Asymmetric Synthesis and Biological Evaluation of Platensilin, Platensimycin, Platencin, and Their Analogs via a Bioinspired Skeletal Reconstruction Approach.

Platensilin, platensimycin, and platencin are potent inhibitors of ß-ketoacyl-acyl carrier protein synthase (FabF) in the bacterial and mammalian fatty acid synthesis system, presenting promising drug leads for both antibacterial and antidiabetic therapies. Herein, a bioinspired skeleton reconstruction approach is reported, which enables the unified synthesis of these three natural FabF inhibitors and their skeletally diverse analogs, all stemming from a common ent-pimarane core. The synthesis features a diastereoselective biocatalytic reduction and an intermolecular Diels-Alder reaction to prepare the common ent-pimarane core. From this intermediate, stereoselective Mn-catalyzed hydrogen atom-transfer hydrogenation and subsequent Cu-catalyzed carbenoid C-H insertion afford platensilin. Furthermore, the intramolecular Diels-Alder reaction succeeded by regioselective ring opening of the newly formed cyclopropane enables the construction of the bicyclo[3.2.1]-octane and bicyclo[2.2.2]-octane ring systems of platensimycin and platencin, respectively. This skeletal reconstruction approach of the ent-pimarane core facilitates the preparation of analogs bearing different polycyclic scaffolds. Among these analogs, the previously unexplored cyclopropyl analog 47 exhibits improved antibacterial activity (MIC80 = 0.0625 µg/mL) against S. aureus compared to platensimycin.

PSMA-Targeted 2-Deoxyglucose-Based Dendrimer Nanomedicine for the Treatment of Prostate Cancer.

Prostate cancer (PC) is the fifth leading cause of cancer-related deaths among men worldwide. Prostate-specific membrane antigen (PSMA), a molecular target of PC, is clinically used for the treatment and diagnosis of PC using radioligand approaches. However, no PSMA-based chemotherapies have yet been approved by the FDA. Here, we present a novel therapeutic approach using PSMA-targeted 2-deoxyglucose-dendrimer (PSMA-2DG-D) for targeted delivery of a potent tyrosine kinase inhibitor, cabozantinib (Cabo), selectively to PC cells. PSMA-2DG-D demonstrates intracellular localization in PSMA (+) PC cells through PSMA-mediated internalization. This PSMA-specific targeting translates to enhanced efficacy of Cabo compared to the free drug when conjugated to PSMA-2DG-D. Furthermore, systemically administered fluorescently labeled PSMA-2DG-D-Cy5 specifically targets PSMA (+) tumors with minimal off-target accumulation in the PC3-PIP tumor xenograft mouse model. This demonstrates that the PSMA-2DG-D platform is a promising new delivery system for potent chemotherapeutics, where systemic side effects are a significant concern.

Anti-inflammatory effect of covalent PPARγ ligands that have a hybrid structure of GW9662 and a food-derived cinnamic acid derivative.

Peroxisome proliferator-activated receptor γ (PPARγ) belongs to the nuclear receptor superfamily and is involved in the inflammatory process. Previously, we synthesized the ligands of PPARγ that possess the hybrid structure of a food-derived cinnamic acid derivative (CA) and GW9662, an irreversible PPARγ antagonist. These ligands activate the transcription of PPARγ through the covalent bond formation with the Cys285 residue of PPARγ, whereas their anti-inflammatory effect has not been examined yet. Here, we show the anti-inflammatory effect of the covalent PPARγ ligands in RAW264 cells, murine macrophage-like cells. GW9662 suppressed the production of nitric oxide (NO) stimulated by lipopolysaccharide and exerted a synergistic effect in combination with CA. The compounds bearing their hybrid structure dramatically inhibited NO production and transcription of proinflammatory cytokines. A comparison study suggested that the 2-chloro-5-nitrobenzoyl group of the ligands is important for anti-inflammation. Furthermore, we synthesized an alkyne-tagged analogue that becomes an activity-based probe for future mechanistic study.

Association mechanism of bicalutamide and human serum albumin for potential clinical implications.

