Drug Discovery Technologies Research Articles

Polymerase-Extension-Based Selection Method for DNA-Encoded Chemical Libraries against Nonimmobilized Protein Targets.

DNA-encoded chemical libraries (DELs) have become an important ligand discovery technology in biomedical research and drug discovery. DELs can be comprised of hundreds of millions to billions of candidate molecules and provide outstanding chemical diversity for discovering novel ligands and inhibitors for a large variety of biological targets. However, in most cases, DELs are selected against purified and immobilized proteins based on binding affinity. The development and application of DELs to more complex biological targets requires selection methods compatible with nonimmobilized and unpurified proteins. Here, we describe an approach using polymerase-based extension and target-directed photo-cross-linking and its application to the interrogation of a solution-phase protein target, carbonic anhydrase II.

Progress in targeting RAS with small molecule drugs.

RAS proteins have traditionally been deemed undruggable, as they do not possess an active site to which small molecules could bind but small molecules that target one form of oncogenic RAS, KRAS G12C, are already in preclinical and clinical trials, and several other compounds that bind to different RAS proteins at distinct sites are in earlier stage evaluation. KRAS is the major clinical target, as it is by far the most significant form of RAS in terms of cancer incidence. Unfortunately, KRAS exists in two isoforms, each with unique biochemical properties. This complicates efforts to target KRAS specifically. KRAS is also a member of a family of closely related proteins, which share similar effector-binding regions and G-domains, further increasing the challenge of specificity. Nevertheless, progress is being made, driven by new drug discovery technologies and creative science.

Cancer Biology Aspects of Computational Methods & Applications in Drug Discovery.

Cancer is one of the most debilitating diseases worldwide; even though advances in molecular and cellular biology have contributed to the decline of mortality associated with cancer, the procedure of drug discovery and development of cancer are time-consuming and expensive. However, with computer-aided drug discovery (CADD) techniques, pharmaceutical firms can save production costs and reduce the time of introducing effective anticancer drugs for clinical trials. CADD strategies like structure-based drug designing, ligandbased drug designing, and combined structure-based and ligand-based approaches also have the advantage of identifying target sites and discovering active compounds with high affinity for the target sites. In this article, research carried out on cancer biology aspect of the computational approaches in drug discovery technology have been reviewed. The main objective of the study is to identify the potential causes and the development of the cancer. In addition to this, its recovery has been discussed briefly. Our findings indicate that only a few studies have been carried out regarding this area. Hence, it is recommended that further researches should be conducted on the computational methods for identifying candidate drugs for breast, pancreatic, colon, prostate, and other types of cancer.

Drug discovery technologies to achieve pain relief through drug repositioning approaches

Drug discovery technologies to achieve pain relief through drug repositioning approaches

Preface: Computational Technologies for Drug Discovery

Preface: Computational Technologies for Drug Discovery

Monash Fragment Platform

The Monash Fragment Platform (MFP) provides academic and commercial researchers with access to fragment-based drug discovery (FBDD) technologies for their therapeutic targets. The facilities are located at the Monash Institute for Pharmaceutical Sciences (MIPS) in Parkville, Australia's largest and most successful pharmaceutical institute. FBDD screening uses our high-quality, in-house fragment library designed to maximise chemical space coverage and enable rapid hit optimisation. Screening cascades are tailored for each target and can be performed using a variety of techniques including NMR spectroscopy and our state-of-the-art surface plasmon resonance (SPR) facility. Structural biology and medicinal chemistry expertise utilising our standardized REFIL strategy enables the rapid elaboration of fragments into leads without the requirement for a large chemistry program.

Modern Approaches for the Discovery of Anti-Infectious Drugs for the Treatment of Neglected Diseases.

Neglected diseases comprise a number of infectious diseases historically endemic to low- and middle-income countries, though recently they have spread to high-income countries due to human migrations. In the past, pharmaceutical companies have shown hesitant to invest in these health conditions, due to the limited return on investment. As a result, the role of the academic sector and not-for-profit organizations in the discovery of new drugs for neglected diseases has been particularly relevant. Here, we review recent applications of modern drug discovery technologies in the field of neglected diseases, including high-throughput screening, in silico screening and computer-aided drug design. The suitability and perspectives of each approach are discussed depending on the context, along with the technology and translational gaps influencing them.

