Small Molecule Drug Development Research Articles

Synthesis of Sialic Acids, Their Derivatives, and Analogs by Using a Whole-Cell Catalyst.

Sialic acids (Sias) are important constituents of cell surface glycans. Ready access to Sias in large quantities would facilitate the development of carbohydrate-based vaccines and small-molecule drugs. We now present a facile method for synthesizing various natural forms and non-natural derivatives or analogs of Sias by using a whole-cell catalyst, which is constructed by adding a plasmid containing necessary enzyme genes into a metabolically engineered strain of Escherichia coli. The flexible substrate tolerance of incorporated enzymes (N-acetylglucosamine 2-epimerase and N-acetylneuraminic acid aldolase) allows the cellular catalyst to convert a wide range of simple and inexpensive sugars into various Sia-related compounds through an easily scalable fermentation process. Further, syntheses using this whole-cell biotransformation in combination with three conventional enzymatic reactions provide a series of complex Sia-containing glycans (sialyloligosaccharides) and their derivatives bearing different substituents. The processes described herein should permit the large-scale and economical production of both Sias and sialyloligosaccharides, and may complement existing chemical and enzymatic strategies.

Development of a Non-Hydroxamate Dual Matrix Metalloproteinase (MMP)-7/-13 Inhibitor.

Matrix metalloproteinase 7 (MMP-7) is a member of the MMP superfamily and is able to degrade extracellular matrix proteins such as casein, gelatin, fibronectin and proteoglycan. MMP-7 is a validated target for the development of small molecule drugs against cancer. MMP-13 is within the enzyme class the most efficient contributor to type II collagen degeneration and is a validated target in arthritis and cancer. We have developed the dual MMP-7/-13 inhibitor ZHAWOC6941 with IC50-values of 2.2 μM (MMP-7) and 1.2 μM (MMP-13) that is selective over a broad range of MMP isoforms. It spares MMP-1, -2, -3, -8, -9, -12 and -14, making it a valuable modulator for targeted polypharmacology approaches.

Recommendations of the Oligonucleotide Safety Working Group's Formulated Oligonucleotide Subcommittee for the Safety Assessment of Formulated Oligonucleotide-Based Therapeutics.

The use of lipid formulations has greatly improved the ability to effectively deliver oligonucleotides and has been instrumental in the rapid expansion of therapeutic development programs using oligonucleotide drugs. However, the development of such complex multicomponent therapeutics requires the implementation of unique, scientifically sound approaches to the nonclinical development of these drugs, based upon a hybrid of knowledge and experiences drawn from small molecule, protein, and oligonucleotide therapeutic drug development. The relative paucity of directly applicable regulatory guidance documents for oligonucleotide therapeutics in general has resulted in the generation of multiple white papers from oligonucleotide drug development experts and members of the Oligonucleotide Safety Working Group (OSWG). The members of the Formulated Oligonucleotide Subcommittee of the OSWG have utilized their collective experience working with a variety of formulations and their associated oligonucleotide payloads, as well as their insights into regulatory considerations and expectations, to generate a series of consensus recommendations for the pharmacokinetic characterization and nonclinical safety assessment of this unique class of therapeutics. It should be noted that the focus of Subcommittee discussions was on lipid nanoparticle and other types of particulate formulations of therapeutic oligonucleotides and not on conjugates or other types of modifications of oligonucleotide structure intended to facilitate delivery.

Analysis of Clinical Dose–Response in Small-Molecule Drug Development: 2009–2014

ABSTRACTA meta-analysis of dose–response studies is reported for small-molecule drugs approved by the U.S. Food and Drug Administration (FDA) between January 2009 and May 2014. Summaries of the study designs are presented, including the number of studies conducted for each drug, dosing range, and the number of doses evaluated. Most drugs were studied on a ⩽ 4-fold range. Most of the study designs and their analyses focused on a small number of pairwise comparisons of dose groups to placebo. For the meta-analysis, efficacy endpoints were evaluated at a single landmark time. Safety endpoints were not collected. The commonly used Emax model was fit for each drug. Due to the limited number of doses and dosing ranges, maximum likelihood estimation applied to drugs separately performed poorly. Bayesian hierarchical models were successfully fit producing Emax curves that represented the data well. The distributions of the Emax model parameters were consistent with previously reported distributions estimated from a sponsor-specific meta-analysis of dose response. Assessment of model fit, which focused on potential nonmonotone loss of efficacy at the highest doses, supported the use of the Emax curves. The meta-analysis provides additional empirical basis for Bayesian prior distributions for model parameters. Supplementary materials for this article are available online.

