Discovery, optimization and biological activity evaluation of genipin derivatives as potential KRAS G12D inhibitors.

A series of genipin derivatives were designed and synthesized as potential inhibitors targeted KRAS G12D mutation. The majority of these compounds demonstrated potential antiproliferative effects against KRAS G12D mutant tumor cells (CT26 and A427). Notably, seven compounds exhibited the anticancer effects with IC50 values ranging from 7.06 to 9.21 µM in CT26 (KRASG12D) and A427 (KRASG12D) cells and effectively suppressed the colony formation of CT26 cells. One representative compound SK12 was selected for further investigation into biological activity and action mechanisms. SK12 markedly induced apoptosis in CT26 cells in a concentration-dependent manner. Moreover, SK12 elevated the levels of reactive oxygen species (ROS) in tumor cells and exhibited a modulatory effect on the KRAS signaling pathway, thereby inhibiting the activation of downstream phosphorylated proteins. The binding affinity of SK12 to KRAS G12D protein was further confirmed by the surface plasmon resonance (SPR) assay with a binding KD of 157 µM. SK12 also exhibited notable anticancer efficacy in a nude mice tumor model. The relative tumor proliferation rate (T/C) of the experimental group (50 mg/kg) was 31.04 % (P < 0.05), while maintaining a commendable safety profile.

Moderation of mitochondrial respiration mitigates metabolic syndrome of aging.

Deregulation of mitochondrial dynamics leads to the accumulation of oxidative stress and unhealthy mitochondria; consequently, this accumulation contributes to premature aging and alterations in mitochondria linked to metabolic complications. We postulate that restrained mitochondrial ATP synthesis might alleviate age-associated disorders and extend healthspan in mammals. Herein, we prepared a previously discovered mitochondrial complex IV moderate inhibitor in drinking water and orally administered to standard-diet-fed, wild-type C57BL/6J mice every day for up to 16 mo. No manifestation of any apparent toxicity or deleterious effect on studied mouse models was observed. The impacts of an added inhibitor on a variety of mitochondrial functions were analyzed, such as respiratory activity, mitochondrial bioenergetics, and biogenesis, and a few age-associated comorbidities, including reactive oxygen species (ROS) production, glucose abnormalities, and obesity in mice. It was found that mitochondrial quality, dynamics, and oxidative metabolism were greatly improved, resulting in lean mice with a specific reduction in visceral fat plus superb energy and glucose homeostasis during their aging period compared to the control group. These results strongly suggest that a mild interference in ATP synthesis through moderation of mitochondrial activity could effectively up-regulate mitogenesis, reduce ROS production, and preserve mitochondrial integrity, thereby impeding the onset of metabolic syndrome. We conclude that this inhibitory intervention in mitochondrial respiration rectified the age-related physiological breakdown in mice by protecting mitochondrial function and markedly mitigated certain undesired primary outcomes of metabolic syndrome, such as obesity and type 2 diabetes. This intervention warrants further research on the treatment of metabolic syndrome of aging in humans.

Design, synthesis and antitumor activities of thiazole-containing mitochondrial targeting agents.

In this study, a novel batch of thiazole-containing mitochondrial targeting agents were designed and synthesized. Four kinds of mitochondrial targeting moieties and six kinds of linkers were designed. Their structures were confirmed by NMR and HR-MS. The screening of antiproliferative activity revealed that most compounds displayed cytotoxicity on HeLa cancer cell. In particular, D1 has an IC50 value of 35.32 µmol·L-1 against HeLa cell. In addition, cellular respiratory activities were also tested on HeLa cancer cells. D1 had a basal oxygen consumption rate of 8.84 pmol·s-1·mL-1. Also, D1 inhibited the mitochondrial respiration of HeLa cell significantly at 5 µmol·L-1, as well as a complete inhibitory of oxygen consumption for cellular ATP coupling. Furthermore, the pKa, logP, and logD under different pH conditions of all the compounds were calculated by the ACD/Percepta-PhysChem Suite, and the results manifested the correlation between physicochemical properties and chemical activity of compounds. The results identify D1 as a promising mitochondria inhibitor and anticancer agent with appropriate physicochemical properties.

Biological evaluation of mitochondria targeting small molecules as potent anticancer drugs.

