studies employing a combination strategy of drug repurposing and NP-based drug delivery systems as a treatment approach against NSCLC are listed. It includes the latest study on nanoparticle-based drug delivery systems loaded with repurposed drugs.

Synergistic combination of carvedilol, amlodipine, amitriptyline, and antibiotics as an alternative treatment approach for the susceptible and multidrug-resistant A. baumannii infections via drug repurposing.

We evaluated in vitro activity of 13 drugs used in the treatment of some non-communicable diseases via repurposing to determine their potential use in the treatment of Acinetobacter baumannii infections caused by susceptible and multidrug-resistant strains. A. baumannii is a multidrug-resistant Gram-negative bacteria causing nosocomial infections, especially in intensive care units. It has been identified in the WHO critical pathogen list and this emphasises urgent need for new treatment options. As the development of new therapeutics is expensive and time consuming, finding new uses of existing drugs via drug repositioning has been favoured. Antimicrobial susceptibility tests were conducted on all 13 drugs according to CLSI. Drugs with MIC values below 128 µg/mL and control antibiotics were further subjected to synergetic effect and bacterial time-kill analysis. Carvedilol-gentamicin (FICI 0.2813) and carvedilol-amlodipine (FICI 0.5625) were determined to have synergetic and additive effect, respectively, on the susceptible A. baumannii strain, and amlodipine-tetracycline (FICI 0.75) and amitriptyline-tetracycline (FICI 0.75) to have additive effect on the multidrug-resistant A. baumannii strain. Most remarkably, both amlodipine and amitriptyline reduced the MIC of multidrug-resistant, including some carbapenems, A. baumannii reference antibiotic tetracycline from 2 to 0.5 µg/mL, for 4-folds. All these results were further supported by bacterial time-kill assay and all combinations showed bactericidal activity, at certain hours, at 4XMIC. Combinations proposed in this study may provide treatment options for both susceptible and multidrug-resistant A. baumannii infections but requires further pharmacokinetics and pharmacodynamics analyses and in vivo re-evaluations using appropriate models.

Heterologous expression, biochemical characterisation and computational analysis of Bacteroides fragilis enolase.

Bacteriodes fragilis is an anaerobic bacterium found in the human intestinal flora. In this study, BfEno was targeted with a structure-based drug design approach because inhibition of this enzyme may prevent both the aerobic and anaerobic pathways due to its role in the glycolytic pathway. First, the gene encoding BfEno was cloned, expressed and the protein produced over 95% purity. The Km and Vmax values of BfEno were determined as 314.9 µM and 256.2 µmol/min.mg, respectively. Drug-like chemicals were retrieved from the ZINC database for high-throughput virtual screening analyses. As a result of screening study, the ZINC91441604 has been proposed to bind to the active site of the enzyme and remain stable. The same compound exhibited weak binding to the human enolases than the bacterial enolase. Hence, ZINC91441604 may be proposed as a novel candidate for further in vitro and in vivo drug analysis towards the treatment of B. fragilis infections.

Targeting SARS-CoV-2 Nsp12/Nsp8 interaction interface with approved and investigational drugs: an in silico structure-based approach.

In this study, the Nsp12-Nsp8 complex of SARS-CoV-2 was targeted with structure-based and computer-aided drug design approach because of its vital role in viral replication. Sequence analysis of RNA-dependent RNA polymerase (Nsp12) sequences from 30,366 different isolates were analysed for possible mutations. FDA-approved and investigational drugs were screened for interaction with both mutant and wild-type Nsp12-Nsp8 interfaces. Sequence analysis revealed that 70.42% of Nsp12 sequences showed conserved P323L mutation, located in the Nsp8 binding cleft. Compounds were screened for interface interaction, any with XP GScores lower than -7.0 kcal/mol were considered as possible interface inhibitors. RX-3117 (fluorocyclopentenyl cytosine) and Nebivolol had the highest binding affinities in both mutant and wild-type enzymes, therefore they were selected and resultant protein-ligand complexes were simulated for analysis of stability over 100 ns. Although the selected ligands had partial mobility in the binding cavity, they were not removed from the binding pocket after 100 ns. The ligand RX-3117 remained in the same position in the binding pocket of the mutant and wild-type enzyme after 100 ns MD simulation. However, the ligand Nebivolol folded and embedded in the binding pocket of mutant Nsp12 protein. Overall, FDA-approved and investigational drugs are able to bind to the Nsp12-Nsp8 interaction interface and prevent the formation of the Nsp12-Nsp8 complex. Interruption of viral replication by drugs proposed in this study should be further tested to pave the way for in vivo studies towards the treatment of COVID-19.Communicated by Ramaswamy H. Sarma.