Shikonin alleviates the biotoxicity produced by pneumococcal pneumolysin

AimsStreptococcus pneumoniae (S. pneumoniae) is a common pathogen that can cause severe infections in humans. Pneumolysin (PLY) is an important virulence trait of S. pneumoniae and has cytotoxicity, genotoxicity and pro-inflammatory activity; it is essential for the pathogenesis of S. pneumoniae pneumonia and is an anti-virulence target of small molecule drug development. The treatment options for this microbe were limit due to the ubiquitous antibiotic resistance; therefore, new drugs and treatment strategies are needed. MethodsShikonin was selected by drug screening based on haemolysis assays, and its mechanism of suppressing PLY toxicity was determined by oligomerization assay. Meanwhile, the in vitro cell viability assays and in vivo experiments were performed to explore the capability of shikonin to protect cells and tissue from S. pneumoniae-mediated damage. Key findingsShikonin was found to significantly decrease PLY-induced haemolytic activity, cytotoxicity and genotoxicity via lessening the formation of oligomers; moreover, the agent can reduce the mortality of mice caused by lethal pneumonia and mitigate the injury of target organs as well. SignificanceWe suggest that shikonin could be a potent candidate for a novel therapeutic or auxiliary substance in the treatment of infections encountering insufficient vaccines and antimicrobial resistance to traditional antibiotics.

Morin Moderates the Biotoxicity of Pneumococcal Pneumolysin by Weakening the Oligomers' Formation.

Streptococcus pneumoniae (pneumococcus) is an important causative agent of acute invasive and non-invasive infections. Pneumolysin is one of a considerable number of virulence traits produced by pneumococcus that exhibits a variety of biological activities, thus making it a target of small molecule drug development. In this study, we aimed to evaluate the effect of morin, a natural compound that has no antimicrobial activity against S. pneumonia, is a potent neutralizer of pneumolysin-mediated cytotoxicity and genotoxicity by impairing oligomer formation, and possesses the capability of mitigating tissue damage caused by pneumococcus. These findings indicate that morin could be a potent candidate for a novel therapeutic or auxiliary substance to treat infections for which there are inadequate vaccines and that are resistant to traditional antibiotics.

Small molecule drug development based on derivatization of natural products

Natural products in a general term refer to endogenous chemical substances or secondary metabolites produced by living organisms such as animals, plants or microorganisms. Historically, natural products have been indispensable resources as medication throughout human civilization worldwide. Crude or semi-purified extracts of these living organisms, especially terrestrial plants, have been described in a variety of ancient Chinese books and utilized to treat various human diseases. With the establishment of modern scientific system, research on individual active principles or secondary metabolites from these living organisms was expanded, initially focusing on the chemistry and pharmacological effects. The isolation, purification, pharmacological validation, and market of a naturally occurring natural product morphine as a painkiller directly led to the foundation of a modern pharmaceutical company Merck in 1893. Since then a number of natural products, such as quinine and penicillin, have been introduced as important medicines for the wellbeing of mankind. Novel natural products have been the gold mine as drug leads in the field of small molecule drug discovery and development to all pharmaceutical companies for over a century. With the great advancement in synthetic organic chemistry and medicinal chemistry large volume of structurally diversified small molecules can now be rapidly produced and screened in a high throughput manner, the role of natural products in modern drug discovery and development have been largely diminished. Very often intrinsic side effects with natural products such as toxicity, limited availability, and unfavorable potency and selectivity render it more and more difficult to utilize them directly as drug candidates. Practically, synthetic transformation or total synthesis of natural products is required due to above-mentioned suboptimal physicochemical and pharmacological properties or limited availability. 2015 Nobel Prize in Physiology or Medicine was awarded to the discoveries of two natural products based novel therapies: i.e. avermectin-based therapy against infections caused by roundworm parasites and artemisinin (Qinghaosu)-based therapy against Malaria. These two natural products technically possessed certain unfavorable properties and the clinically utilized medicines were indeed their synthetic derivatives. This review is intended to illustrate the importance and significance of structural determination and synthetic transformations on natural products in modern drug discovery and development.

