Natural‐Product‐Like Spiroketals and Fused Bicyclic Acetals as Potential Therapeutic Agents for B‐Cell Chronic Lymphocytic Leukaemia

B‐cell chronic lymphocytic leukaemia (CLL) is the most common form of leukaemia in the Western world for which no curative treatments are currently available. Purine nucleotide analogues and alkylating agents feature frequently in combination regimens to treat the malignant state, but their use has not led to any significant improvement in patient survival. Consequently, there still remains a need for alternative small‐molecule chemotherapeutics. Natural products are an unparalleled source of drug leads, and an unending inspiration for the design of small‐molecule libraries for drug discovery. The screening of focused libraries of natural‐product‐like spiroketal and fused bicyclic acetal small molecules against primary CLL cells has led to the identification of a small series of novel and potent cytotoxic agents towards primary CLL cells. The validation of the activity of these molecules is delineated through a series of synthesis and screening iterations, whereas preliminary mode of action studies positively indicate their ability to induce cell death via an apoptotic pathway with no evidence of necrosis to further support their potential as novel chemotherapeutic agents.     

Anti‐HIV Small‐Molecule Binding in the Peptide Subpocket of the CXCR4:CVX15 Crystal Structure

The CXC chemokine receptor 4 (CXCR4) is involved in chemotaxis and serves as a coreceptor for T‐tropic HIV‐1 viral entry, thus making this receptor an attractive drug target. Recently, crystal structures of CXCR4 were reported as complexes with the small molecule IT1t and the CVX15 peptide. Follow‐up efforts to model different antagonists into the small molecule CXCR4:IT1t crystal structure did not generate poses consistent with either the X‐ray crystal structure or site‐directed mutagenesis (SDM). Here, we compare the binding pockets of the two CXCR4 crystal structures, revealing differences in helices IV, V, VI, and VII, with major differences for the His203 residue buried in the binding pocket. The small molecule antagonist AMD11070 was docked into both CXCR4 crystal structures. An AMD11070 pose identified from the CXCR4:CVX15 model presented interactions with Asp171, Glu288, Trp94, and Asp97, consistent with published SDM data, thus suggesting it is the bioactive pose. A CXCR4 receptor model was optimized around this pose of AMD11070, and the resulting model correlated HIV‐1 inhibition with MM‐GBSA docking scores for a congeneric AMD11070‐like series. Subsequent NAMFIS NMR results successfully linked the proposed binding pose to an independent experimental structure. These results strongly suggest that not all small molecules will bind to CXCR4 in a similar manner as IT1t. Instead, the CXCR4:CVX15 crystal structure may provide a binding locus for small organic molecules that is more suitable than the secondary IT1t site. This work is expected to provide modeling insights useful for future CXCR4 antagonist and X4‐tropic HIV‐1 based drug design efforts.     

Advancing Small‐Molecule‐Based Chemical Biology with Next‐Generation Sequencing Technologies

Next‐generation‐sequencing (NGS) technologies enable us to obtain extensive information by deciphering millions of individual DNA sequencing reactions simultaneously. The new DNA‐sequencing strategies exceed their precursors in output by many orders of magnitude, resulting in a quantitative increase in valuable sequence information that could be harnessed for qualitative analysis. Sequencing on this scale has facilitated significant advances in diverse disciplines, ranging from the discovery, design, and evaluation of many small molecules and relevant biological mechanisms to maturation of personalized therapies. NGS technologies that have recently become affordable allow us to gain in‐depth insight into small‐molecule‐triggered biological phenomena and empower researchers to develop advanced versions of small molecules. In this review we focus on the overlooked implications of NGS technologies in chemical biology, with a special emphasis on small‐molecule development and screening.     

Ahp Cyclodepsipeptides: The Impact of the Ahp Residue on the “Canonical Inhibition” of S1 Serine Proteases

S1 serine proteases are by far the largest and most diverse family of proteases encoded in the human genome. Although recent decades have seen an enormous increase in our knowledge, the biological functions of most of these proteases remain to be elucidated. Chemical inhibitors have proven to be versatile tools for studying the functions of proteases, but this approach is hampered by the limited availability of inhibitor scaffold structures with the potential to allow rapid discovery of selective, noncovalent small‐molecule protease inhibitors. The natural product class of Ahp cyclodepsipeptides is an unusual class of small‐molecule canonical inhibitors; the incorporation of protease cleavage sequences into their molecular scaffolds enables the design of specific small‐molecule inhibitors that simultaneously target the S and S′ subsites of the protease through noncovalent mechanisms. Their synthesis is tedious, however, so in this study we have investigated the relevance of the Ahp moiety for achieving potent inhibition. We found that although the Ahp residue plays an important role in inhibition potency, appropriate replacement with β‐hydroxy amino acids results in structurally less complex derivatives that inhibit serine proteases in the low micromolar range.     

Discovery of Rogaratinib (BAY 1163877): a pan‐FGFR Inhibitor

Rogaratinib (BAY 1163877) is a highly potent and selective small‐molecule pan‐fibroblast growth factor receptor (FGFR) inhibitor (FGFR1–4) for oral application currently being investigated in phase 1 clinical trials for the treatment of cancer. In this publication, we report its discovery by de novo structure‐based design and medicinal chemistry optimization together with its pharmacokinetic profile.     