Bronchopulmonary dysplasia and wnt pathway-associated single nucleotide polymorphisms.

AIM: Genetic variants contribute to the pathogenesis of bronchopulmonary dysplasia (BPD). The aim of this study is to evaluate the association of 45 SNPs with BPD susceptibility in a Turkish premature infant cohort. METHODS: Infants with gestational age <32 weeks were included. Patients were divided into BPD or no-BPD groups according to oxygen need at 28 days of life, and stratified according to the severity of BPD. We genotyped 45 SNPs, previously identified as BPD risk factors, in 192 infants. RESULTS: A total of eight SNPs were associated with BPD risk at allele level, two of which (rs4883955 on KLF12 and rs9953270 on CHST9) were also associated at the genotype level. Functional relationship maps suggested an interaction between five of these genes, converging on WNT5A, a member of the WNT pathway known to be implicated in BPD pathogenesis. Dysfunctional CHST9 and KLF12 variants may contribute to BPD pathogenesis through an interaction with WNT5A. CONCLUSIONS: We suggest investigating the role of SNPs on different genes which are in relation with the Wnt pathway in BPD pathogenesis. We identified eight SNPs as risk factors for BPD in this study. In-silico functional maps show an interaction of the genes harboring these SNPs with the WNT pathway, supporting its role in BPD pathogenesis. TRIAL REGISTRATION: NCT03467828. IMPACT: It is known that genetic factors may contribute to the development of BPD in preterm infants. Further studies are required to identify specific genes that play a role in the BPD pathway to evaluate them as a target for therapeutic interventions. Our study shows an association of BPD predisposition with certain polymorphisms on MBL2, NFKBIA, CEP170, MAGI2, and VEGFA genes at allele level and polymorphisms on CHST9 and KLF12 genes at both allele and genotype level. In-silico functional mapping shows a functional relationship of these five genes with WNT5A, suggesting that Wnt pathway disruption may play a role in BPD pathogenesis.

Genomic chronicle of SARS-CoV-2: a mutational analysis with over 1 million genome sequences.

Use of information technologies to analyse big data on SARS-CoV-2 genome provides an insight for tracking variations and examining the evolution of the virus. Nevertheless, storing, processing, alignment and analyses of these numerous genomes are still a challenge. In this study, over 1 million SARS-CoV-2 genomes have been analysed to show distribution and relationship of variations that could enlighten development and evolution of the virus. In all genomes analysed in this study, a total of over 215M SNVs have been detected and average number of SNV per isolate was found to be 21.83. Single nucleotide variant (SNV) average is observed to reach 31.25 just in March 2021. The average variation number of isolates is increasing and compromising with total case numbers around the world. Remarkably, cytosine deamination, which is one of the most important biochemical processes in the evolutionary development of coronaviruses, accounts for 46% of all SNVs seen in SARS-CoV-2 genomes within 16 months. This study is one of the most comprehensive SARS-CoV-2 genomic analysis study in terms of number of genomes analysed in an academic publication so far, and reported results could be useful in monitoring the development of SARS-CoV-2.

In vitro inhibition studies of coumarin derivatives on Bos taurus enolase and elucidating their interaction by molecular docking, molecular dynamics simulations and MMGB(PB)SA binding energy calculation.