A Promising but Challenging Strategy for Cancer Treatment: Disruption of Myc-Max Heterodimerization

c-Myc (Myc) is oncoprotein and nuclear transcription factor which plays a crucial role in several central cellular processes, involving cell proliferation, transformation, inhibition of differentiation and apoptosis. These functions result from the transcriptional activity of Myc. To be a functional form, Myc protein binds to the Max which recruits the E-box. Dysregulated over-expression of Myc is observed in many human malignancies including breast cancer, small-cell lung cancer, osteosarcomas, glioblastomas, and myeloid leukemias. Therefore, inhibition of the Myc-Max dimerization is a promising strategy for anticancer therapy. In this review, we summarize the Myc-Max disruption by small molecules targeting the oncogene. These information will contribute to the research and development of Myc-targeted smallmolecule drugs.

The promise of epigenomic therapeutics in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is often viewed to arise primarily by genetic alterations. However, today we know that many aspects of the cancer phenotype require a crosstalk among these genetic alterations with epigenetic changes. Indeed, aberrant gene expression patterns, driven by epigenetics are fixed by altered signaling from mutated oncogenes and tumor suppressors to define the PDAC phenotype. This conceptual framework may have significant mechanistic value and could offer novel possibilities for treating patients affected with PDAC. In fact, extensive investigations are leading to the development of small molecule drugs that reversibly modify the epigenome. These new ‘epigenetic therapeutics’ discussed herein are promising to fuel a new era of studies, by providing the medical community with new tools to treat this dismal disease.

Allostery and Biased Agonism at Class B G Protein-Coupled Receptors.

Class B G protein-coupled receptors (GPCRs) respond to paracrine or endocrine peptide hormones involved in control of bone homeostasis, glucose regulation, satiety, and gastro-intestinal function, as well as pain transmission. These receptors are targets for existing drugs that treat osteoporosis, hypercalcaemia, Paget's disease, type II diabetes, and obesity and are being actively pursued as targets for numerous other diseases. Exploitation of class B receptors has been limited by difficulties with small molecule drug discovery and development and an under appreciation of factors governing optimal therapeutic efficacy. Recently, there has been increasing awareness of novel attributes of GPCR function that offer new opportunity for drug development. These include the presence of allosteric binding sites on the receptor that can be exploited as drug binding pockets and the ability of individual drugs to enrich subpopulations of receptor conformations to selectively control signaling, a phenomenon termed biased agonism. In this review, current knowledge of biased signaling and small molecule allostery within class B GPCRs is discussed, highlighting areas that have progressed significantly over the past decade, in addition to those that remain largely unexplored with respect to these phenomena.

Is Science the Only Driver in Species Selection? An Internal Study to Evaluate Compound Requirements in the Minipig Compared to the Dog in Preclinical Studies.

Dogs have been often chosen as a nonrodent species for preclinical development of small molecule drugs mainly due to availability and relative ease of handling. Recently, focus has increased on the minipig as a potential alternative to the dog, based on either scientific rationale or public opinion concerns. There are, however, other factors influencing nonrodent choices, in particular drug amount and synthesis time, which differ between species and therefore may impact the milestones of a drug development program. To assess the magnitude of compound need, a retrospective internal survey was conducted on drug amounts used in dog studies which were translated into the requirements for minipigs. Compound need approximately doubles if minipigs are used. Costs of compound are accordingly higher, and synthesis times are slightly increased. In our company, the differences were not considered significant enough to preclude the use of minipigs if the later preclinical program might benefit from improved human risk prediction.

