Discovery of a Class of Potent and Selective Non-competitive Sentrin-Specific Protease 1 Inhibitors

Sentrin-specific proteases (SENPs) are responsible for the maturation of small ubiquitin-like modifiers (SUMOs) and the deconjugation of SUMOs from their substrate proteins. Studies on prostate cancer revealed an overexpression of SENP1, which promotes prostate cancer progression as well as metastasis. Therefore, SENP1 has been identified as a novel drug target against prostate cancer. Herein, we report the discovery and biological evaluation of potent and selective SENP1 inhibitors. A structure-activity relationship (SAR) of the newly identified pyridone scaffold revealed allosteric inhibitors with very attractive in vitro ADMET properties regarding plasma binding and plasma stability for this challenging target. This study also emphasizes the importance of biochemical mode of inhibition studies for de novo designed inhibitors.     

N‐Arylsulfonyl Indolines as Retinoic Acid Receptor‐Related Orphan Receptor γ (RORγ) Agonists

The nuclear retinoic acid receptor‐related orphan receptor γ (RORγ; NR1F3) is a key regulator of inflammatory gene programs involved in T helper 17 (T_H17) cell proliferation. As such, synthetic small‐molecule repressors (inverse agonists) targeting RORγ have been extensively studied for their potential as therapeutic agents for various autoimmune diseases. Alternatively, enhancing T_H17 cell proliferation through activation (agonism) of RORγ may boost an immune response, thereby offering a potentially new approach in cancer immunotherapy. Herein we describe the development of N‐arylsulfonyl indolines as RORγ agonists. Structure–activity studies reveal a critical linker region in these molecules as the major determinant for agonism. Hydrogen/deuterium exchange coupled to mass spectrometry (HDX‐MS) analysis of RORγ–ligand complexes help rationalize the observed results.     

Discovery of Potent Vascular Endothelial Growth Factor Receptor‐2 Inhibitors

Substantial evidence over the last decades has implicated uncontrolled angiogenesis with various pathological states, including cancer. Vascular endothelial growth factor (VEGF) plays a critical role in its regulation. Because the tyrosine kinase VEGF receptor‐2 (VEGFR‐2) is the major mediator of the mitogenic, angiogenic, and permeability‐enhancing effects of VEGF, it has become one of the most profound anti‐angiogenesis targets. Inspired by the anthranilamide class of VEGFR‐2 inhibitors, we performed a computational analysis of some potent representative members, using docking and molecular dynamics calculations. Based on the observations drawn from introducing the effect of the receptor's flexibility in implicit aqueous environment, we designed, synthesized, and characterized several new analogues of related scaffolds with modifications in their steric and electronic characteristics. In vitro evaluation of these compounds revealed several novel VEGFR‐2 inhibitors that are less cytotoxic and more potent than the parent compounds.     

N‐Benzyl‐4‐((heteroaryl)methyl)benzamides: A New Class of Direct NADH‐Dependent 2‐trans Enoyl–Acyl Carrier Protein Reductase (InhA) Inhibitors with Antitubercular Activity

Isoniazid (INH) remains one of the cornerstones of antitubercular chemotherapy for drug‐sensitive strains of M. tuberculosis bacteria. However, the increasing prevalence of multidrug‐resistant (MDR) and extensively drug‐resistant (XDR) strains containing mutations in the KatG enzyme, which is responsible for the activation of INH into its antitubercular form, have rendered this drug of little or no use in many cases of drug‐resistant tuberculosis. Presented herein is a novel family of antitubercular direct NADH‐dependent 2‐trans enoyl–acyl carrier protein reductase (InhA) inhibitors based on an N‐benzyl‐4‐((heteroaryl)methyl)benzamide template; unlike INH, these do not require prior activation by KatG. Given their direct InhA target engagement, these compounds should be able to circumvent KatG‐related resistance in the clinic. The lead molecules were shown to be potent inhibitors of InhA and showed activity against M. tuberculosis bacteria. This new family of inhibitors was found to be chemically tractable, as exemplified by the facile synthesis of analogues and the establishment of structure–activity relationships. Furthermore, a co‐crystal structure of the initial hit with the enzyme is disclosed, providing valuable information toward the design of new InhA inhibitors for the treatment of MDR/XDR tuberculosis.     

O‐(Triazolyl)methyl Carbamates as a Novel and Potent Class of Fatty Acid Amide Hydrolase (FAAH) Inhibitors

Inhibition of fatty acid amide hydrolase (FAAH) activity is under investigation as a valuable strategy for the treatment of several disorders, including pain and drug addiction. A number of potent FAAH inhibitors belonging to different chemical classes have been disclosed to date; O‐aryl carbamates are one of the most representative families. In the search for novel FAAH inhibitors, a series of O‐(1,2,3‐triazol‐4‐yl)methyl carbamate derivatives were designed and synthesized exploiting a copper‐ catalyzed [3+2] cycloaddition reaction between azides and alkynes (click chemistry). Exploration of the structure–activity relationships within this new class of compounds identified potent inhibitors of both rat and human FAAH with IC₅₀ values in the single‐digit nanomolar range. In addition, these derivatives showed improved stability in rat plasma and kinetic solubility in buffer with respect to the lead compound. Based on the results of the study, the novel analogues identified can be considered to be promising starting point for the development of new FAAH inhibitors with improved drug‐like properties.     