Tropical theileriosis is among the most common vector-borne diseases and caused by Theileria parasites. Theileria annulata is an obligate intracellular protozoan parasite and transmitted to especially Bos taurus and Bos indicus by Hyalomma tick vectors. C8 ([4-(3,4-dimethoxyphenyl)-6,7-dihydroxy-2H-chromen-2-one); C9 (4-(3,4-dihydroxyphenyl)-7,8 dihydroxy-2H-chromen-2-one); C21 (4-(3,4-dihydroxyphenyl)-6,7-dihydroxy-2H-chromen-2 one) were identified as potent Theileria annulata enolase (TaEno) inhibitors in our previous studies. An ideal drug compound must inhibit the target parasite enzyme without inhibiting its homolog in the host. In this study, the inhibitory effect of the compounds previously evaluated on TaEno were tested on the host Bos taurus enolase (BtEno3) by in vitro studies. The interactions of enzyme-coumarin and enzyme-coumarin-substrate by in silico studies were also performed. All of the coumarin derivatives tested showed very low inhibitory effects on B. taurus enolase; 36,87% inhibition at 100 µM concentration for C8, 8,13% inhibition at 100 µM concentration for C9 and 77,69 µM of IC50 value for C21. In addition, these three coumarin derivatives and substrate 2PG were docked into the BtEno3 using molecular docking methods. Molecular interactions between enolase-coumarin and enolase-coumarin-substrate complexes were analyzed using molecular dynamics simulation methods for 100 ns. Estimated free energy of bindings of the substrate 2PG and coumarin derivatives to the BtEno3 were calculated by MM-GB(PB)SA methods. In comparison to the inhibition studies performed on TaEno, C8 and C9 coumarin derivatives remain the possible inhibitor candidates as they inhibit the host enolase at very high concentrations. These two promising compounds will be further analyzed by in vitro and in vivo studies towards developing an alternative drug against tropical theileriosis.

Hit identification against peptidyl-prolyl isomerase of Theileria annulata by combined virtual high-throughput screening and molecular dynamics simulation approach.

Theileria annulata secretes peptidyl prolyl isomerase enzyme (TaPIN1) to manipulate the host cell oncogenic signaling pathway by disrupting the tumor suppressor F-box and WD repeat domain-containing 7 (FBW7) protein level leading to an increased level of c-Jun proto-oncogene. Buparvaquone is a hydroxynaphthoquinone anti-theilerial drug and has been used to treat theileriosis. However, TaPIN1 contains the A53 P mutation that causes drug resistance. In this study, potential TaPIN1 inhibitors were investigated using a library of naphthoquinone derivatives. Comparative models of mutant (m) and wild type (wt) TaPIN1 were predicted and energy minimization was followed by structure validation. A naphthoquinone (hydroxynaphthalene-1,2-dione, hydroxynaphthalene-1,4-dione) and hydroxynaphthalene-2,3-dione library was screened by Schrödinger Glide HTVS, SP and XP docking methodologies and the docked compounds were ranked by the Glide XP scoring function. The two highest ranked docked compounds Compound 1 (4-hydroxy-3-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxynaphthalene-1,2-dione) and Compound 2 (6-acetyl-1,4,5,7,8-pentahydroxynaphthalene-2,3-dione) were used for further molecular dynamics (MD) simulation studies. The MD results showed that ligand Compound 1 was located in the active site of both mTaPIN1 and wtTaPIN1 and could be proposed as a potential inhibitor by acting as a substrate antagonist. However, ligand Compound 2 was displaced away from the binding pocket of wtTaPIN1 but was located near the active site binding pocket of mTaPIN1 suggesting that could be selectively evaluated as a potential inhibitor against the mTaPIN1. Compound 1 and Compound 2 ligands are potential inhibitors but Compound 2 is suggested as a better inhibitor for mTaPIN1. These ligands could also further evaluated as potential inhibitors against human peptidyl prolyl isomerase which causes cancer in humans by using the same mechanism as TaPIN1.

Evaluation of the potency of FDA-approved drugs on wild type and mutant SARS-CoV-2 helicase (Nsp13).

SARS-CoV-2 has caused COVID-19 outbreak with nearly 2 M infected people and over 100K death worldwide, until middle of April 2020. There is no confirmed drug for the treatment of COVID-19 yet. As the disease spread fast and threaten human life, repositioning of FDA approved drugs may provide fast options for treatment. In this aspect, structure-based drug design could be applied as a powerful approach in distinguishing the viral drug target regions from the host. Evaluation of variations in SARS-CoV-2 genome may ease finding specific drug targets in the viral genome. In this study, 3458 SARS-CoV-2 genome sequences isolated from all around the world were analyzed. Incidence of C17747T and A17858G mutations were observed to be much higher than others and they were on Nsp13, a vital enzyme of SARS-CoV-2. Effect of these mutations was evaluated on protein-drug interactions using in silico methods. The most potent drugs were found to interact with the key and neighbor residues of the active site responsible from ATP hydrolysis. As result, cangrelor, fludarabine, folic acid and polydatin were determined to be the most potent drugs which have potency to inhibit both the wild type and mutant SARS-CoV-2 helicase. Clinical data supporting these findings would be important towards overcoming COVID-19.

An updated analysis of variations in SARS-CoV-2 genome.

