FRAUNHOFER IME SCREENINGPORT Design of screening projects General remarks
Introduction Screening is done to identify new chemical substances against molecular mechanisms of a disease It is a question of what chemical matter is used Five different approaches; virtual library, diversity focused library, natural product library, fragment library, biologicals It is a question of the disease Molecular mechanism has to be known and understood Three approaches, Target-based screening, phenotypic screening, combinatorial screening It is a question of a process Minimize cost and time while retain the chance of identifying new drugs
Introduction of chemical matter natural products Problems of natural products Extraction and identification are complicated Sourcing is complicated (resynthesis is a mayor challenge) Convention on Biological Diversity and Nagoya Protocol protect the countries and the biodiversity (not ratified by USA) Chemically often complicated, resulting in stability issues Advantage of natural products Predesigned for biological activity Especially in the area of antibiotic research nearly all targets originate from natural product discovery Anticancer agent The re-emergence of natural products for drug discovery in the genomics era; doi:10.1038/nrd4510
Comparing chemical spaces
Lipinski s Rule of 5 2,000 drugs and clinical candidates were analysed and a concept for membrane permeability was found: Molecular weight below 500 Lipophilicity (lopp [partitioning between water and 1-octanol]) below 5 Groups in the molecule that can donate hydrogen bonds (hydrogen plus amine) less than 5 Groups in the molecule that can accept hydrogen bonds (oxygen plus nitrogen) less than 10 Rules are based on the 90-percent quantile Only passive diffusion is taken into account; active transport (e.g. vitamins) is not regarded Instead of absorption these rules are now regarded as drug-likeness parameters
Typical in silico approach Compounds were preselected; 90,000 compounds were docked; 5,000 were reexamined, 30 compounds were tested manually
Fragments and virtual screening Movie 1. The dynamic nature of the small molecule-binding site is illustrated in this movie (rigimol, www.delanoscientific.com), which cycles through two unliganded IL-2 structures (3INK and 1M47) and a structure of Compound 1 bound (1M48). The transitions reveal the differences that exist between the wild-type structures and the structure of Compound 1 bound. Movie 2. The structural changes that occur in a sequence of wild-type IL-2 (1M47), compound 1 (1M48), indole glyoxylate fragment (1M4A), and guanidine fragment (1M4B), ending with wild-type IL-2 (1M47). The transitions are illustrated by using rigimol (www.delanoscientific.com). Fragments are smaller (below 200 g/mol) and have to be screened at higher concentrations ( around mm) using very sensitive techniques (frequently NMR) Frequently, fragments are screened as mixtures Virtual screening enables the analysis of vast libraries, but the problem of the biological relevant chemical space is critical M. R. Arkin, M. Randal, W. L. DeLano, J. Hyde, T. N. Luong, J. D. Oslob, D. R. Raphael, L. Taylor, J. Wang, R. S. McDowell, J. A. Wells, A. C. Braisted, PNAS, 2003, 100, 1603
Summary chemical matter All approaches have their merit Natural products designed to interact with life, but complicated to find, analyse and synthesise Diverse chemical libraries enourmous diversity and good starting points for synthesis, but an enourmous effort to identify the actives and a high failure rate Virtual screening unlimited posibilities, but the problem of flexibility (compound and protein) Fragemt libraries easy to synthesise, but enourmous efforts have to made to identify the binding molecules
Number of drug targets 29,679 genes were found in the human genome (June06) The number of successful drug targets is fairly limited Some gene-families are severely overrepresented FDA approved in total 1357 drugs, 1204 small molecules, 166 biologicals
Pharmaceutical innovation
Pharmaceutical innovation part II Drug discovery is working hard and has quite some success
The relevance of the best model system The right model system is critical (primary cells, stem cells, organoid, animal) Overexpression of CLTA and DNM2 is not representative of physiological conditions
Terms used in typical screening programms Screen High throughput Focused screen Fragment screen Structural aided drug design Virtual screen Physiological screen NMR screen Description Large numbers of compounds analysed in a assay generally designed to run in plates of 384 wells and above Compounds previously identified as hitting specific classes of targets (e.g. kinases) and compounds with similar structures Soak small compounds into crystals to obtain compounds with low mm activity which can then be used as building blocks for larger Molecules Use of crystal structures to help design molecules Docking models: interogation of a virtual compound library with the X-ray structure of the protein or, if have a known ligand, as a base to develop further compounds on A tissue-based approach for determination of the effects of a drug at the tissue rather than the cellular or subcellular level, for example, muscle contractility Screen small compounds (fragments) by soaking into protein targets of known crystal or NMR structure to look for hits with low mm activity which can then be used as building blocks for larger molecules
Drug discovery cascade
Phenotype vs. Target based screening
Phenotype vs. Target based screening part II Currently phenotypic screening is performed in the majority of cases Drug discovery is associated with a high degree of failure Current understanding is that assay systems with a higher relevance reduce this attrition; more complex cellular models; better and more disease relevant animal models
Phenotype vs. Target based screening part III Two diametral opposing dogmas clash when phenotypic vs. target-based screening is considered
Steps at every point of the cascade Target-to-hit phase Assay development: Assay automatisation, miniaturisation, reagent development Screening: marker library screening (conc. finding), primary screening (hit finding), confirmational screening (hit conf., same conc., n=3), counter screening (interference), specificity screening (pathogen vs. host protein)
Steps at every point of the cascade Hit-to-Lead phase (proof of in-vitro biology) Hit expansion to improve activity and specificity Target deconvolution In-vitro pharmacology (toxicity (cell lines, genotox, herg), plasma protein binding, drug metabolism (liver; phase I and II)) Medicinal chemistry
Steps at every point of the cascade Lead optimisation phase (proof of in-vivo biology) In-vivo pharmacology ADMET (exretion (renal, biliary), teratogenicity) Chemistry scale-up In-vivo animal models Preclinical (clinical candidate) Formulation Toxicology, clinical pharmacology
Steps at every point of the cascade Phase I (first in man) Small group (20-100 people), only healthy people; safety, tolerability and pharmacodynamic assessment; dose finding Phase II Medium sized group (100-300 people), efficiency of the drug is evaluated Phase IIa check dosing regime, often checked with genetic background Phase IIb used to study efficiency Phase III Double blind, placebo controlled trials; definite answer about efficiency
Summary biology and processes R&D productivity is relatively low, but constant (despite the efforts) Better Screening and disease models are the current trend (stem cells, organoids, CRISPR/Cas) The fight between phenotypic and target based is currently favouring phenotypic screening The overall process is very diverse but well structured