Nanoliter Centrifugal Liquid Dispenser Coupled with Superhydrophobic Microwell Array Chips for High-Throughput Cell Assays.

Microfluidic systems have been regarded as a potential platform for high-throughput screening technology in drug discovery due to their low sample consumption, high integration, and easy operation. The handling of small-volume liquid is an essential operation in microfluidic systems, especially in investigating large-scale combination conditions. Here, we develop a nanoliter centrifugal liquid dispenser (NanoCLD) coupled with superhydrophobic microwell array chips for high-throughput cell-based assays in the nanoliter scale. The NanoCLD consists of a plastic stock block with an array of drilled through holes, a reagent microwell array chip (reagent chip), and an alignment bottom assembled together in a fixture. A simple centrifugation at 800 rpm can dispense ~160 nL reagents into microwells in 5 min. The dispensed reagents are then delivered to cells by sandwiching the reagent chip upside down with another microwell array chip (cell chip) on which cells are cultured. A gradient of doxorubicin is then dispensed to the cell chip using the NanoCLD for validating the feasibility of performing drug tests on our microchip platform. This novel nanoliter-volume liquid dispensing method is simple, easy to operate, and especially suitable for repeatedly dispensing many different reagents simultaneously to microwells.

Tissue engineered bone mimetics to study bone disorders ex vivo: Role of bioinspired materials

Recent advances in materials development and tissue engineering has resulted in a substantial number of bioinspired materials that recapitulate cardinal features of bone extracellular matrix (ECM) such as dynamic inorganic and organic environment(s), hierarchical organization, and topographical features. Bone mimicking materials, as defined by its self-explanatory term, are developed based on the current understandings of the natural bone ECM during development, remodeling, and fracture repair. Compared to conventional plastic cultures, biomaterials that resemble some aspects of the native environment could elicit a more natural molecular and cellular response relevant to the bone tissue. Although current bioinspired materials are mainly developed to assist tissue repair or engineer bone tissues, such materials could nevertheless be applied to model various skeletal diseases in vitro. This review summarizes the use of bioinspired materials for bone tissue engineering, and their potential to model diseases of bone development and remodeling ex vivo. We largely focus on biomaterials, designed to re-create different aspects of the chemical and physical cues of native bone ECM. Employing these bone-inspired materials and tissue engineered bone surrogates to study bone diseases has tremendous potential and will provide a closer portrayal of disease progression and maintenance, both at the cellular and tissue level. We also briefly touch upon the application of patient-derived stem cells and introduce emerging technologies such as organ-on-chip in disease modeling. Faithful recapitulation of disease pathologies will not only offer novel insights into diseases, but also lead to enabling technologies for drug discovery and new approaches for cell-based therapies.

Development and Evaluation of Antibody Proteomics Technology for Rapid and Comprehensive Identification of Potential Biomarkers and Therapeutic Targets.

Proteomics-based analyses are powerful means of identifying potentially useful proteins in the initial stage of drug development. Technological developments in the field of proteomics, and increases in the sensitivity of MS analyses, now facilitate identification and examination of increasingly small amounts of proteins that are differentially expressed in diseased versus normal tissues and can be candidate biomarkers or therapeutic targets. However, the current approach is for candidate proteins to be prioritized by research interest and then validated one by one; this is very inefficient. To address this issue, we have developed what we refer to as "antibody proteomics technology," which uses a phage antibody library and tissue microarray analysis to rapidly and comprehensively isolate monoclonal antibodies against candidate proteins for the identification of potential biomarkers and therapeutic targets. In our validation of this technology, we successfully identified oxysterol binding protein-like 5 and calumenin as potential biomarkers related to metastasis in lung cancer, annexin A4 as a potential biomarker related to cisplatin resistance in malignant mesothelioma, and Eph receptor A10 as a potential therapeutic target in breast cancer, including refractory breast cancer. These findings suggest that antibody proteomics technology has the potential to become a fundamental technology in drug discovery for the development of novel biomarkers and therapeutic targets.

In Vitro Tissue-Engineered Skeletal Muscle Models for Studying Muscle Physiology and Disease.