The binding mechanism of molecular interaction between bicalutamide and human serum albumin (HSA) in a pH 7.4 phosphate buffer was studied using various spectroscopic techniques in combination with molecular modeling. Fluorescence data revealed that the fluorescence quenching of HSA by bicalutamide was a static quenching procedure. The binding constants and number of binding sites were evaluated at different temperatures. The thermodynamic parameters, ΔH and ΔS, were calculated to be 4.30 × 104 J·mol-1 and 245 J·mol-1·K-1, respectively, suggesting that the binding of bicalutamide to HSA was driven mainly by hydrophobic interactions and hydrogen bonds. The displacement studies indicated neither Sudlow's site I nor II but subdomain IB as the main binding site for bicalutamide on HSA. The binding distance between bicalutamide and HSA was determined to be 3.54 nm based on the Förster theory. Analysis of circular dichroism, synchronous, and 3D fluorescence spectra demonstrated that HSA conformation was slightly altered in the presence of bicalutamide.

Influence of Solvent Polarity on the Conformer Ratio of Bicalutamide in Saturated Solutions: Insights from NOESY NMR Analysis and Quantum-Chemical Calculations.

The study presents a thorough and detailed analysis of bicalutamide's structural and conformational properties. Quantum chemical calculations were employed to explore the conformational properties of the molecule, identifying significant energy differences between conformers. Analysis revealed that hydrogen bonds stabilise the conformers, with notable variations in torsion angles. Conformers were classified into 'closed' and 'open' types based on the relative orientation of the cyclic fragments. NOE spectroscopy in different solvents (CDCl3 and DMSO-d6) was used to study the conformational preferences of the molecule. NOESY experiments provided the predominance of 'closed' conformers in non-polar solvents and a significant presence of 'open' conformers in polar solvents. The proportions of open conformers were 22.7 ± 3.7% in CDCl3 and 59.8 ± 6.2% in DMSO-d6, while closed conformers accounted for 77.3 ± 3.7% and 40.2 ± 6.2%, respectively. This comprehensive study underscores the solvent environment's impact on its structural behaviour. The findings significantly contribute to a deeper understanding of conformational dynamics, stimulating further exploration in drug development.

Structural basis of Smoothened regulation by its extracellular domains.

Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzledclass G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked a top the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD-linker domain-TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains.

Decatungstate-Catalyzed C(sp3)-H Sulfinylation: Rapid Access to Diverse Organosulfur Functionality.

Here we report the direct conversion of strong, aliphatic C(sp3)-H bonds into the corresponding alkyl sulfinic acids via decatungstate photocatalysis. This transformation has been applied to a diverse range of C(sp3)-rich scaffolds, including natural products and approved pharmaceuticals, providing efficient access to complex sulfur-containing products. To demonstrate the broad potential of this methodology for the divergent synthesis of pharmaceutically relevant molecules, procedures for the diversification of the sulfinic acid products into a range of medicinally relevant functional groups have been developed.

Synthesis, biological evaluation and X-ray analysis of bicalutamide sulfoxide analogues for the potential treatment of prostate cancer.

The androgen receptor (AR) is a pivotal target for the treatment of prostate cancer (PC) even when the disease progresses toward androgen-independent or castration-resistant forms. In this study, a series of sulfoxide derivatives were prepared and their antiproliferative activity evaluated in vitro against four different human prostate cancer cell lines (22Rv1, DU-145, LNCaP and VCap). Bicalutamide and enzalutamide were used as positive controls. Compound 28 displayed significant enhancement in anticancer activity across the four PC cell lines with IC50 = 9.09 - 31.11 µM compared to the positive controls: bicalutamide (IC50 = 45.20 -51.61 µM) and enzalutamide (IC50 = 11.47 - 53.04 µM). Sulfoxide derivatives of bicalutamide were prepared efficiently from the corresponding sulfides using only one equivalent of mCPBA, limiting the reaction time to 15-30 min and maintaining the temperature at 0 °C. Interestingly, three pairs of sulfoxide diastereomers were separated and NMR comparison of their diastereotopic methylene (CH2) group is presented. X-ray diffraction crystal structure analysis provided relative configuration assignment at the chiral sulfur and carbon centres. Molecular modelling study of the four diastereoisomers of compound 28 is described.