Design and Synthesis of A‐Ring Simplified Pyripyropene A Analogues as Potent and Selective Synthetic SOAT2 Inhibitors

Currently, pyripyropene A, which is isolated from the culture broth of Aspergillus fumigatus FO‐1289, is the only compound known to strongly and selectively inhibit the isozyme sterol O‐acyltransferase 2 (SOAT2). To aid in the development of new cholesterol‐lowering or anti‐atherosclerotic agents, new A‐ring simplified pyripyropene A analogues have been designed and synthesized based on total synthesis, and the results of structure–activity relationship studies of pyripyropene A. Among the analogues, two A‐ring simplified pyripyropene A analogues exhibited equally efficient SOAT2 inhibitory activity to that of natural pyripyropene A. These new analogues are the most potent and selective SOAT2 inhibitors to be used as synthetic compounds and attractive seed compounds for the development of drug for dyslipidemia, including atherosclerotic disease and steatosis.     

Identification of Novel Histone Deacetylase 6-Selective Inhibitors Bearing 3,3,3-Trifluorolactic Amide (TFLAM) Motif as a Zinc Binding Group

Pharmacological inhibition of histone deacetylase 6 (HDAC6) is an effective therapeutic strategy for cancer and immunological diseases. Most of the previously reported HDAC6 inhibitors have a hydroxamate group as a zinc binding group (ZBG), which coordinates to the catalytic zinc ion of HDAC6. The hydroxamate group is liable to metabolically generate mutagenetic hydroxylamine; therefore, non-hydroxamate HDAC6 inhibitors would be advantageous. In this study, to identify novel non-hydroxamate HDAC6-selective inhibitors, screening of a chemical library and the subsequent structural optimization were performed, which led to the identification of HDAC6-selective inhibitors with 3,3,3-trifluorolactic amide (TFLAM) as a novel ZBG. The identified inhibitor showed potent and selective HDAC6-inhibitory activity in cells and induced regulatory T (Treg) cell differentiation.     

Alzheimer’s Disease: Identification and Development of β‐Secretase (BACE‐1) Binding Fragments and Inhibitors by Dynamic Ligation Screening (DLS)

The application of dynamic ligation screening (DLS), a methodology for fragment‐based drug discovery (FBDD), to the aspartic protease β‐secretase (BACE‐1) is reported. For this purpose, three new fluorescence resonance energy transfer (FRET) substrates were designed and synthesized. Their kinetic parameters (V_max, K_M, and k_cat) were determined and compared with a commercial substrate. Secondly, a peptide aldehyde was designed as a chemically reactive inhibitor (CRI) based on the Swedish mutation substrate sequence. Incubation of this CRI with the protease, a FRET substrate, and one amine per well taken from an amine library, which was assembled by a maximum common substructure (MCS) approach, revealed the fragment 3‐(3‐aminophenyl)‐2H‐chromen‐2‐one ( 1 ) to be a competitive BACE‐1 inhibitor that enhanced the activity of the CRI. Irreversibly formed fragment combination products of 1 with the initial peptide sequence were active and confirmed the targeting of the active site through the ethane‐1,2‐diamine isostere. Finally, structure‐assisted combination of fragment 1 with secondary fragments that target the S1 site in hit optimization yielded novel, entirely fragment‐based BACE‐1 inhibitors with up to 30‐fold improved binding affinity. Interactions with the protein were explained by molecular modeling studies, which indicate that the new fragment combinations interact with the catalytic aspartic acid dyad, as well as with the adjacent binding sites required for potency.     

Towards Dynamic Drug Design: Identification and Optimization of β‐Galactosidase Inhibitors from a Dynamic Hemithioacetal System

A discovery strategy relying on the identification of fragments through resolution of a constitutional dynamic system, coupled to subsequent static ligand design and optimization, is demonstrated. The strategic design and synthesis of the best molecular fragments identified from a dynamic hemithioacetal system into static ligand structures yielded a range of β‐galactosidase inhibitors. Two series of structures mimicking the hemithioacetal motif were envisaged: thioglycosides and C‐glycosides. Inhibition studies provided important structural information for the two groups, and 1‐thiobenzyl‐β‐D ‐galactopyranoside demonstrated the best inhibitory effects.     