Healthy skeletal muscle possesses the extraordinary ability to regenerate in response to small-scale injuries; however, this self-repair capacity becomes overwhelmed with aging, genetic myopathies, and large muscle loss. The failure of small animal models to accurately replicate human muscle disease, injury and to predict clinically-relevant drug responses has driven the development of high fidelity in vitro skeletal muscle models. Herein, the progress made and challenges ahead in engineering biomimetic human skeletal muscle tissues that can recapitulate muscle development, genetic diseases, regeneration, and drug response is discussed. Bioengineering approaches used to improve engineered muscle structure and function as well as the functionality of satellite cells to allow modeling muscle regeneration in vitro are also highlighted. Next, a historical overview on the generation of skeletal muscle cells and tissues from human pluripotent stem cells, and a discussion on the potential of these approaches to model and treat genetic diseases such as Duchenne muscular dystrophy, is provided. Finally, the need to integrate multiorgan microphysiological systems to generate improved drug discovery technologies with the potential to complement or supersede current preclinical animal models of muscle disease is described.

Chemical Cleavage of an Asp-Cys Sequence Allows Efficient Production of Recombinant Peptides with an N-Terminal Cysteine Residue.

Peptides with an N-terminal cysteine residue allow site-specific modification of proteins and peptides and chemical synthesis of proteins. They have been widely used to develop new strategies for imaging, drug discovery, diagnostics, and chip technologies. Here we present a method to produce recombinant peptides with an N-terminal cysteine residue as a convenient alternative to chemical synthesis. The method is based on the release of the desired peptide from a recombinant fusion protein by mild acid hydrolysis of an Asp-Cys sequence. To test the general validity of the method we prepared four fusion proteins bearing three different peptides (20-37 amino acid long) at the C-terminus of a ketosteroid isomerase-derived and two Onconase-derived carriers for the production of toxic peptides in E. coli. The chosen peptides were (C)GKY20, an antimicrobial peptide from the C-terminus of human thrombin, (C)ApoBL, an antimicrobial peptide from an inner region of human Apolipoprotein B, and (C)p53pAnt, an anticancer peptide containing the C-terminal region of the p53 protein fused to the cell penetrating peptide Penetratin. Cleavage efficiency of Asp-Cys bonds in the four fusion proteins was studied as a function of pH, temperature, and incubation time. In spite of the differences in the amino acid sequence (GTGDCGKY, GTGDCHVA, GSGTDCGSR, SQGSDCGSR) we obtained for all the proteins a cleavage efficiency of about 70-80% after 24 h incubation at 60 °C and pH 2. All the peptides were produced with very good yield (5-16 mg/L of LB cultures), high purity (>96%), and the expected content of free thiol groups (1 mol per mole of peptide). Furthermore, (C)GKY20 was modified with PyMPO-maleimide, a commercially available fluorophore bearing a thiol reactive group, and with 6-hydroxy-2-cyanobenzothiazole, a reagent specific for N-terminal cysteines, with yields of 100% thus demonstrating that our method is very well suited for the production of fully reactive peptides with an N-terminal cysteine residue.

PROTACs: An Emerging Targeting Technique for Protein Degradation in Drug Discovery.

Proteolysis-targeting chimeric molecules (PROTACs) represent an emerging technique that is receiving much attention for therapeutic intervention. The mechanism is based on the inhibition of protein function by hijacking a ubiquitin E3 ligase for protein degradation. The hetero-bifunctional PROTACs contain a ligand for recruiting an E3 ligase, a linker, and another ligand to bind with the protein targeted for degradation. Thus, PROTACs have profound potential to eliminate "undruggable" protein targets, such as transcription factors and non-enzymatic proteins, which are not limited to physiological substrates of the ubiquitin-proteasome system. These findings indicate great prospects for PROTACs in the development of therapeutics. However, there are several limitations related to poor stability, biodistribution, and penetrability in vivo. This review provides an overview of the main PROTAC-based approaches that have been developed and discusses the promising opportunities and considerations for the application of this technology in therapies and drug discovery.

Colloidal shuttles for programmable cargo transport

The active transport of cargo molecules within cells is essential for life. Developing synthetic strategies for cargo control in living or inanimate thermal systems could lead to powerful tools to manipulate chemical gradients at the microscale and thus drive processes out of equilibrium to realize work. Here we demonstrate a colloidal analog of the complex biological shuttles responsible for molecular trafficking in cells. Our colloidal shuttles consist of magneto-dielectric particles that are loaded with cargo particles or living cells through size-selective dielectrophoretic trapping using electrical fields. The loaded colloidal shuttle can be transported with magnetic field gradients before cargo is released at the target location by switching off the electrical field. Such spatiotemporal control over the distribution of chemically active cargo in a reversible fashion can be potentially exploited for fundamental biological research or for the development of novel technologies for advanced cell culturing, drug discovery and medical diagnosis.