A novel pathogen, named SARS-CoV-2, has caused an unprecedented worldwide pandemic in the first half of 2020. As the SARS-CoV-2 genome sequences have become available, one of the important focus of scientists has become tracking variations in the viral genome. In this study, 30366 SARS-CoV-2 isolate genomes were aligned using the software developed by our group (ODOTool) and 11 variations in SARS-CoV-2 genome over 10% of whole isolates were discussed. Results indicated that, frequency rates of these 11 variations change between 3.56%-88.44 % and these rates differ greatly depending on the continents they have been reported. Despite some variations being in low frequency rate in some continents, C14408T and A23403G variations on Nsp12 and S protein, respectively, observed to be the most prominent variations all over the world, in general, and both cause missense mutations. It is also notable that most of isolates carry C14408T and A23403 variations simultaneously and also nearly all isolates carrying the G25563T variation on ORF3a, also carry C14408T and A23403 variations, although their location distributions are not similar. All these data should be considered towards development of vaccine and antiviral treatment strategies as well as tracing diversity of virus in all over the world.

An insight into the epitope-based peptide vaccine design strategy and studies against COVID-19.

SARS-CoV-2 is a new member of the coronavirus family and caused the pandemic of coronavirus disease 2019 (COVID-19) in 2020. It is crucial to design and produce an effective vaccine for the prevention of rapid transmission and possible deaths wcaused by the disease. Although intensive work and research are being carried out all over the world to develop a vaccine, an effective and approved formulation that can prevent the infection and limit the outbreak has not been announced yet. Among all types of vaccines, epitope-based peptide vaccines outshine with their low-cost production, easy modification in the structure, and safety. In this review, vaccine studies against COVID-19 have been summarized and detailed information about the epitope-based peptide vaccines against COVID-19 has been provided. We have not only compared the peptide vaccine with other types of vaccines but also presented comprehensive literature information about development steps for an effective and protective formulation to give an insight into on-going peptide vaccine studies against SARS-CoV-2.

Discovery and evaluation of inhibitory activity and mechanism of arylcoumarin derivatives on Theileria annulata enolase by in vitro and molecular docking studies.

In this study, the inhibition potential of 3- and 4-arylcoumarin derivatives on Theileria annulata enolase (TaENO) was assessed for the first time in the literature. Firstly, protein stabilization analyses of TaENO were performed and it was found that the enzyme remains stable with the addition of 6 M ethylene glycol at + 4 °C. Inhibitor screening analyses were carried out using 25 coumarin derivatives on highly purified TaENO (> 95%), and four coumarin derivatives [4-(3,4-dimethoxyphenyl)-6,7-dihydroxy-2H-chromen-2-one (C8); 4-(3,4-dihydroxyphenyl)-7,8 dihydroxy-2H-chromen-2-one (C9); 4-(3,4-dihydroxyphenyl)-6,7-dihydroxy-2H-chromen-2 one (C21); and 3-(3,4-dihydroxyphenyl)-7,8-dihydroxy-2H-chromen-2-one (C23)] showed the highest inhibitory effects with the IC50 values of 10.450, 13.170, 8.871 and 10.863 µM, respectively. The kinetic results indicated that these compounds inhibited the enzyme by uncompetitive inhibition. In addition, the successful binding of the most potent inhibitor (C21) into TaENO was confirmed by using MALDI-TOF mass spectrophotometry. Molecular docking analyses have predicted that C8 and C21 coumarin derivatives which showed high inhibitory effects on TaENO were interacted with high affinity to the potential regions out of the active site. Taken together, these coumarin derivatives (C8, C9, C21 and C23) are first known potent, nonsubstrate, uncompetitive inhibitors of TaENO and these results will facilitate further in vitro and in vivo analysis toward structure-based drug design studies.

Functional analyses of dipeptide and pentapeptide insertions on Theileria annulata enolase by site-directed mutagenesis and in silico approaches.

Theileria annulata enolase (TaENO) could be assessed as a druggable target for tropical theileriosis treatment. The parasite enzyme plays an important role in many cellular functions and carries some structural differences like dipeptide (262EK263) and pentapeptide (103EWGYC107) insertions from the host enzyme, Bos taurus enolase. In this study, the functional effects of these insertions on TaENO activity were analyzed by in vitro site-directed mutagenesis and in silico molecular docking analyses for the first time in the literature. In vitro results showed that, although the deletion of the pentapeptide insertion (TaENOΔEWGYC) reduced the enzyme activity slightly, the removal of the dipeptide insertion (TaENOΔEK) halted it. Also, molecular docking results revealed that the deletion of these insertions affected the substrate binding affinity of the mutant enzymes. The active site of TaENOΔEK exhibited a small decrease of substrate binding affinity compared to the active site of TaENOΔEWGYC relative to the wild type TaENO. Although we conclude that both regions could be evaluated as possible drug-binding sites to inhibit TaENO in further studies, these results indicate that the dipeptide insertion could be a more promising drug binding site than the pentapeptide insertion.