Computational Study of the C5-Hydroxylation Mechanism Catalyzed by the Diiron Monooxygenase PtmU3 as Part of the Platensimycin Biosynthesis.

PtmU3 is a newly identified nonheme diiron monooxygenase, which installs a C-5 ß-hydroxyl group into the C-19 CoA-ester intermediate involved in the biosynthesis of unique diterpene-derived scaffolds of platensimycin and platencin. PtmU3 possesses a noncanonical diiron active site architecture of a saturated six-coordinate iron center and lacks the µ-oxo bridge. Although the hydroxylation process is a simple reaction for nonheme mononuclear iron-dependent enzymes, how PtmU3 employs the diiron center to catalyze the H-abstraction and OH-rebound is still unknown. In particular, the electronic characteristic of diiron is also unclear. To understand the catalytic mechanism of PtmU3, we constructed two reactant models in which both the Fe1II-Fe2III-superoxo and Fe1II-Fe2IV═O are considered to trigger the H-abstraction and performed a series of quantum mechanics/molecular mechanics calculations. Our calculation results reveal that PtmU3 is a special monooxygenase, that is, both atoms of the dioxygen molecule can be incorporated into two molecules of the substrate by the successive reactions. In the first-round reaction, PtmU3 uses the Fe1II-Fe2III-superoxo to install a hydroxyl group into the substrate, generating the high-reactive Fe1II-Fe2IV═O complex. In the second-round reaction, the Fe1II-Fe2IV═O species is responsible for the hydroxylation of another molecule of the substrate. In the diiron center, Fe2 adopts the high spin state (S = 5/2) during the catalysis, whereas for Fe1, in addition to its structural role, it may also play an assistant role for Fe1 catalysis. In the two successive OH-installing steps, the H-abstraction is always the rate-liming step. E241 and D308 not only act as bridging ligands to connect two Fe ions but also take part in the electron reorganization. Owing to the high reactivity of Fe1II-Fe2IV═O compared to Fe1II-Fe2III-superoxo, besides the C5-hydroxylation, the C3- or C18-hydroxylation was also calculated to be feasible.

Rapid, Selective, and Sensitive Method for Semitargeted Discovery of Congeneric Natural Products by Liquid Chromatography Tandem Mass Spectrometry.

Natural product congeners serve a useful role in the understanding of natural product biosynthesis and structure-activity relationships. A minor congener with superior activity, selectivity, and modifiable functional groups could serve as a more effective lead structure and replace even the original lead molecule that was used for medicinal chemistry modifications. Currently, no effective method exists to discover targeted congeners rapidly, specifically, and selectively from producing sources. Herein, a new method based on liquid-chromatography tandem-mass spectrometry combination is evaluated for targeted discovery of congeners of platensimycin and platencin from the extracts of Streptomyces platensis. By utilizing a precursor-ion searching protocol, tandem mass spectrometry not only confirmed the presence of known congeners but also provided unambiguous detection of many previously unknown congeners of platensimycin and platencin. This high-throughput and quantitative method can be rapidly and broadly applied for dereplication and congener discovery from a variety of producing sources, even when the targeted compounds are obscured by the presence of unrelated natural products.

Design, synthesis and biological evaluation of anilide (dicarboxylic acid) shikonin esters as antitumor agents through targeting PI3K/Akt/mTOR signaling pathway.

