Identification of predictive genetic signatures of Cytarabine responsiveness using a 3D acute myeloid leukaemia model.

This study reports the establishment of a bone marrow mononuclear cell (BMMC) 3D culture model and the application of this model to define sensitivity and resistance biomarkers of acute myeloid leukaemia (AML) patient bone marrow samples in response to Cytarabine (Ara-C) treatment. By mimicking physiological bone marrow microenvironment, the growth conditions were optimized by using frozen BMMCs derived from healthy donors. Healthy BMMCs are capable of differentiating into major hematopoietic lineages and various types of stromal cells in this platform. Cryopreserved BMMC samples from 49 AML patients were characterized for ex vivo growth and sensitivity to Ara-C. RNA sequencing was performed for 3D and 2D cultures to determine differential gene expression patterns. Specific genetic mutations and/or gene expression signatures associated with the ability of the ex vivo expansion and response to Ara-C were elucidated by whole-exome and RNA sequencing. Data analysis identified unique gene expression signatures and novel genetic mutations associated with sensitivity to Ara-C treatment of proliferating AML specimens and can be used as predictive therapeutic biomarkers to determine the optimal treatment regimens. Furthermore, these data demonstrate the translational value of this ex vivo platform which should be widely applicable to evaluate other therapies in AML.

Effect of cryopreservation on delineation of immune cell subpopulations in tumor specimens as determinated by multiparametric single cell mass cytometry analysis.

BACKGROUND: Comprehensive understanding of cellular immune subsets involved in regulation of tumor progression is central to the development of cancer immunotherapies. Single cell immunophenotyping has historically been accomplished by flow cytometry (FC) analysis, enabling the analysis of up to 18 markers. Recent advancements in mass cytometry (MC) have facilitated detection of over 50 markers, utilizing high resolving power of mass spectrometry (MS). This study examined an analytical and operational feasibility of MC for an in-depth immunophenotyping analysis of the tumor microenvironment, using the commercial CyTOF™ instrument, and further interrogated challenges in managing the integrity of tumor specimens. RESULTS: Initial longitudinal studies with frozen peripheral blood mononuclear cells (PBMCs) showed minimal MC inter-assay variability over nine independent runs. In addition, detection of common leukocyte lineage markers using MC and FC detection confirmed that these methodologies are comparable in cell subset identification. An advanced multiparametric MC analysis of 39 total markers enabled a comprehensive evaluation of cell surface marker expression in fresh and cryopreserved tumor samples. This comparative analysis revealed significant reduction of expression levels of multiple markers upon cryopreservation. Most notably myeloid derived suppressor cells (MDSC), defined by co-expression of CD66b+ and CD15+, HLA-DRdim and CD14- phenotype, were undetectable in frozen samples. CONCLUSION: These results suggest that optimization and evaluation of cryopreservation protocols is necessary for accurate biomarker discovery in frozen tumor specimens.

Evaluation of JAK3 Biology in Autoimmune Disease Using a Highly Selective, Irreversible JAK3 Inhibitor.

Reversible janus associated kinase (JAK) inhibitors such as tofacitinib and decernotinib block cytokine signaling and are efficacious in treating autoimmune diseases. However, therapeutic doses are limited due to inhibition of other JAK/signal transducer and activator of transcription pathways associated with hematopoiesis, lipid biogenesis, infection, and immune responses. A selective JAK3 inhibitor may have a better therapeutic index; however, until recently, no compounds have been described that maintain JAK3 selectivity in cells, as well as against the kinome, with good physicochemical properties to test the JAK3 hypothesis in vivo. To quantify the biochemical basis for JAK isozyme selectivity, we determined that the apparent Km value for each JAK isozyme ranged from 31.8 to 2.9 µM for JAK1 and JAK3, respectively. To confirm compound activity in cells, we developed a novel enzyme complementation assay that read activity of single JAK isozymes in a cellular context. Reversible JAK3 inhibitors cannot achieve sufficient selectivity against other isozymes in the cellular context due to inherent differences in enzyme ATP Km values. Therefore, we developed irreversible JAK3 compounds that are potent and highly selective in vitro in cells and against the kinome. Compound 2, a potent inhibitor of JAK3 (0.15 nM) was 4300-fold selective for JAK3 over JAK1 in enzyme assays, 67-fold [interleukin (IL)-2 versus IL-6] or 140-fold [IL-2 versus erythropoietin or granulocyte-macrophage colony-stimulating factor (GMCSF)] selective in cellular reporter assays and >35-fold selective in human peripheral blood mononuclear cell assays (IL-7 versus IL-6 or GMCSF). In vivo, selective JAK3 inhibition was sufficient to block the development of inflammation in a rat model of rheumatoid arthritis, while sparing hematopoiesis.