Small‐Molecule Mechanism of Action Studies in Caenorhabditis elegans

A general protocol for exogenous small‐molecule pull‐down experiments with Caenorhabditis elegans is described; it provides a link between small‐molecule screens in worms and existing mutant and RNAi technologies, thereby enabling organismal mechanism of action studies for the natural product clovanemagnolol. Forward chemical genetic screens followed by mechanism of action studies with C. elegans, when coupled with genetic validation of identified targets to reproduce the small molecule's phenotypic effects, provide a unique platform for discovering the biological targets of compounds that affect multicellular processes. First, the use of an immobilized FK506 derivative and soluble competition experiments with optimally prepared soluble C. elegans proteome successfully identified interactions with FK506 binding proteins 1 to 6. This approach was used to determine an unknown mechanism of action for clovanemagnolol, a small molecule that promotes axonal branching in both primary neuronal cultures and in vivo in C. elegans. Following the synthesis of an appropriately functionalized solid‐phase reagent bearing a clovanemagnolol analogue pull‐down experiments employing soluble competition identified kinesin light chain‐1 (KLC‐1), a protein involved in axonal cargo transport, as a putative target. This was corroborated through the use of mutant worms lacking klc‐1 and possessing GFP neuronal labeling, reproducing the axonal branching phenotype induced by the small molecule clovanemagnolol.     

Hedgehog Pathway Agonism: Therapeutic Potential and Small‐Molecule Development

The Hedgehog (Hh) pathway is a developmental signaling pathway that plays multiple roles during embryonic development and in adult tissues. Constitutive Hh signaling has been linked to the development and progression of several forms of cancer, and the application of small‐molecule pathway inhibitors as anticancer chemotherapeutics is well studied and clearly defined. Activation of the Hh pathway as a therapeutic strategy for a variety of degenerative or ischemic disorders has also been proposed; however, the development of small‐molecule Hh agonists has received less attention. The goal of this review is to highlight the recent evidence supporting the therapeutic potential of Hh pathway activators and to provide a comprehensive overview of small‐molecule pathway agonists.     

Improving the Solubility of Artificial Ligands of Streptavidin to Enable More Practical Reversible Switching of Protein Localization in Cells

Chemical inducers that can control target‐protein localization in living cells are powerful tools to investigate dynamic biological systems. We recently reported the retention using selective hook or “RUSH” system for reversible localization change of proteins of interest by addition/washout of small‐molecule artificial ligands of streptavidin (ALiS). However, the utility of previously developed ALiS was restricted by limited solubility in water. Here, we overcame this problem by X‐ray crystal structure‐guided design of a more soluble ALiS derivative (ALiS‐3), which retains sufficient streptavidin‐binding affinity for use in the RUSH system. The ALiS‐3–streptavidin interaction was characterized in detail. ALiS‐3 is a convenient and effective tool for dynamic control of α‐mannosidase II localization between ER and Golgi in living cells.     

Improving the Potency of Cancer Immunotherapy by Dual Targeting of IDO1 and DNA

Herein we report the first exploration of a dual‐targeting drug design strategy to improve the efficacy of small‐molecule cancer immunotherapy. New hybrids of indoleamine 2,3‐dioxygenase 1 (IDO1) inhibitors and DNA alkylating nitrogen mustards that respectively target IDO1 and DNA were rationally designed. As the first‐in‐class examples of such molecules, they were found to exhibit significantly enhanced anticancer activity in vitro and in vivo with low toxicity. This proof‐of‐concept study has established a critical step toward the development of a novel and effective immunotherapy for the treatment of cancers.     

Force applied to a single molecule at a single nanogate molecule filter

We have investigated the origin of molecule filtering system based on a chemical potential barrier produced by thermodynamically driven molecular flow in a nanoscopic space at nanogates. Single molecule tracking experiments prove that the highly localized potential barrier allows for selective manipulation of the target molecule. We propose the presence of a force, a few fN per molecule, to decelerate the molecule's movement at the nanogate, which is comparable to or larger than the force applied by conventional electrophoretic operation. The present force can be tuned by changing the nanogate width at the nanometre level. These findings allow us to propose an accurate design of novel devices for molecular manipulation on an ultra small scale using a very small number of molecules without any external biases.     

Chemical Genetics Approach to Engineer Kinesins with Sensitivity towards a Small‐Molecule Inhibitor of Eg5

Due to their fast and often reversible mode of action, small molecules are ideally suited to dissect biological processes. Yet, the validity of small‐molecule studies is intimately tied to the specificity of the applied compounds, thus imposing a great challenge to screens for novel inhibitors. Here, we applied a chemical‐genetics approach to render kinesin motor proteins sensitive to inhibition by the well‐characterized small molecule S‐Trityl‐l ‐cysteine (STLC). STLC specifically inhibits the kinesin Eg5 through binding to a known allosteric site within the motor domain. Transfer of this allosteric binding site into the motor domain of the human kinesins Kif3A and Kif4A sensitizes them towards STLC. Single‐molecule microscopy analyses confirmed that STLC inhibits the movement of chimeric but not wild‐type Kif4A along microtubules. Thus, our proof‐of‐concept study revealed that this chemical‐genetic approach provides a powerful strategy to specifically inhibit kinesins in vitro for which small‐molecule inhibitors are not yet available.     