Triple-negative breast cancer (TNBC) has an unfavorable prognosis attribute to its low differentiation, rapid proliferation and high distant metastasis rate. PI3K/Akt/mTOR as an intracellular signaling pathway plays a key role in the cell proliferation, migration, invasion, metabolism and regeneration. In this work, we designed and synthesized a series of anilide (dicarboxylic acid) shikonin esters targeting PI3K/Akt/mTOR signaling pathway, and assessed their antitumor effects. Through three rounds of screening by computer-aided drug design method (CADD), we preliminarily obtained sixteen novel anilide (dicarboxylic acid) shikonin esters and identified them as excellent compounds. CCK-8 assay results demonstrated that compound M9 exhibited better antiproliferative activities against MDA-MB-231, A549 and HeLa cell lines than shikonin (SK), especially for MDA-MB-231 (M9: IC50 = 4.52 ± 0.28 µM; SK: IC50 = 7.62 ± 0.26 µM). Moreover, the antiproliferative activity of M9 was better than that of paclitaxel. Further pharmacological studies showed that M9 could induce apoptosis of MDA-MB-231 cells and arrest the cell cycle in G2/M phase. M9 also inhibited the migration of MDA-MB-231 cells by inhibiting Wnt/ß-catenin signaling pathway. In addition, western blot results showed that M9 could inhibit cell proliferation and migration by down-regulating PI3K/Akt/mTOR signaling pathway. Finally, a three-dimensional quantitative structure-activity relationship (3D-QSAR) model was also constructed to provide a basis for further development of shikonin derivatives as potential antitumor drugs through structure-activity relationship analysis. To sum up, M9 could be a potential candidate for TNBC therapy.

Magnetic nanocarriers as a therapeutic drug delivery strategy for promoting pain-related motor functions in a rat model of cartilage transplantation.

Cartilage is an avascular tissue with low cellularity and insufficient self-repair response. In clinical practice, a large articular cartilage defect is usually fixed by cartilage transplantation. Importantly, the fast repair process has been demanded postoperatively in the area between the host cartilage and the transplanted cartilage. In the past few years, magnetic nanoparticles have drawn great attention due to their biocompatible, biodegradable, and nontoxic properties. In addition, the nanoparticles can easily pass through the cell plasma membrane and increase the cellular uptake efficiency. Here, a therapeutic drug delivery strategy was proposed for cartilage repair. The prepared kartogenin (KGN)-conjugated magnetic nanocarriers (KGN@NCs) promoted the viability of chondrocytes in vitro. In a rat model of cartilage transplantation, intra-articularly delivered KGN@NCs generated cartilage with a flat surface and a high level of aggrecan in vivo. Notably, KGN@NCs were also capable of improving the pain-related motor functions. They promoted the motor functional parameters including the print area and intensity to restore to a normal level compared with the single KGN. Therefore, these therapeutic drug nanocarriers provided the potential for cartilage repair.

Evaluation of Class IIa Histone Deacetylases Expression and In Vivo Epigenetic Imaging in a Transgenic Mouse Model of Alzheimer's Disease.

Epigenetic regulation by histone deacetylase (HDAC) is associated with synaptic plasticity and memory formation, and its aberrant expression has been linked to cognitive disorders, including Alzheimer's disease (AD). This study aimed to investigate the role of class IIa HDAC expression in AD and monitor it in vivo using a novel radiotracer, 6-(tri-fluoroacetamido)-1-hexanoicanilide ([18F]TFAHA). A human neural cell culture model with familial AD (FAD) mutations was established and used for in vitro assays. Positron emission tomography (PET) imaging with [18F]TFAHA was performed in a 3xTg AD mouse model for in vivo evaluation. The results showed a significant increase in HDAC4 expression in response to amyloid-ß (Aß) deposition in the cell model. Moreover, treatment with an HDAC4 selective inhibitor significantly upregulated the expression of neuronal memory-/synaptic plasticity-related genes. In [18F]TFAHA-PET imaging, whole brain or regional uptake was significantly higher in 3xTg AD mice compared with WT mice at 8 and 11 months of age. Our study demonstrated a correlation between class IIa HDACs and Aßs, the therapeutic benefit of a selective inhibitor, and the potential of using [18F]TFAHA as an epigenetic radiotracer for AD, which might facilitate the development of AD-related neuroimaging approaches and therapies.

Synthesis, Characterization and HPLC Analysis of the (1S,2S,5R)-Diastereomer and the Enantiomer of the Clinical Candidate AR-15512.