Synthesis and Preclinical Evaluation of Sulfonamido-based [(11)C-Carbonyl]-Carbamates and Ureas for Imaging Monoacylglycerol Lipase.

Monoacylglycerol lipase (MAGL) is a 33 kDa member of the serine hydrolase superfamily that preferentially degrades 2-arachidonoylglycerol (2-AG) to arachidonic acid in the endocannabinoid system. Inhibition of MAGL is not only of interest for probing the cannabinoid pathway but also as a therapeutic and diagnostic target for neuroinflammation. Limited attempts have been made to image MAGL in vivo and a suitable PET ligand for this target has yet to be identified and is urgently sought to guide small molecule drug development in this pathway. Herein we synthesized and evaluated the physiochemical properties of an array of eleven sulfonamido-based carbamates and ureas with a series of terminal aryl moieties, linkers and leaving groups. The most potent compounds were a novel MAGL inhibitor, N-((1-(1H-1,2,4-triazole-1-carbonyl)piperidin-4-yl) methyl)-4-chlorobenzenesulfonamide (TZPU; IC50 = 35.9 nM), and the known inhibitor 1,1,1,3,3,3-hexafluoropropan-2-yl 4-(((4-chlorophenyl)sulfonamido) methyl)piperidine-1-carboxylate (SAR127303; IC50 = 39.3 nM), which were also shown to be selective for MAGL over fatty acid amide hydrolase (FAAH), and cannabinoid receptors (CB1 & CB2). Both of these compounds were radiolabeled with carbon-11 via [11C]COCl2, followed by comprehensive ex vivo biodistribution and in vivo PET imaging studies in normal rats to determine their brain permeability, specificity, clearance and metabolism. Whereas TZPU did not show adequate specificity to warrant further evaluation, [11C]SAR127303 was advanced for preliminary PET neuroimaging studies in nonhuman primate. The tracer showed good brain permeability (ca. 1 SUV) and heterogeneous regional brain distribution which is consistent with the distribution of MAGL.

Biology, Chemistry, and Pharmacology of Sirtuins.

Sirtuins are a family of protein deacylases related by amino acid sequence and cellular function to the yeast Saccharomyces cerevisiae protein Sir2 (Silent Information Regulator-2), the first of this class of enzymes to be identified and studied in detail. Based on its initially discovered activity, Sir2 was classified as a histone deacetylase that removes acetyl groups from histones H3 and H4. The acetylation/deacetylation of these particular substrates leads to changes in transcriptional silencing at specific loci in the yeast genome, hence its name. Sirtuins, however, have been shown to regulate a wide variety of cellular processes beyond transcriptional repression in varied subcellular compartments and in different cell types. Mechanistically distinct from Zn(2+)-dependent deacylases, sirtuins use nicotinamide adenine dinucleotide as a cofactor in the removal of acetyl and other acyl groups linking metabolic status and posttranslational modification. Sirtuins' unique position has made them attractive targets for small-molecule drug development. In this chapter, we describe the biological roles, therapeutic areas in which sirtuins may play a role and development of small-molecule inhibitors of sirtuins employing phenotypic screening technologies ranging from assays in yeast, as well as biochemical screens to yield lead drug development candidates targeting a broad spectrum of human diseases.

Crystal structure of Streptococcus pneumoniae pneumolysin provides key insights into early steps of pore formation.

Pore-forming proteins are weapons often used by bacterial pathogens to breach the membrane barrier of target cells. Despite their critical role in infection important structural aspects of the mechanism of how these proteins assemble into pores remain unknown. Streptococcus pneumoniae is the world’s leading cause of pneumonia, meningitis, bacteremia and otitis media. Pneumolysin (PLY) is a major virulence factor of S. pneumoniae and a target for both small molecule drug development and vaccines. PLY is a member of the cholesterol-dependent cytolysins (CDCs), a family of pore-forming toxins that form gigantic pores in cell membranes. Here we present the structure of PLY determined by X-ray crystallography and, in solution, by small-angle X-ray scattering. The crystal structure reveals PLY assembles as a linear oligomer that provides key structural insights into the poorly understood early monomer-monomer interactions of CDCs at the membrane surface.