Inhibitory effects of arylcoumarin derivatives on Bacteroides fragilisd­lactate dehydrogenase.

Bacteroides fragilis is an anaerobic bacterium naturally hosted in the human colon flora. B. fragilisd­lactate dehydrogenase (Bfd­LDH) is an important enzyme which catalyzes the conversion of d­lactate to pyruvate and regulates anaerobic glycolysis. In this study Bfd­LDH has been targeted for structure based drug design. B. fragilisd­lactate dehydrogenase has been expressed, purified and inhibitory activities of 25 coumarin derivatives previously synthetize for their antioxidant activity were evaluated. Among the 25 coumarin derivatives, compound 6a, 5l, and 6b exhibited the highest inhibitory activity with IC50 values of 0,47 µM, 0,57 µM ve 0,057 µM, respectively. The results indicate that the mechanism by which 6a, 5l and 6b coumarin derivatives inhibit Bfd­LDH by reversible non-competitive inhibition. Docking experiments were carried out to further explain the results and compare the theoretical and experimental affinity of these compounds to the Bfd­LDH protein. According to docking results, all coumarins bind to the site occupied by pyruvate and the nicotinamide ring of NADH.

A study of Bos taurus muscle specific enolase; biochemical characterization, homology modelling and investigation of molecular interaction using molecular docking and dynamics simulations.

Tropical theileriosis caused by Theileria annulata obligate parasite that infect ruminant animals, including Bos taurus. The disease results massive economic losses in livestock production worldwide. Here we describe cloning, expression and both biochemical and structural characterization of beta enolase from Bos taurus in vitro and in silico. The interconversion of 2­phosphoglycerate to phosphoenolpyruvate was catalyzed by enolase is a metalloenzyme in glycolytic pathway and gluconeogenesis. Enolase from Bos taurus was cloned, expressed and the protein was purified at 95% purity using cobalt column by affinity chromatography. The optimum enzymatic activity was calculated at pH 6.5. For the first time in the literature, the kinetic parameters of the enzyme, Vmax and Km, were measured as 0.1141 mM/min and 0.514 mM, respectively. Besides, Bos taurus enolase 3-dimensional structure was built by homology modelling to be used in silico analyses. The interactions of the enzyme-substrate complex were elucidated by molecular dynamics simulations for 100 ns. These interactions were found to be the same as experimentally determined interactions in yeast. These results would enable further structure based drug design studies with the biochemical characterization of the host organism Bos taurus enolase enzyme in vitro and the elucidation of behavior of enzyme-substrate complex in silico.

Comprehensive structural analysis of the open and closed conformations of Theileria annulata enolase by molecular modelling and docking.

Theileria annulata is an apicomplexan parasite which is responsible for tropical theileriosis in cattle. Due to resistance of T. annulata against commonly used antitheilerial drug, new drug candidates should be identified urgently. Enolase might be a druggable protein candidate which has an important role in glycolysis, and could also be related to several cellular functions as a moonlight protein. In this study; we have described three-dimensional models of open and closed conformations of T. annulata enolase by homology modeling method for the first time with the comprehensive domain, active site and docking analyses. Our results show that the enolase has similar folding patterns within enolase superfamily with conserved catalytic loops and active site residues. We have described specific insertions, possible plasminogen binding sites, electrostatic potential surfaces and positively charged pockets as druggable regions in T. annulata enolase.

Biochemical and in silico Characterization of Recombinant L-Lactate Dehydrogenase of Theileria annulata.