Lead optimization of 4,4-biaryl piperidine amides as γ-secretase inhibitors.

Alzheimer's disease is a major unmet medical need with pathology characterized by extracellular proteinaceous plaques comprised primarily of ß-amyloid. γ-Secretase is a critical enzyme in the cellular pathway responsible for the formation of a range of ß-amyloid peptides; one of which, Aß42, is believed to be responsible for the neuropathological features of the disease. Herein, we report 4,4 disubstituted piperidine γ-secretase inhibitors that were optimized for in vitro cellular potency and pharmacokinetic properties in vivo. Key agents were further characterized for their ability to lower cerebral Aß42 production in an APP-YAC mouse model. This structural series generally suffered from sub-optimal pharmacokinetics but hypothesis driven lead optimization enabled the discovery of γ-secretase inhibitors capable of lowering cerebral Aß42 production in mice.

Discovery of MK-1454: A Potent Cyclic Dinucleotide Stimulator of Interferon Genes Agonist for the Treatment of Cancer.

Stereochemically and structurally complex cyclic dinucleotide-based stimulator of interferon genes (STING) agonists were designed and synthesized to access a previously unexplored chemical space. The assessment of biochemical affinity and cellular potency, along with computational, structural, and biophysical characterization, was applied to influence the design and optimization of novel STING agonists, resulting in the discovery of MK-1454 as a molecule with appropriate properties for clinical development. When administered intratumorally to immune-competent mice-bearing syngeneic tumors, MK-1454 exhibited robust tumor cytokine upregulation and effective antitumor activity. Tumor shrinkage in mouse models that are intrinsically resistant to single-agent therapy was further enhanced when treating the animals with MK-1454 in combination with a fully murinized antimouse PD-1 antibody, mDX400. These data support the development of STING agonists in combination with pembrolizumab (humanized anti-PD-1 antibody) for patients with tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 therapy.

Discovery of PDK1 kinase inhibitors with a novel mechanism of action by ultrahigh throughput screening.

The phosphoinositide 3-kinase/AKT signaling pathway plays a key role in cancer cell growth, survival, and angiogenesis. Phosphoinositide-dependent protein kinase-1 (PDK1) acts at a focal point in this pathway immediately downstream of phosphoinositide 3-kinase and PTEN, where it phosphorylates numerous AGC kinases. The PDK1 kinase domain has at least three ligand-binding sites: the ATP-binding pocket, the peptide substrate-binding site, and a groove in the N-terminal lobe that binds the C-terminal hydrophobic motif of its kinase substrates. Based on the unique PDK1 substrate recognition system, ultrahigh throughput TR-FRET and Alphascreen screening assays were developed using a biotinylated version of the PDK1-tide substrate containing the activation loop of AKT fused to a pseudo-activated hydrophobic motif peptide. Using full-length PDK1, K(m) values were determined as 5.6 mum for ATP and 40 nm for the fusion peptide, revealing 50-fold higher affinity compared with the classical AKT(Thr-308)-tide. Kinetic and biophysical studies confirmed the PDK1 catalytic mechanism as a rapid equilibrium random bireactant reaction. Following an ultrahigh throughput screen of a large library, 2,000 compounds were selected from the reconfirmed hits by computational analysis with a focus on novel scaffolds. ATP-competitive hits were deconvoluted by dose-response studies at 1x and 10x K(m) concentrations of ATP, and specificity of binding was assessed in thermal shift assay. Inhibition studies using fusion PDK1-tide1 substrate versus AKT(Thr-308)-tide and kinase selectivity profiling revealed a novel selective alkaloid scaffold that evidently binds to the PDK1-interacting fragment pocket. Molecular modeling suggests a structural paradigm for the design of inhibitory versus activating allosteric ligands of PDK1.

