Biomarkers for Drug Discovery: Important Aspects of in vitro Assay Design for HTS and HCS Bioassays

High throughput screening (HTS) is the foundation of current drug discovery to assay drug candidates for toxicity and biological effects (i.e. off-target and on-target responses, respectively). HTS is typically based on measuring thousands of drug candidates per day with a single endpoint assay on a limited number of doses or even a single dose of compound. The assays can be either based on absorbance or fluorescence measurements (i.e. Alamar Blue, MTT, Fluo-4 for calcium, etc.). Conversely, high content screening (HCS) is based on measuring a limited number of drugs per day, but measuring up to eight different assays simultaneously with multiple drug doses and even kinetic measurements. HCS assays typically are based on fluorescence microscopy and automatic image analysis algorithms. With HCS technology, tedious and time consuming assays can now be automated (i.e. nuclear size, micronucleus assay, lysosomal mass, mitochondrial membrane potential, neurite outgrowth, etc.). Multi-channel FACS (fluorescence activated cell sorting) can also be considered to be »high content« analysis. The purpose of this essay is to review important aspects of in vitro assay design common to both HTS and HCS screening technologies

Three-dimensional cell cultures as a new tool in drug discovery

Background and purpose: Producing of reliable information about pharmacological activity of new chemical entities is essential in early stages of drug discovery and development. There is a continuous need for improvement of existing in vitro technologies, in order to get more accurate and more predictive biological data (and for compounds selection) in pre-clinical screening methods and models. Materials and methods: Two-dimensional (2D) cell cultures, in comparison with original tissues, does not fully reproduce in vivo cell growth and differentiation. Therefore, significant efforts have been made toward the development of more realistic three-dimensional (3D) in vitro cell culture models that would better mimic tissue physiology. Results: Two-dimensional (2D) cell cultures, in comparison with original tissues, does not fully reproduce in vivo cell growth and differentiation. Therefore, significant efforts have been made toward the development of more realistic three-dimensional (3D) in vitro cell culture models that would better mimic tissue physiology. Basic concepts and advantages of 3D cell cultures, as well as different approaches in technologies that enable the cell growth in 3D will be presented here. Possible applications of 3D cell culture in drug discovery will be discussed, and example of formation of spherical growth of three different human breast cancer cells (MDA-MB-231, SK-BR-3 and T-47D cells) in 3D format will be shown. Conclusions: Although biological significance of obtained data from 2D and 3D cell cultures is still poorly understood, discrepancy of compunds activity illustrated importance of implementation 3D cell culture assays in early part of drug discovery process

Macromolecular Databases – A Background of Bioinformatics

We propose a novel quasi‐Bayesian Metropolis‐within‐Gibbs algorithm that can be used to estimate drifts in the shock volatilities of a linearized dynamic stochastic general equilibrium (DSGE) model. The resulting volatility estimates differ from the existing approaches in two ways. First, the time variation enters non‐parametrically, so that our approach ensures consistent estimation in a wide class of processes, thereby eliminating the need to specify the volatility law of motion and alleviating the risk of invalid inference due to mis‐specification. Second, the conditional quasi‐posterior of the drifting volatilities is available in closed form, which makes inference straightforward and simplifies existing algorithms. We apply our estimation procedure to a standard DSGE model and find that the estimated volatility paths are smoother compared to alternative stochastic volatility estimates. Moreover, we demonstrate that our procedure can deliver statistically significant improvements to the density forecasts of the DSGE model compared to alternative methods.PostprintPeer reviewe

Kombinirani in silico i in vitro pristup pronalaženju spojeva s mogućim ljekovitim djelovanjem