AR-15512 (formerly known as AVX-012 and WS-12) is a TRPM8 receptor agonist currently in phase 2b clinical trials for the treatment of dry eye. This bioactive compound with menthol-like cooling activity has three stereogenic centers, and its final structure and absolute configuration, (1R,2S,5R), have been previously solved by cryo-electron microscopy. The route of synthesis of AR-15512 has also been reported, revealing that epimerization processes at the C-1 can occur at specific stages of the synthesis. In order to confirm that the desired configuration of AR-15512 does not change throughout the process and to discard the presence of the enantiomer in the final product due to possible contamination of the initial starting material, both the enantiomer of AR-15512 and the diastereomer at the C-1 were synthesized and fully characterized. In addition, the absolute configuration of the (1S,2S,5R)-diastereomer was determined by X-ray crystallographic analysis, and new HPLC methods were designed and developed for the identification of the two stereoisomers and their comparison with the clinical candidate AR-15512.

Enriching the Arsenal of Pharmacological Tools against MICAL2.

Molecule interacting with CasL 2 (MICAL2), a cytoskeleton dynamics regulator, are strongly expressed in several human cancer types, especially at the invasive front, in metastasizing cancer cells and in the neo-angiogenic vasculature. Although a plethora of data exist and stress a growing relevance of MICAL2 to human cancer, it is worth noting that only one small-molecule inhibitor, named CCG-1423 (1), is known to date. Herein, with the aim to develop novel MICAL2 inhibitors, starting from CCG-1423 (1), a small library of new compounds was synthetized and biologically evaluated on human dermal microvascular endothelial cells (HMEC-1) and on renal cell adenocarcinoma (786-O) cells. Among the novel compounds, 10 and 7 gave interesting results in terms of reduction in cell proliferation and/or motility, whereas no effects were observed in MICAL2-knocked down cells. Aside from the interesting biological activities, this work provides the first structure-activity relationships (SARs) of CCG-1423 (1), thus providing precious information for the discovery of new MICAL2 inhibitors.

Chemical Degradation of Androgen Receptor (AR) Using Bicalutamide Analog-Thalidomide PROTACs.

A series of PROTACs (PROteolysis-TArgeting Chimeras) consisting of bicalutamide analogs and thalidomides were designed, synthesized, and biologically evaluated as novel androgen receptor (AR) degraders. In particular, we found that PROTAC compound 13b could successfully demonstrate a targeted degradation of AR in AR-positive cancer cells and might be a useful chemical probe for the investigation of AR-dependent cancer cells, as well as a potential therapeutic candidate for prostate cancers.

Leveraging a large microbial strain collection for natural product discovery.

Throughout history, natural products have significantly contributed to the discovery of novel chemistry, drug leads, and tool molecules to probe and address complex challenges in biology and medicine. Recent microbial genome sequencing efforts have uncovered many microbial biosynthetic gene clusters without an associated natural product. This means that the natural products isolated to date do not fully reflect the biosynthetic potential of microbial strains. This observation has rejuvenated the natural product community and inspired a return to microbial strain collections. Mining large microbial strain collections with the most current technologies in genome sequencing, bioinformatics, and high-throughput screening techniques presents new opportunities in natural product discovery. In this review, we report on the newly expanded microbial strain collection at The Scripps Research Institute, which represents one of the largest and most diverse strain collections in the world. Two complementary approaches, i.e. structure-centric and function-centric, are presented here to showcase how to leverage a large microbial strain collection for natural product discovery and to address challenges and harness opportunities for future efforts. Highlighted examples include the discovery of alternative producers of known natural products with superior growth characteristics and high titers, novel analogs of privileged scaffolds, novel natural products, and new activities of known and new natural products. We anticipate that this large microbial strain collection will facilitate the discovery of new natural products for many applications.

Molecular basis of drug resistance in smoothened receptor: An in silico study of protein resistivity and specificity.