Establishing and Validating Cellular Functional Target Engagement Assay for Selective IRAK4 Inhibitor Discovery.

One of the main reasons for the lack of drug efficacy in late-stage clinical trials is the lack of specific and selective target engagement. To increase the likelihood of success of new therapeutics, one approach is to conduct proximal target engagement testing during the early phases of preclinical drug discovery. To identify and optimize selective IRAK4 inhibitors, a kinase that has been implicated in multiple inflammatory and autoimmune diseases, we established an electrochemiluminescence (ECL)-based cellular endogenous IRAK1 activation assay as the most proximal functional evaluation of IRAK4 engagement to support structure-activity relationship (SAR) studies. Since IRAK1 activation is dependent on both the IRAK4 scaffolding function in Myddosome formation and IRAK4 kinase activity for signal transduction, this assay potentially captures inhibitors with different mechanisms of action. Data from this IRAK1 assay with compounds representing different structural classes showed statistically significant correlations when compared with results from both IRAK4 biochemical kinase activity and functional peripheral blood mononuclear cell (PBMC)-derived tumor necrosis factor α (TNFα) secretion assays, validating the biological relevancy of the IRAK1 target engagement as a biomarker of the IRAK4 activity. Plate uniformity and potency reproducibility evaluations demonstrated that this assay is amenable to high throughput. Using Bland-Altman assay agreement analysis, we demonstrated that incorporating such proximal pharmacological assessment of cellular target engagement to an in vitro screening funnel for SAR studies can prevent compound optimization toward off-target activity.

Development of High-Throughput Assays for Evaluation of Hematopoietic Progenitor Kinase 1 Inhibitors.

Hematopoietic progenitor kinase 1 (HPK1), also referred to as mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1), is a serine/threonine kinase that negatively regulates T-cell signaling by phosphorylating Ser376 of Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76), a critical mediator of T-cell receptor activation. HPK1 loss of function mouse models demonstrated enhanced immune cell activation and beneficial antitumor activity. To enable discovery and functional characterization of high-affinity small-molecule HPK1 inhibitors, we have established high-throughput biochemical, cell-based, and novel pharmacodynamic (PD) assays. Kinase activity-based time-resolved fluorescence energy transfer (TR-FRET) assays were established as the primary biochemical approach to screen for potent inhibitors and assess selectivity against members of MAP4K and other closely related kinases. A proximal target engagement (TE) assay quantifying pSLP-76 levels as a readout and a distal assay measuring IL-2 secretion as a functional response were established using human peripheral blood mononuclear cells (PBMCs) from two healthy donors. Significant correlations between biochemical and cellular assays as well as excellent correlation between the two donors for the cellular assays were observed. pSLP-76 levels were further used as a PD marker in the preclinical murine model. This effort required the development of a novel ultrasensitive single-molecule array (SiMoA) assay to monitor pSLP-76 changes in mouse spleen.

An orally available non-nucleotide STING agonist with antitumor activity.

Pharmacological activation of the STING (stimulator of interferon genes)-controlled innate immune pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, an orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous and oral MSA-2 regimens were well tolerated and stimulated interferon-ß secretion in tumors, induced tumor regression with durable antitumor immunity, and synergized with anti-PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution, but only dimers bind and activate STING. This model was validated by using synthetic covalent MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular acidification, which mimics the tumor microenvironment. These properties appear to underpin the favorable activity and tolerability profiles of effective systemic administration of MSA-2.

Design and implementation of an automated compound management system in support of lead optimization.

To meet the needs of the increasingly rapid and parallelized lead optimization process, a fully integrated local compound storage and liquid handling system was designed and implemented to automate the generation of assay-ready plates directly from newly submitted and cherry-picked compounds. A key feature of the system is the ability to create project- or assay-specific compound-handling methods, which provide flexibility for any combination of plate types, layouts, and plate bar-codes. Project-specific workflows can be created by linking methods for processing new and cherry-picked compounds and control additions to produce a complete compound set for both biological testing and local storage in one uninterrupted workflow. A flexible cherry-pick approach allows for multiple, user-defined strategies to select the most appropriate replicate of a compound for retesting. Examples of custom selection parameters include available volume, compound batch, and number of freeze/thaw cycles. This adaptable and integrated combination of software and hardware provides a basis for reducing cycle time, fully automating compound processing, and ultimately increasing the rate at which accurate, biologically relevant results can be produced for compounds of interest in the lead optimization process.