Theileria annulata is a parasite that causes theileriosis in cattle. Reports about drug resistance made essential to develop new drug. LDH of Theileria schizonts is the vital enzyme for its anaerobic metabolism. TaLDH gene was first cloned into pGEM-T cloning vector with two introns in our previous study. Here we report cloning of TaLDH without introns into pLATE 31 vector in E. coli BL21(DE3). Protein was in an inactive form. Two mutations were fixed to express the active protein. Protein was purified by affinity chromatography and evaluated by SDS-PAGE and size exclusion chromatography. Optimum pH of enzyme was performed in pH 7.5, and enzyme was stabilized at 20-40 °C. Enzyme kinetics of recombinant TaLDH were found to be in the direction of pyruvate to lactate K m 0.1324 and K i 4.295 mM, k cat, 44.55/s and k cat /K m, 3.3693 × 10(5)/M/s. 3D structure of TaLDH was predicted, and possible drug binding sites were determined by homology modelling.

Cloning of intron-removed enolase gene and expression, purification, kinetic characterization of the enzyme from Theileria annulata.

Tropical theileriosis is a disease caused by infection with an apicomplexan parasite, Theileria annulata, and giving rise to huge economic losses. In recent years, parasite resistance has been reported against the most effective antitheilerial drug used for the treatment of this disease. This emphasizes the need for alternative methods of treatment. Enolase is a key glycolytic enzyme and can be selected as a macromolecular target of therapy of tropical theileriosis. In this study, an intron sequence present in T. annulata enolase gene was removed by PCR-directed mutagenesis, and the gene was first cloned into pGEM-T Easy vector and then subcloned into pLATE31 vector, and expressed in Escherichia coli cells. The enzyme was purified by affinity chromatography using Ni-NTA agarose column. Steady-state kinetic parameters of the enzyme were determined using GraFit 3.0. High quantities (~65 mg/l of culture) of pure recombinant T. annulata enolase have been obtained in a higly purified form (>95 %). Homodimer form of purified protein was determined from the molecular weights obtained from a single band on SDS-PAGE (48 kDa) and from size exclusion chromatography (93 kDa). Enzyme kinetic measurements using 2-PGA as substrate gave a specific activity of ~40 U/mg, K m: 106 µM, kcat: 37 s(-1), and k cat/K m: 3.5 × 10(5) M(-1) s(-1). These values have been determined for the first time from this parasite enzyme, and availability of large quantities of enolase enzyme will facilitate further kinetic and structural characterization toward design of new antitheilerial drugs.

Single mutation in Shine-Dalgarno-like sequence present in the amino terminal of lactate dehydrogenase of Plasmodium effects the production of an eukaryotic protein expressed in a prokaryotic system.

One of the most important step in structure-based drug design studies is obtaining the protein in active form after cloning the target gene. In one of our previous study, it was determined that an internal Shine-Dalgarno-like sequence present just before the third methionine at N-terminus of wild type lactate dehydrogenase enzyme of Plasmodium falciparum prevent the translation of full length protein. Inspection of the same region in P. vivax LDH, which was overproduced as an active enzyme, indicated that the codon preference in the same region was slightly different than the codon preference of wild type PfLDH. In this study, 5'-GGAGGC-3' sequence of P. vivax that codes for two glycine residues just before the third methionine was exchanged to 5'-GGAGGA-3', by mimicking P. falciparum LDH, to prove the possible effects of having an internal SD-like sequence when expressing an eukaryotic protein in a prokaryotic system. Exchange was made by site-directed mutagenesis. Results indicated that having two glycine residues with an internal SD-like sequence (GGAGGA) just before the third methionine abolishes the enzyme activity due to the preference of the prokaryotic system used for the expression. This study emphasizes the awareness of use of a prokaryotic system to overproduce an eukaryotic protein.

Electrophoretic analysis of total protein profiles of some Lathyrus L. (Sect. Cicercula) grown in Turkey.

In this study, total 8 species (Section Cicercula) belong to 14 different geographic distributions collected from Turkey have been studied for the analysis of seed storage protein profiles to examine their relationship by Sodium Dodecyl Sulphate Polyacrilamide Gel Electrophoresis (SDS-PAGE) technique. Hierarchical cluster analysis and Euclidean distance were used for comparison between species and calculating the genetic resemblance respectively. Dendogram was formed using average linkage. Electrophoretic protein profiles of seed cotyledons were showed that all species formed two clusters. The first one consisted of the L. stenophyllus, L. hirsutus, L. chloranthus, L. cicera and L. sativus second one by three species (L. annuus, L. cassius and L. phaselitanus). In addition, very little differences were observed in total protein profiles of the examined sample species (L. cicera, L. hirsutus and L. chloranthus) from nine geographical regions belong to section Cicercula. Protein amount was found to be highest in L. hirsutus and lowest in L. cicera.