An Fc–Small Molecule Conjugate for Targeted Inhibition of the Adenosine 2A Receptor

The adenosine A_2A receptor (A_2AR) is expressed in immune cells, as well as brain and heart tissue, and has been intensively studied as a therapeutic target for multiple disease indications. Inhibitors of the A_2AR have the potential for stimulating immune response, which could be valuable for cancer immune surveillance and mounting a response against pathogens. One well‐established potent and selective small molecule A_2AR antagonist, ZM‐241385 (ZM), has a short pharmacokinetic half‐life and the potential for systemic toxicity due to A_2AR effects in the brain and the heart. In this study, we designed an analogue of ZM and tethered it to the Fc domain of the immunoglobulin IgG3 by using expressed protein ligation. The resulting protein–small molecule conjugate, Fc–ZM, retained high affinity for two Fc receptors: FcγRI and the neonatal Fc receptor, FcRn. In addition, Fc–ZM was a potent A_2AR antagonist, as measured by a cell‐based cAMP assay. Cell‐based assays also revealed that Fc–ZM could stimulate interferon γ production in splenocytes in a fashion that was dependent on the presence of A_2AR. We found that Fc–ZM, compared with the small molecule ZM, was a superior A_2AR antagonist in mice, consistent with the possibility that Fc attachment can improve pharmacokinetic and/or pharmacodynamic properties of the small molecule.     

Glycan‐Mediated,Ligand‐Controlled Click Chemistry for Drug‐Target Identification

Membrane‐bound proteins are important pharmaceutical drug targets, yet few strategies exist for the identification of small‐molecule‐targeted membrane proteins in live‐cell systems. By exploiting metabolic glycan engineering of cell membrane proteins, we have developed an in situ glycan‐mediated ligand‐controlled click (“GLiCo‐Click”) chemistry methodology that enables the attachment of small‐molecule chemical probes to their receptor protein through glycans on live cells. In addition to enabling receptor enrichment from cell lysates, this strategy can be used to demonstrate target receptor engagement and enables the molecular characterization of receptors.     

Dissipative particle dynamic simulation on the assembly and release of siRNA/polymer/gold nanoparticles based polyplex

Dissipative particle dynamics simulation is used to reveal the loading/release of small interfering RNA (siRNA) in pH‐sensitive polymers/gold nanoparticles (AuNPs) polyplex. The conformation dynamics of these polyplex at various Au/siRNA mass ratios, the original AuNPs sizes, polymer types, and pH values are simulated and compared to experimental results. At neutral conditions (pH = 7.4), spherical micelles with a multilayer structure are formed in siRNA/polyethyleneimine/cis‐aconitic anhydride functionalized poly(allylamine)/polyethyleneimine/11‐mercaptoundecanoic acid‐gold nanoparticle (siRNA/PEI/PAH‐Cit/PEI/MUA‐AuNP) polyplex. Large polyplex are obtained with high Au/siRNA mass ratio and/or small original AuNPs size. The release dynamics of siRNA from AuNPs‐polyplex systems were also simulated in the intracellular environment (pH = 5.0). A swelling‐demicellization‐releasing mechanism is followed while the release of siRNA is found much faster for polyplex involving charge‐reversal PAH‐Cit. These findings are qualitatively consistent with the experimental results and may provide valuable guidance in later design and optimization of delivery carriers for siRNA or other molecule probes. © 2017 American Institute of Chemical Engineers AIChE J, 64: 810–821, 2018     

Substrate Selection Influences Molecular Recognition in a Screen for Lymphoid Tyrosine Phosphatase Inhibitors

Assay design is an important variable that influences the outcome of an inhibitor screen. Here, we have investigated the hypothesis that protein tyrosine phosphatase inhibitors with improved biological activity could be identified from a screen by using a biologically relevant peptide substrate, rather than traditional phosphotyrosine mimetic substrates. A 2000‐member library of drugs and drug‐like compounds was screened for inhibitors of lymphoid tyrosine phosphatase (LYP) by using both a peptide substrate (Ac‐ARLIEDNE‐pCAP‐TAREG‐NH₂, peptide 1) and a small‐molecule phosphotyrosine mimetic substrate (difluoromethyl umbelliferyl phosphate, DiFMUP). The results demonstrate that compounds that inhibited enzyme activity on the peptide substrate had greater biological activity than compounds that only inhibited enzyme activity on DiFMUP. Finally, epigallocatechin‐3,5‐digallate was identified as the most potent inhibitor of lymphoid tyrosine phosphatase activity to date, with an IC₅₀ of 50 nM and significant activity in T‐cells. Molecular docking simulations provided a first model for binding of this potent inhibitor to LYP; this will constitute the platform for ongoing lead optimization efforts.