A simplified scintillation proximity assay for fatty acid synthase activity: development and comparison with other FAS activity assays.

Fatty acid synthase (FAS), an essential enzyme for de novo lipogenesis, has been implicated in a number of disease states, including obesity, dyslipidemia, and cancer. To identify small-molecule inhibitors of FAS, the authors developed a bead-based scintillation proximity assay (SPA) to detect the fatty acid products of FAS enzymatic activity. This homogeneous SPA assay discriminates between a radiolabeled hydrophilic substrate of FAS (acetyl-coenzyme A) and the labeled lipophilic products of FAS (fatty acids), generating signal only when labeled fatty acids are present. The assay requires a single addition of unmodified polystyrene imaging SPA beads and can be miniaturized to 384- or 1536-well density with appropriate assay statistics for high-throughput screening. High-potency FAS inhibitors were used to compare the sensitivity of the SPA bead assay with previously described assays that measure FAS reaction intermediates (CoA-SH and NADP+). The advantages and disadvantages of these different FAS assays in small-molecule inhibitor discovery are discussed.

Development of a cell-based assay for measurement of c-Met phosphorylation using AlphaScreen technology and high-content imaging analysis.

c-Met is a receptor tyrosine kinase (RTK) with a critical role in many fundamental cellular processes, including cell proliferation and differentiation. Deregulated c-Met signaling has been implicated in both the initiation and progression of human cancers and therefore represents an attractive target for anticancer therapy. Monitoring the phosphorylation status of relevant tyrosine residues provides an important method of assessing c-Met kinase activity. This report describes a novel assay to monitor c-Met phosphorylation in cells using Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen) technology. Using AlphaScreen, the authors were able to detect both global and site-specific phosphorylation of c-Met in transformed cell lines. Data obtained from the AlphaScreen assay were compared to data obtained from a high-content imaging (HCI) method developed in parallel to monitor c-Met phosphorylation at the single cell level. The AlphaScreen assay was miniaturized to a 384-well format with acceptable signal-to-background ratio (S/B) and Z' statistics and was employed to measure c-Met kinase activity in situ after treatment with potent c-Met-specific kinase inhibitors. The authors discuss the utility of quantifying endogenous cellular c-Met phosphorylation in lead optimization and how the modular design of the AlphaScreen assay allows its adaptation to measure cellular activity of other kinases.

High-throughput analysis of HGF-stimulated cell scattering.

Historically, only relatively low-throughput or expensive methods have been available to measure cell migration. Hepatocyte growth factor (HGF) is a ligand for the tyrosine kinase receptor Met that, in addition to mediating proliferation and survival, increases cell motility and metastasis. The authors have developed a high-throughput imaging assay for measuring inhibition of HGF-induced scattering in human HPAF-II pancreatic adenocarcinoma cells. Following treatment with test compounds and HGF for 24 h, cells are labeled with a nuclear stain and imaged at 10x magnification. The proximity of neighboring nuclei is measured, and the distribution of internuclear distances across each field of view is used to calculate the fraction of scattered cells. This method of analysis can be extended to other cell types and signaling pathways and, compared with other membrane-based migration assays currently available, the assay is significantly lower in cost, is less labor intensive, and provides higher throughput.

From rigid cyclic templates to conformationally stabilized acyclic scaffolds. Part I: the discovery of CCR3 antagonist development candidate BMS-639623 with picomolar inhibition potency against eosinophil chemotaxis.

Conformational analysis of trans-1,2-disubstituted cyclohexane CCR3 antagonist 2 revealed that the cyclohexane linker could be replaced by an acyclic syn-alpha-methyl-beta-hydroxypropyl linker. Synthesis and biological evaluation of mono- and disubstituted propyl linkers support this conformational correlation. It was also found that the alpha-methyl group to the urea lowered protein binding and that the beta-hydroxyl group lowered affinity for CYP2D6. Ab initio calculations show that the alpha-methyl group governs the spatial orientation of three key functionalities within the molecule. alpha-Methyl-beta-hydroxypropyl urea 31 with a chemotaxis IC(50)=38 pM for eosinophils was chosen to enter clinical development for the treatment of asthma.