The purpose of High Throughput Screening (HTS) in pharmaceutical industry is to identify, as soon as possible, compounds that are good starting points for successful new drug development process. Experts from this area study the chemical structures of so called »hit« compounds that have been found to interact with the target protein, interfere with proliferation of different types of cells or stop bacterial or fungal growth. Hypotheses to design related structures with improved biological properties are than builded. Each idea is then tested by the iterative synthesis and testing of novel compounds in various biological assays, searching for hits with better properties and defining useful and promising »lead« molecules. In parallel, molecular modeling and chemoinformatics experts can increase efficiency and decrease experimental costs by using different database filtering methods. In such a way, hits from HTS may be assessed before committing significant resource for chemical optimization. Joint efforts of these HTS experimental and modeling groups are the best way to speed up the process of finding a new useful hits and promising leads.Svrha HTS-a u farmaceutskoj industriji je identifikacija spojeva koji mogu poslužiti kao dobre polazne molekule u procesa razvoja lijeka iz novih kemijskih entiteta. Proučavanjem kemijske strukture takvih »hit« spojeva koji interagiraju s proteinom – metom, stručnjaci iz tog područja tragaju za strukturama poboljšanih bioloških svojstava. Svaka se ideja kasnije provjerava iterativnim postupkom sinteze i testiranja novih spojeva uporabom različitih metoda bioloških probira, kako bi se došlo do hitova s boljim svojstvima i do uporabivih i obećavajućih »lead« molekula. Istovremeno, molekularno modeliranje i kemoinformatika mogu povećati učinkovitosti i smanjenja troškova eksperimenata uporabom različitih metoda filtriranja baza spojeva. Na taj način, »hitovi« iz HTS-a mogu virtualno biti procijenjeni prije značajnog ulaganja resursa u kemijsku optimizaciju. Udruženi napori eksperimentalnih HTS grupa i grupa koje se bave molekularnim modeliranjem najbolji su način ubrzavanja procesa pronalaženja novih, uporabivih »hitova« i obećavajućih »leadova«

Kombinirani in silico i in vitro pristup pronalaženju spojeva s mogućim ljekovitim djelovanjem

The purpose of High Throughput Screening (HTS) in pharmaceutical industry is to identify, as soon as possible, compounds that are good starting points for successful new drug development process. Experts from this area study the chemical structures of so called »hit« compounds that have been found to interact with the target protein, interfere with proliferation of different types of cells or stop bacterial or fungal growth. Hypotheses to design related structures with improved biological properties are than builded. Each idea is then tested by the iterative synthesis and testing of novel compounds in various biological assays, searching for hits with better properties and defining useful and promising »lead« molecules. In parallel, molecular modeling and chemoinformatics experts can increase efficiency and decrease experimental costs by using different database filtering methods. In such a way, hits from HTS may be assessed before committing significant resource for chemical optimization. Joint efforts of these HTS experimental and modeling groups are the best way to speed up the process of finding a new useful hits and promising leads.Svrha HTS-a u farmaceutskoj industriji je identifikacija spojeva koji mogu poslužiti kao dobre polazne molekule u procesa razvoja lijeka iz novih kemijskih entiteta. Proučavanjem kemijske strukture takvih »hit« spojeva koji interagiraju s proteinom – metom, stručnjaci iz tog područja tragaju za strukturama poboljšanih bioloških svojstava. Svaka se ideja kasnije provjerava iterativnim postupkom sinteze i testiranja novih spojeva uporabom različitih metoda bioloških probira, kako bi se došlo do hitova s boljim svojstvima i do uporabivih i obećavajućih »lead« molekula. Istovremeno, molekularno modeliranje i kemoinformatika mogu povećati učinkovitosti i smanjenja troškova eksperimenata uporabom različitih metoda filtriranja baza spojeva. Na taj način, »hitovi« iz HTS-a mogu virtualno biti procijenjeni prije značajnog ulaganja resursa u kemijsku optimizaciju. Udruženi napori eksperimentalnih HTS grupa i grupa koje se bave molekularnim modeliranjem najbolji su način ubrzavanja procesa pronalaženja novih, uporabivih »hitova« i obećavajućih »leadova«

In vitro Evaluation of Aldoxime Interactions with Human Acetylcholinesterase

We related the ability of eleven pyridinium and imidazolium aldoximes to reactivate tabun-inhibited human erythrocyte acetylcholinesterase with their molecular properties. Using molecular mechanics we performed conformational analysis to determine the flexibility of the aldoximes. Semi-empirical calculations show that differences in reactivation rates probably do not origin from different electron density on the oxygen of the oxime group, but can be explained by the steric hindrance within the aldoxime molecule. Tabun-inhibited acetylcholinesterase was efficiently reactivated by flexible bispyridinium para-aldoximes with propylene or butylene linker. Although pyridinium/imidazolium aldoximes with the oxime group in ortho-position did not show significant reactivation ability, they protected acetylcholinesterase against phosphorylation by tabun due to their high affinity for the native acetylcholinesterase. The aldoximes were examined for cytotoxicity on different cell lines and no cytotoxic effect was observed for doses of up to 400 µmol dm–3