Smoothened (SMO) antagonist Vismodegib effectively inhibits the Hedgehog pathway in proliferating cancer cells. In early stage of treatment, Vismodegib exhibited promising outcomes to regress the tumors cells, but ultimately relapsed due to the drug resistive mutations in SMO mostly occurring before (primary mutations G497W) or after (acquired mutations D473H/Y) anti-SMO therapy. This study investigates the unprecedented insights of structural and functional mechanism hindering the binding of Vismodegib with sensitive and resistant mutant variants of SMO (SMOMut ). Along with the basic dynamic understanding of Vismodegib-SMO complexes, network propagation theory based on heat diffusion principles is first time applied here to identify the modules of residues influenced by the individual mutations. The allosteric modulation by GLY497 residue in Vismodegib bound SMO wild-type (SMOWT ) conformation depicts the interconnections of intermediate residues of SMO with the atom of Vismodegib and identify two important motifs (E-X-P-L) and (Q-A-N-V-T-I-G) mediating this allosteric regulation. In this study a novel computational framework based on the heat diffusion principle is also developed, which identify significant residues of allosteric site causing drug resistivity in SMOMut . This framework could also be useful for assessing the potential allosteric sites of different other proteins. Moreover, previously reported novel inhibitor "ZINC12368305," which is proven to make an energetically favorable complex with SMOWT is chosen as a control sample to assess the impact of receptor mutation on its binding and subsequently identify the important factors that govern binding disparity between Vismodegib and ZINC12368305 bound SMOWT/Mut conformations.

Platensimycin-Encapsulated Poly(lactic-co-glycolic acid) and Poly(amidoamine) Dendrimers Nanoparticles with Enhanced Anti-Staphylococcal Activity in Vivo.

Serious bacterial infections by multi-drug-resistant pathogens lead to human losses and endanger public health. The discovery of antibiotics with new modes of action, in combination with nanotechnology, might offer a promising route to combat multi-drug-resistant pathogens. Platensimycin (PTM), a potent inhibitor of FabB/FabF for bacterial fatty acid biosynthesis, is a promising drug lead against many drug-resistant bacteria. However, the clinical development of PTM is hampered by its poor pharmacokinetics. Herein, we report a nanostrategy that encapsulated PTM in two types of nanoparticles (NPs) poly(lactic-co-glycolic acid) (PLGA) and poly(amidoamine) (PAMAM) dendrimer to enhance its antibacterial activity in vitro and in vivo. The PTM-encapsulated NPs were effective to inhibit Staphylococcus aureus biofilm formation, and killed more S. aureus in a macrophage cell infection model over free PTM. The pharmacokinetic studies showed that PTM-loaded PLGA and PAMAM NPs exhibited increased AUC0-t (area under the curve) (∼4- and 2-fold) over free PTM. In a mouse peritonitis model, treatment of methicillin-resistant S. aureus infected mice using both PTM-loaded NPs (10 mg/kg) by intraperitoneal injection led to their full survival, while all infected mice died when treated by free PTM (10 mg/kg). These results not only suggest that PTM-loaded NPs may hold great potential to improve the poor pharmacokinetic properties of PTM, but support the rationale to develop bacterial fatty acid synthase inhibitors as promising antibiotics against drug-resistant pathogens.

Design and Characterization of Injectable Poly(Lactic-Co-Glycolic Acid) Pastes for Sustained and Local Drug Release.

PURPOSE: We describe the preparation of injectable polymeric paste (IPP) formulations for local and sustained release of drugs. Furthermore, we include the characterization and possible applications of such pastes. Particular attention is paid to characteristics relevant to the successful clinical formulation development, such as viscosity, injectability, degradation, drug release, sterilization, stability performance and pharmacokinetics. METHODS: Paste injectability was characterized using measured viscosity and the Hagen-Poiseuille equation to determine injection forces. Drug degradation, release and formulation stability experiments were performed in vitro and drug levels were quantified using HPLC-UV methods. Pharmacokinetic evaluation of sustained-release lidocaine IPPs used five groups of six rats receiving increasing doses subcutaneously. An anti-cancer formulation was evaluated in a subcutaneous tumor xenograft mouse model. RESULTS: The viscosity and injectability of IPPs could be controlled by changing the polymeric composition. IPPs demonstrated good long-term stability and tunable drug-release with low systemic exposure in vivo in rats. Preliminary data in a subcutaneous tumor model points to a sustained anticancer effect. CONCLUSIONS: These IPPs are tunable platforms for local and sustained delivery of drugs and have potential for further clinical development to treat a number of diseases.