Microfluidic sorting of mammalian cells by optical force switching.

Microfluidic-based devices have allowed miniaturization and increased parallelism of many common functions in biological assays; however, development of a practical technology for microfluidic-based fluorescence-activated cell sorting has proved challenging. Although a variety of different physical on-chip switch mechanisms have been proposed, none has satisfied simultaneously the requirements of high throughput, purity, and recovery of live, unstressed mammalian cells. Here we show that optical forces can be used for the rapid (2-4 ms), active control of cell routing on a microfluidic chip. Optical switch controls reduce the complexity of the chip and simplify connectivity. Using all-optical switching, we have implemented a fluorescence-activated microfluidic cell sorter and evaluated its performance on live, stably transfected HeLa cells expressing a fused histone-green fluorescent protein. Recovered populations were verified to be both viable and unstressed by evaluation of the transcriptional expression of two genes, HSPA6 and FOS, known indicators of cellular stress.

Determination of EPAC2 function using EPAC2 null Min6 sublines generated through CRISPR-Cas9 technology.

Min6 cells, a mouse ß cell line derived from transgenic mouse expressing the large T-antigen of SV40 in pancreatic beta cells, are commonly utilized as an in vitro cellular model for investigating targets involved in insulin secretion. Epac2, an exchange protein that can be directly activated by cyclic AMP (cAMP), is critical for pharmacologic stimuli-induced insulin secretion and has been hypothesized to be a direct target of sulfonylurea. Previous loss of function studies only specifically knocked out EPAC2 isoform A, leaving the other two isoforms intact. In this study, we investigated the function of EPAC2 in Min6 cells by generating EPAC2 knock-out sublines using CRISPR-Cas9 technology, by removing all three isoforms of EPAC2. Our results indicate that Min6 cells can be successfully cloned from a single cell after electroporation with plasmids expressing EPAC2 specific guide RNA, Cas9 and GFP, followed by sorting for GFP expressing single cells. Two clones were found to have a single nucleotide deletion in targeted site of EPAC2 gene by sequencing, therefore creating a frame shift in exon 13. The EPAC2 null clones have an unexpectedly increased secretion of insulin at basal level and an elevated total intracellular insulin content. However, EPAC2 deficiency impaires glucose and sulfonylurea induced insulin secretion without affecting sulfonylurea binding to cells. Potassium chloride induced insulin secretion remains intact. Interestingly, cAMP levels remained unchanged in EPAC2 null cells during these processes. To understand the global function of EPAC2, RNA Seq study was performed, which reveals that EPAC2 deficiency affects expression of multiple previously unrecognized genes, suggesting that EPAC2 can function through multiple pathways in addition to being a cAMP sensor.

Quality control procedures for dose-response curve generation using nanoliter dispense technologies.

With the advancement of high-throughput biomolecular screening techniques to the lead optimization stage, there is a critical need to quality control (QC) dose-response curves generated by robotic liquid handlers to ensure accurate affinity determinations. One challenge in evaluating the performance of liquid handlers is identifying and validating a robust method for testing dispense volumes across different instruments. Although traditional automated liquid handlers are still considered the standard platform in many laboratories, nanoliter dispensers are becoming more common and pose new challenges for routine quality control procedures. For example, standard gravimetric measurements are unreliable for testing the accuracy of nanoliter liquid dispenses. However, nanoliter dispensing technology allows for the conservation of compound, reduces compound carryover from well to well through discrete dispenses, and eliminates the need for intermediate compound dilution steps to achieve a low final DMSO assay concentration. Moreover, an intermediate dilution step in aqueous solution might result in compound precipitation at high concentrations. This study compared representative automation procedures done on a variety of liquid dispensers, including manual, traditional, and nanodispense volumes. The data confirmed the importance of establishing robust QC procedures for dose-response generation in addition to accuracy and precision determinations for each instrument, and they validated the use of nanoliter pipettors for dose-response testing. The results of this study also support the requirement for thorough mixing during serial compound dilutions prepared for high-throughput lead optimization strategies using traditional liquid handlers.