Pet godina utiskivanja CO2 za povećanje iscrpka nafte na polju Ivanić i Žutica – Iskustva i rezultati

Petogodišnje iskustvo utiskivanja CO2 u ležišta proizvodnih polja Ivanić i Žutica donijelo je iznimno dobre rezultate – primijenjene metodologije i znanja koja su vrijedan kapital i podloga za projektiranje takvih i sličnih projekata u bližoj budućnosti. S obzirom na to da INA u svom portfelju ima mnogo djelomično ili potpuno iscrpljenih plinskih i naftnih ležišta, EOR projekt i njegovi rezultati biti će ugrađeni u svaki novi manji ili veći pothvat u području tercijarnih metoda crpljenja. Važnost EOR projekta ne podrazumijeva samo razmatranje količina dodatnog iscrpka ugljiko-vodika, već ima i veoma važan okolišni aspekt projekta – izdvajanje, upotrebu i skladištenja CO2 Od listopada 2014. do listopada 2019. na EPU Ivanić proizvedeno je ukupno 1 579 429 boe nafte i plina, od čega je 677 129 boe isključivo doprinos EOR projekta. Izraženo u postotcima, to je povećanje iscrpka od 35% u odnosu na predviđenu proizvodnju koja bi se ostvarila da nije primijenjeno utiskivanje ugljičnog dioksida za povećanje iscrpka nafte u sklopu EOR projekta. Današnja dnevna proizvodnja nafte i plina odgovarala bi proizvodnji nafte iz 2000., dok je udio EOR projekta u dnevnoj proizvodnji 65%. Tijekom četiri godine utiskivanja ugljičnog diok-sida na EPU Žutica dnevna proizvodnja nafte i plina povećana je više od sedam puta, a ukupno je proizve-deno 390 136 boe nafte i plina. Od toga je 300 534 boe isključivo doprinos EOR projekta. U postotcima to je povećanje iscrpka od 77%

Pet godina utiskivanja CO2 za povećanje iscrpka nafte na polju Ivanić i Žutica – Iskustva i rezultati

Petogodišnje iskustvo utiskivanja CO2 u ležišta proizvodnih polja Ivanić i Žutica donijelo je iznimno dobre rezultate – primijenjene metodologije i znanja koja su vrijedan kapital i podloga za projektiranje takvih i sličnih projekata u bližoj budućnosti. S obzirom na to da INA u svom portfelju ima mnogo djelomično ili potpuno iscrpljenih plinskih i naftnih ležišta, EOR projekt i njegovi rezultati biti će ugrađeni u svaki novi manji ili veći pothvat u području tercijarnih metoda crpljenja. Važnost EOR projekta ne podrazumijeva samo razmatranje količina dodatnog iscrpka ugljiko-vodika, već ima i veoma važan okolišni aspekt projekta – izdvajanje, upotrebu i skladištenja CO2 Od listopada 2014. do listopada 2019. na EPU Ivanić proizvedeno je ukupno 1 579 429 boe nafte i plina, od čega je 677 129 boe isključivo doprinos EOR projekta. Izraženo u postotcima, to je povećanje iscrpka od 35% u odnosu na predviđenu proizvodnju koja bi se ostvarila da nije primijenjeno utiskivanje ugljičnog dioksida za povećanje iscrpka nafte u sklopu EOR projekta. Današnja dnevna proizvodnja nafte i plina odgovarala bi proizvodnji nafte iz 2000., dok je udio EOR projekta u dnevnoj proizvodnji 65%. Tijekom četiri godine utiskivanja ugljičnog diok-sida na EPU Žutica dnevna proizvodnja nafte i plina povećana je više od sedam puta, a ukupno je proizve-deno 390 136 boe nafte i plina. Od toga je 300 534 boe isključivo doprinos EOR projekta. U postotcima to je povećanje iscrpka od 77%