Unraveling the biology of bipolar disorder using induced pluripotent stem-derived neurons.

Bipolar disorder has been studied from numerous angles, from pathological studies to large-scale genomic studies, overall making moderate gains toward an understanding of the disorder. With the advancement of induced pluripotent stem (iPS) cell technology, in vitro models based on patient samples are now available that inherently incorporate the complex genetic variants that largely are the basis for this disorder. A number of groups are starting to apply iPS technology to the study of bipolar disorder. We selectively reviewed the literature related to understanding bipolar disorder based on using neurons derived from iPS cells. So far, most work has used the prototypical iPS cells. However, others have been able to transdifferentiate fibroblasts directly to neurons. Others still have utilized olfactory epithelium tissue as a source of neural-like cells that do not need reprogramming. In general, iPS and related cells can be used for studies of disease pathology, drug discovery, or stem cell therapy. Published studies have primarily focused on understanding bipolar disorder pathology, but initial work is also being done to use iPS technology for drug discovery. In terms of disease pathology, some evidence is pointing toward a differentiation defect with more ventral cell types being prominent. Additionally, there is evidence for a calcium signaling defect, a finding that builds on the genome-wide association study results. Continued work with iPS cells will certainly help us understand bipolar disorder and provide a way forward for improved treatments.

An Automated Platform for Assessment of Congenital and Drug-Induced Arrhythmia with hiPSC-Derived Cardiomyocytes.

The ability to produce unlimited numbers of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) harboring disease and patient-specific gene variants creates a new paradigm for modeling congenital heart diseases (CHDs) and predicting proarrhythmic liabilities of drug candidates. However, a major roadblock to implementing hiPSC-CM technology in drug discovery is that conventional methods for monitoring action potential (AP) kinetics and arrhythmia phenotypes in vitro have been too costly or technically challenging to execute in high throughput. Herein, we describe the first large-scale, fully automated and statistically robust analysis of AP kinetics and drug-induced proarrhythmia in hiPSC-CMs. The platform combines the optical recording of a small molecule fluorescent voltage sensing probe (VoltageFluor2.1.Cl), an automated high throughput microscope and automated image analysis to rapidly generate physiological measurements of cardiomyocytes (CMs). The technique can be readily adapted on any high content imager to study hiPSC-CM physiology and predict the proarrhythmic effects of drug candidates.

Efficacy of Bioactive Cyclic Peptides in Rheumatoid Arthritis: Translation from In Vitro to In Vivo Models.

Using a novel drug discovery technology reported in previous issues of this journal cyclic peptides have been created which are able to down-regulate secretion of inflammatory cytokines, in vitro, by stimulated cells of the macrophage cell line J774. The cytokines in question, TNF-alpha and IL-6, are strongly implicated in etiology of diseases such as rheumatoid arthritis. Studies are reported here using the CAIA animal model for rheumatoid arthritis, which show that the peptides identified are indeed able to impact on inflammation of joints, induced in vivo. The results suggest that these peptides are effective at a dose which could be viable in man, and at which no adverse side effects are evident in the short term.

DruGAN: An Advanced Generative Adversarial Autoencoder Model for de Novo Generation of New Molecules with Desired Molecular Properties in Silico.

Deep generative adversarial networks (GANs) are the emerging technology in drug discovery and biomarker development. In our recent work, we demonstrated a proof-of-concept of implementing deep generative adversarial autoencoder (AAE) to identify new molecular fingerprints with predefined anticancer properties. Another popular generative model is the variational autoencoder (VAE), which is based on deep neural architectures. In this work, we developed an advanced AAE model for molecular feature extraction problems, and demonstrated its advantages compared to VAE in terms of (a) adjustability in generating molecular fingerprints; (b) capacity of processing very large molecular data sets; and (c) efficiency in unsupervised pretraining for regression model. Our results suggest that the proposed AAE model significantly enhances the capacity and efficiency of development of the new molecules with specific anticancer properties using the deep generative models.

Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers.