Use of optophoresis as an in vitro predictor of cell response to chemotherapy for chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia [CLL] is characterized by active accumulation of clonal CD5+/CD19+/CD23+ B cells. Individualized characterization of patient cell resistance/sensitivity to specific agents can provide important information to guide therapy selection. We have utilized optophoresis, which is a technique for the analysis of the motion of cells within a moving optical gradient field. It detects the broad cellular changes associated with apoptosis based on physical characteristics of the cell, such as morphology, size, refractive index, density, and surface properties. We analyzed peripheral blood samples from 62 CLL patients in the presence of varying concentrations of chemotherapeutic agents. Optophoresis and a more conventional measurement of cell death were utilized. The outcome of ex vivo drug resistance using optophoresis was compared to clinical response in 30 patients for which there was clinical outcome data available. The overall accuracy of optophoresis in reflecting clinical response was 80%. It has advantages over alternative methods of determining chemoresistance including the ability to evaluate very small sample sizes and ability to work in mixed-cell populations. Changes in cell physical characteristics in response to chemotherapy, as measured by optophoresis is an accurate method for predicting chemosensitivity ex vivo in CLL.

Integration of Affinity Selection-Mass Spectrometry and Functional Cell-Based Assays to Rapidly Triage Druggable Target Space within the NF-κB Pathway.

The primary objective of early drug discovery is to associate druggable target space with a desired phenotype. The inability to efficiently associate these often leads to failure early in the drug discovery process. In this proof-of-concept study, the most tractable starting points for drug discovery within the NF-κB pathway model system were identified by integrating affinity selection-mass spectrometry (AS-MS) with functional cellular assays. The AS-MS platform Automated Ligand Identification System (ALIS) was used to rapidly screen 15 NF-κB proteins in parallel against large-compound libraries. ALIS identified 382 target-selective compounds binding to 14 of the 15 proteins. Without any chemical optimization, 22 of the 382 target-selective compounds exhibited a cellular phenotype consistent with the respective target associated in ALIS. Further studies on structurally related compounds distinguished two chemical series that exhibited a preliminary structure-activity relationship and confirmed target-driven cellular activity to NF-κB1/p105 and TRAF5, respectively. These two series represent new drug discovery opportunities for chemical optimization. The results described herein demonstrate the power of combining ALIS with cell functional assays in a high-throughput, target-based approach to determine the most tractable drug discovery opportunities within a pathway.

Improvement of "hit-to-lead" optimization by integration of in vitro HTS experimental models for early determination of pharmacokinetic properties.

Development of predictive in vitro surrogate methods for traditional approaches assessing bioavailability and pharmacokinetics of lead compounds must be made to both keep pace with high-throughput (HT) lead identification and to mitigate the high costs associated with progression of compounds with poor chances of developmental success. Indeed opportunities for improvement still exist in the lead optimization phase versus the lead identification phase, where HT methodologies have been nearly optimized. Review of examples, limitations, and development of high-throughput microtiterplate-based assays for evaluating metabolic liabilities, such as in vitro radiometric and fluorometric assays for inhibition of cytochrome p450 (CYP) activity, determination of stability of a compound in liver microsomes, or cloned CYPs coupled to reconstituting systems are described. Parallel approaches to improve speed, resolution, sample preparation, as well as data analysis using LC/MS and LC/MS/MS approaches and technologies to assess compound integrity and biotransformation by automation and multiplexing are also discussed. Realization of the benefits in automation of cell-based models for determining drug permeability to predict drug absorption are still hampered by bottlenecks in analytical analysis of compounds. The implementation and limitations of surrogate physiochemical methods for passive adsorption such as immobilized artificial membranes (IAM) and parallel artificial membrane permeation assays (PAMPA), and compound solubility by laser nephelometry are reviewed as well. Additionally, data from a high-throughput 96-well equilibrium dialysis device, showing good correlation to classical methods, is presented. Finally, the impact of improvements in these downstream bottlenecks in lead optimization and preclinical drug discovery are discussed in this review.