The inhibitory effects of a RANKL-binding peptide on articular and periarticular bone loss in a murine model of collagen-induced arthritis: a bone histomorphometric study

IntroductionWe designed OP3-4 (YCEIEFCYLIR), a cyclic peptide, to mimic the soluble osteoprotegerin (OPG), and was proven to bind to RANKL (receptor activator of NF-κB ligand), thereby inhibiting osteoclastogenesis. We recently found that another RANKL binding peptide, W9, could accelerate bone formation by affecting RANKL signaling in osteoblasts. We herein demonstrate the effects of OP3-4 on bone formation and bone loss in a murine model of rheumatoid arthritis.MethodsTwenty-four seven-week-old male DBA/1J mice were used to generate a murine model of collagen-induced arthritis (CIA). Then, vehicle or OP3-4 (9 mg/kg/day or 18 mg/kg/day) was subcutaneously infused using infusion pumps for three weeks beginning seven days after the second immunization. The arthritis score was assessed, and the mice were sacrificed on day 49. Thereafter, radiographic, histological and biochemical analyses were performed.ResultsThe OP3-4 treatment did not significantly inhibit the CIA-induced arthritis, but limited bone loss. Micro-CT images and quantitative measurements of the bone mineral density revealed that 18 mg/kg/day OP3-4 prevented the CIA-induced bone loss at both articular and periarticular sites of tibiae. As expected, OP3-4 significantly reduced the CIA-induced serum CTX levels, a marker of bone resorption. Interestingly, the bone histomorphometric analyses using undecalcified sections showed that OP3-4 prevented the CIA-induced reduction of bone formation-related parameters at the periarticular sites.ConclusionThe peptide that mimicked OPG prevented inflammatory bone loss by inhibiting bone resorption and stimulating bone formation. It could therefore be a useful template for the development of small molecule drugs for inflammatory bone loss.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0753-8) contains supplementary material, which is available to authorized users.

Discovery of novel nonpeptide allosteric inhibitors interrupting the interaction of CDK2/cyclin A3 by virtual screening and bioassays.

Serine/threonine-specific cyclin-dependent kinases (CDKs) are key regulatory elements in eukaryotic cell cycle progression, and the dysregulation of CDKs has been implicated in cancers. Therefore, CDKs have been identified as anti-cancer targets for the development of small-molecule drugs. In this Letter, virtual screening and biological evaluation were performed to identify novel lead structures that allosterically disrupt the interaction between CDK2 and cyclin A3, which are directed toward a noncatalytic binding pocket of CDK2. Ultimately, B2 was identified as exhibiting superior CDK2/cyclin A3 inhibition activity. In addition, our results indicated that B2 exhibited antiproliferative activities against a broad spectrum of human cancer cell lines. Significantly, B2 certainly interrupted the interaction between CDK2 and cyclin A3 and exhibited a concentration-dependent trend. In summary, our results suggest that B2 is the first effective allosteric chemical small-molecule CDK2 inhibitor to be discovered, and further lead optimization may result in a series of novel anti-CDK2 agents.

Abstract 4932: Development of a highly sensitive library prep method for identifying pathogenic variants from FFPE tumor samples

Abstract The application of genomics in cancer has led to an improved understanding of the disease. To date, 125 known driver genes have been discovered*. These genes and their pathways have become the focus of small-molecule drug development. While the number of available targeted therapies is limited, an estimated 800 oncology drugs are in development*, many of which target specific mutations. Single-analyte companion diagnostics exist for the approved therapies, and more might be developed as new therapeutics become available, but the need to assay each variant individually can be ineffective.. An additional, well-known challenge is the degradation and damage caused to DNA in formalin-fixed, paraffin-embedded (FFPE) samples. For these reasons, Illumina is developing a novel method for simultaneous detection of multiple somatic variants from FFPE specimens. This method leverages a multiplexed PCR approach with amplicons designed to a 15-gene panel that can detect somatic variants in 14 genes, and copy number changes in 3 genes. In our experiments, this multiplexed PCR assay has been demonstrated to be highly sensitive, yielding results from as little as 20 ng of DNA extracted from FFPE tissue, even if the DNA is highly damaged and degraded. Somatic variants can be detected down to 5% allele fraction, and gene amplifications can be detected for ≥ 2.2-fold change in copy number. This method, incorporating newly designed software, will be deployed as part of the Illumina Universal Oncology Test System on the MiSeqDx™ (under development), analyzing the sequencing results, and generating reports of mutations and copy number changes in cancer samples. Citation Format: Amanda G. Young, Charles Lin. Development of a highly sensitive library prep method for identifying pathogenic variants from FFPE tumor samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4932. doi:10.1158/1538-7445.AM2015-4932