Receptor‐Based Virtual Screening and Biological Characterization of Human Apurinic/Apyrimidinic Endonuclease (Ape1) Inhibitors

The endonucleolytic activity of human apurinic/apyrimidinic endonuclease (AP endo, Ape1) is a major factor in maintaining the integrity of the genome. Conversely, as an undesired effect, Ape1 overexpression has been linked to resistance to radio‐ and chemotherapeutic treatments in several human tumors. Inhibition of Ape1 using siRNA or the expression of a dominant negative form of the protein has been shown to sensitize cells to DNA‐damaging agents, including various chemotherapeutic agents. Therefore, inhibition of the enzymatic activity of Ape1 might result in a potent antitumor therapy. A number of small molecules have been described as Ape1 inhibitors; however, those compounds are in the early stages of development. Herein we report the identification of new compounds as potential Ape1 inhibitors through a docking‐based virtual screening technique. Some of the compounds identified have in vitro activities in the low‐to‐medium micromolar range. Interaction of these compounds with the Ape1 protein was observed by mass spectrometry. These molecules also potentiate the cytotoxicity of the chemotherapeutic agent methyl methanesulfonate in fibrosarcoma cells. This study demonstrates the power of docking and virtual screening techniques as initial steps in the design of new drugs, and opens the door to the development of a new generation of Ape1 inhibitors.     

We Need 2C but Not 2B: Developing Serotonin 2C (5‐HT2C) Receptor Agonists for the Treatment of CNS Disorders

The serotonin 2C (5‐HT_2C) receptor has been identified as a potential drug target for the treatment of a variety of central nervous system (CNS) disorders, such as obesity, substance abuse, and schizophrenia. In this Viewpoint article, recent progress in developing selective 5‐HT_2C agonists for use in treating these disorders is summarized, including the work of our group. Challenges in this field and the possible future directions are described. Homology modeling as a method to predict the binding modes of 5‐HT_2C ligands to the receptor is also discussed. Compared to known ligands, the improved pharmacological profiles of the 2‐phenylcyclopropylmethylamine‐based 5‐HT_2C agonists make them preferred candidates for further studies.     

Recent Advances in Inhibitors of Bacterial Fatty Acid Synthesis Type II (FASII) System Enzymes as Potential Antibacterial Agents

Bacterial infections are a constant and serious threat to human health. With the increase of multidrug resistance of clinically pathogenic bacteria, common antibiotic therapies have been less effective. Fatty acid synthesis type II (FASII) system enzymes are essential for bacterial membrane lipid biosynthesis and represent increasingly promising targets for the discovery of antibacterial agents with new mechanisms of action. This review highlights recent advances in inhibitors of bacterial FASII as potential antibacterial agents, paying special attention to the activities, mechanisms, and structure–activity relationships of those inhibitors that mainly target β‐ketoacyl‐ACP synthase, β‐ketoacyl‐ACP reductase, β‐hydroxyacyl‐ACP dehydratase, and enoyl‐ACP reductase. Although inhibitors with low nanomolar and selective activity against various bacterial FASII have entered clinical trials, further research is needed to expand upon both available and yet unknown scaffolds to identify new FASII inhibitors that may have antibacterial potential, particularly against resistant bacterial strains.     

Development of Selective Bisubstrate‐Based Inhibitors Against Protein Kinase C (PKC) Isozymes By Using Dynamic Peptide Microarrays

Kinase inhibitors are increasingly important in drug development. Because the majority of current inhibitors target the conserved ATP‐binding site, selectivity might become an important issue. This could be particularly problematic for the potential drug target protein kinase C (PKC), of which twelve isoforms with high homology exist in humans. A strategy to increase selectivity is to prepare bisubstrate‐based inhibitors that target the more selective peptide‐binding site in addition to the ATP‐binding site. In this paper a generally applicable, rapid methodology is presented to discover such bisubstrate‐based leads. Dynamic peptide microarrays were used to find peptide‐binding site inhibitors. These were linked with chemoselective click chemistry to an ATP‐binding site inhibitor, and this led to novel bisubstrate structures. The peptide microarrays were used to evaluate the resulting inhibitors. Thus, novel bisubstrate‐based inhibitors were obtained that were both more potent and selective compared to their constituent parts. The most promising inhibitor has nanomolar affinity and selectivity towards PKCθ amongst three isozymes.     

Discovery of Diverse Small‐Molecule Inhibitors of Mammalian Sterile20‐like Kinase 3 (MST3)

Increasing evidence suggests key roles for members of the mammalian Sterile20‐like (MST) family of kinases in many aspects of biology. MST3 is a member of the STRIPAK complex, the deregulation of which has recently been associated with cancer cell migration and metastasis. Targeting MST3 with small‐molecule inhibitors may be beneficial for the treatment of certain cancers, but little information exists on the potential of kinase inhibitor scaffolds to engage with MST3. In this study we screened MST3 against a library of 277 kinase inhibitors using differential scanning fluorimetry and confirmed 14 previously unknown MST3 inhibitors by X‐ray crystallography. These compounds, of which eight are in clinical trials or FDA approved, comprise nine distinct chemical scaffolds that inhibit MST3 enzymatic activity with IC₅₀ values between 0.003 and 23 μm . The structure–activity relationships explain the differential inhibitory activity of these compounds against MST3 and the structural basis for high binding potential, the information of which may serve as a framework for the rational design of MST3‐selective inhibitors as potential therapeutics and to interrogate the function of this enzyme in diseased cells.