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise regulation of gene activity. In particular, novel stimuli-responsive materials with improved spatiotemporal control over gene expression would unlock translatable platforms in drug discovery and regenerative medicine technologies. Furthermore, an enhanced ability to control nucleic acid release from materials would enable the development of streamlined methods to predict nanocarrier efficacy a priori, leading to expedited screening of delivery vehicles. Herein, we present a protocol for predicting gene silencing efficiencies and achieving spatiotemporal control over gene expression through a modular photo-responsive nanocarrier system. Small interfering RNA (siRNA) is complexed with mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) polymers to form stable nanocarriers that can be controlled with light to facilitate tunable, on/off siRNA release. We outline two complementary assays employing fluorescence correlation spectroscopy and gel electrophoresis for the accurate quantification of siRNA release from solutions mimicking intracellular environments. Information gained from these assays was incorporated into a simple RNA interference (RNAi) kinetic model to predict the dynamic silencing responses to various photo-stimulus conditions. In turn, these optimized irradiation conditions allowed refinement of a new protocol for spatiotemporally controlling gene silencing. This method can generate cellular patterns in gene expression with cell-to-cell resolution and no detectable off-target effects. Taken together, our approach offers an easy-to-use method for predicting dynamic changes in gene expression and precisely controlling siRNA activity in space and time. This set of assays can be readily adapted to test a wide variety of other stimuli-responsive systems in order to address key challenges pertinent to a multitude of applications in biomedical research and medicine.

Abstract 4023: The SABRE platform: A novel, unbiased technology for drug discovery and prioritization

Abstract Targeted therapies designed to inhibit hyperactive oncogenic signaling have demonstrated some encouraging clinical responses. However, most of the time these responses are not durable as tumors “work around” pathway inhibition, often reactivating the inhibited pathway, leading to drug resistance and disease relapse. New technologies that enable more efficient discovery and development of “suites” of drugs that comprehensively inhibit multiple pathway nodes are needed. The SABRE (Splice Acceptor Brilliant Reporter) platform was developed to provide an unbiased method to screen large compound libraries and dramatically improve the speed and efficiency of which novel targeted therapeutics can be identified. SABRE is built off the premise that oncogenic output is exerted through changes in gene transcription and that such changes can be harnessed as powerful reporters of pathway activation status. SABRE utilizes the power of gene trap technology coupled with a drug selection process to isolate cells that generate a robust “off to on” signal in response to target or pathway inhibition. Via massively parallel comparative analysis, multiple traps let nature provide the best reporter for further analysis and drug discovery. In this proof-of-concept study, we employed the SABRE technology to identify insertion sites that are specifically regulated by the MAPK pathway. Experiments were performed using the human BRAFV600E mutant melanoma cell line A375. SABRE lentiviral transduced A375 cells were treated with trametinib, a MEK inhibitor, and clones were isolated that emitted a positive luciferase signal upon drug treatment. To determine if these reporters were MAPK pathway specific, the platform was miniaturized to a 1536 well format and used to screen a 6000+ compound library. Results from the screen found that 70% (28/40) of the top drug hits were known to directly modulate the MAPK pathway. Since drug resistance is a common occurrence in melanoma, we generated a “resistant” A375 SABRE reporter line to increasing concentrations of vemurafenib and interrogated the top drug candidates identified in the screen. As expected, the resistant SABRE line failed to respond to BRAFV600E specific inhibitors but continued to respond to downstream MEK inhibitors, which is helping to delineate the mechanism of resistance. We were also interested in the screening hits that were not previously known to influence the MAPK pathway including bafetinib and a Tie2 inhibitor. In follow-up testing, we found that these drugs induced robust and titratable reporter signals in both the SABRE A375 vemurafenib sensitive and resistant lines. In addition, treatment of either bafetinib or the Tie2 inhibitor reduced ERK phosphorylation in the vemurafenib resistant cells, suggesting that these drugs could be useful in the treatment of drug resistant melanomas. Studies are currently underway to determine the in vivo efficacy of these drugs in A375 SABRE reporter xenograft models. Citation Format: Shelli M. Morris, Andrew J. Mhyre, Savanna S. Carmack, Connor Burns, Marc Ferrer, Wenjuan Ye, James M. Olson, Richard A. Klinghoffer. The SABRE platform: A novel, unbiased technology for drug discovery and prioritization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4023. doi:10.1158/1538-7445.AM2017-4023