A Dual Receptor and Reporter for Multi-Modal Cell Surface Engineering.

The rapid development of new small molecule drugs, nanomaterials, and genetic tools to modulate cellular function through cell surface manipulation has revolutionized the diagnosis, study, and treatment of disorders in human health. Since the cell membrane is a selective gateway barrier that serves as the first line of defense/offense and communication to its environment, new approaches that molecularly engineer or tailor cell membrane surfaces would allow for a new era in therapeutic design, therapeutic delivery, complex coculture tissue construction, and in situ imaging probe tracking technologies. In order to develop the next generation of multimodal therapies, cell behavior studies, and biotechnologies that focus on cell membrane biology, new tools that intersect the fields of chemistry, biology, and engineering are required. Herein, we develop a liposome fusion and delivery strategy to present a novel dual receptor and reporter system at cell surfaces without the use of molecular biology or metabolic biosynthesis. The cell surface receptor is based on bio-orthogonal functional groups that can conjugate a range of ligands while simultaneously reporting the conjugation through the emission of fluorescence. We demonstrate this dual receptor and reporter system by conjugating and tracking various cell surface ligands for temporal control of cell fluorescent signaling, cell-cell interaction, and tissue assembly construction.

Charting Immune Signaling Proteomes En Route to New Therapeutic Strategies.

The activation state of an antitumor effector T cell in a tumor depends on the sum of all stimulatory signals and inhibitory signals that it receives in the tumor microenvironment. Accumulating data address the increasing complexity of these signals produced by a myriad of immune checkpoint molecules, cytokines, and metabolites. While reductionist experiments have identified key molecules and their importance in signaling, less clear is the integration of all these signals that allows T cells to guide their responses in health and in disease. Mass spectrometry-based proteomics is well poised to offer such insights, including monitoring emergence of resistance mechanisms to immunotherapeutics during treatments. A major application of this technology is in the discovery and characterization of small-molecule agents capable of enhancing the response to immunotherapeutic agents. Such an approach would reinvigorate small-molecule drug development aimed not at tumor cells but rather at tumor-resident T cells capable of producing dramatic and durable antitumor responses.

Small-molecule compounds exhibiting target-mediated drug disposition - A case example of ABT-384.

Nonlinearities are frequently encountered in pharmacokinetics, and they can occur when 1 or more processes of absorption, distribution, metabolism, and excretion are saturable. One special source of nonlinearity that has been noticed recently is the saturable binding of the drug to a high-affinity-low-capacity target, a phenomenon known as target-mediated drug disposition (TMDD). Although TMDD can occur in both small-molecule compounds and large-molecule compounds, the latter has received much more attention because of its high prevalence. With the development of more potent small-molecule drugs acting on highly specific targets and the availability of increasingly sensitive analytical techniques, small-molecule compounds exhibiting TMDD have been increasingly reported in the past several years. ABT-384 is a small-molecule drug candidate that exhibited significant nonlinear pharmacokinetics, potentially imparted by TMDD, in a first-in-human clinical trial conducted in healthy volunteers. Compared with published small-molecule compounds exhibiting TMDD, ABT-384 pharmacokinetic characteristics are more consistent with TMDD. To expand current knowledge of TMDD of small-molecule compounds and increase awareness of this interesting and clinically important phenomenon, in this review the general features of small-molecule compounds exhibiting TMDD are highlighted, with ABT-384 provided as an example.