Cancer therapy targets specific metabolic pathways or a single gene. This may result in low therapeutic effects due to drug selectivity and drug resistance. Recent studies revealed that the mitochondrial membrane potential and transmembrane permeability of cancerous mitochondria are differed from normal mitochondria. Thus, chemotherapy targeting cancerous mitochondria could be an innovative and competent strategy for cancer therapy. Previously, our work with a novel group of mitochondria targeting small molecules presented promising inhibitory capability toward various cancer cell lines and suppressed adenosine triphosphate (ATP) generation. Therefore, it is critical to understand the anticancer effect and targeting mechanism of these small molecules. This study investigated the inhibitory activity of mitochondria targeting small molecules with human cervical cancer cells - HeLa to further explore their therapeutic potential. HeLa cells were exposed to 10 µM of synthesized compounds and presented elevation in intracellular reactive oxygen species (ROS) level, impaired mitochondrial membrane potential and upregulation of apoptosis as well as necrosis. In vivo, HeLa cell tumor-bearing BALB/c nude mice were treated with mitochondria targeting small molecules for 12 days consecutively. Throughout this chemotherapy study, no deleterious side effects nor the appearance of toxicity was observed. Furthermore, mitochondria targeting small molecules treated groups exhibited significant down-regulation with both tumor volume and tumor weight compared to the Doxorubicin (DOX) treated group. Thus, inhibition of mitochondrial ATP synthesis, activation of intracellular ROS production, down-regulation of mitochondrial membrane potential and upregulation of apoptosis and necrosis rates are the indications of cancer therapy. In this work, we examined the anticancer capability of four mitochondria targeting small molecules in vitro and in vivo, and demonstrated a novel therapeutic approach in cancer therapy with tremendous potential.

Design, synthesis and biological evaluation of novel thiazole-derivatives as mitochondrial targeting inhibitors of cancer cells.

Mitochondria are pivotal energy production sources for cells to maintain necessary metabolism activities. Targeting dysfunctional mitochondrial features has been a hotspot for mitochondrial-related disease researches. Investigation with cancerous mitochondrial metabolism is a continuing concern within tumor therapy. Herein, we set out to assess the anti-cancer activities of a novel family of TPP-thiazole derivatives based on our earlier research on mitochondrial targeting agents. Specifically, we designed and synthesized a series of TPP-thiazole derivatives and revealed by the MTT assay that most synthesized compounds effectively inhibited three cancer cell lines (HeLa, PC3 and MCF-7). After structure modifications, we explored the SAR relationships and identified the most promising compound R13 (IC50 of 5.52 µM) for further investigation. In the meantime, we performed ATP production assay to assess the selected compounds inhibitory effect on HeLa cells energy production. The results displayed the test compounds significantly restrained ATP production of cancer cells. Overall, we have designed and synthesized a series of compounds which exhibited significant cytotoxicity against cancer cells and effectively inhibited mitochondrial energy production.

Drug discovery approaches targeting the incretin pathway.

Incretin pathway plays an important role in the development of diabetes medications. Interventions in DPP-4 and GLP-1 receptor have shown remarkable efficacy in experimental and clinical studies and imperatively become one of the most promising therapeutic approaches in the T2DM drug discovery pipeline. Herein, we analyzed the actionmechanismsof DPP-4 and GLP-1 receptor targeting the incretin pathway in T2DM treatment. We gave an insight into the structural requirements for the potent DPP-4 inhibitors and revealed a classification of DPP-4 inhibitors by stressing on the binding modes of these ligands to the enzyme. We then reviewed the drug discovery strategies for the development of peptide and non-peptide GLP-1 receptor agonists (GLP-1 RAs). Furthermore, the drug design strategies for DPP-4 inhibitors and GLP-1R agonists were detailed accurately. This review might provide an efficient evidence for the highly potent and selective DPP-4 inhibitors and the GLP-1 RAs, as novel medicines for patients suffering from T2DM.

Phosphatase activity of small C-terminal domain phosphatase 1 (SCP1) controls the stability of the key neuronal regulator RE1-silencing transcription factor (REST).

The RE1-silencing transcription factor (REST) is the major scaffold protein for assembly of neuronal gene silencing complexes that suppress gene transcription through regulating the surrounding chromatin structure. REST represses neuronal gene expression in stem cells and non-neuronal cells, but it is minimally expressed in neuronal cells to ensure proper neuronal development. Dysregulation of REST function has been implicated in several cancers and neurological diseases. Modulating REST gene silencing is challenging because cellular and developmental differences can affect its activity. We therefore considered the possibility of modulating REST activity through its regulatory proteins. The human small C-terminal domain phosphatase 1 (SCP1) regulates the phosphorylation state of REST at sites that function as REST degradation checkpoints. Using kinetic analysis and direct visualization with X-ray crystallography, we show that SCP1 dephosphorylates two degron phosphosites of REST with a clear preference for phosphoserine 861 (pSer-861). Furthermore, we show that SCP1 stabilizes REST protein levels, which sustains REST's gene silencing function in HEK293 cells. In summary, our findings strongly suggest that REST is a bona fide substrate for SCP1 in vivo and that SCP1 phosphatase activity protects REST against degradation. These observations indicate that targeting REST via its regulatory protein SCP1 can modulate its activity and alter signaling in this essential developmental pathway.

Identification of lipid-like salicylic acid-based derivatives as potent and membrane-permeable PTP1B inhibitors.

Developing protein tyrosine phosphatase-1B (PTP1B) inhibitors is an important strategy to treat type 2 diabetes mellitus (T2DM). Most existing ionic PTP1B inhibitors aren't of clinical useful due to their low cell-permeability, however. Herein, we introduced a series of lipid-like acid-based (salicylic acid) modules to prepare PTP1B inhibitors, and demonstrated a marked improvement of cell-permeability while maintaining excellent PTP1B inhibitory activity (e.g. compound B12D, IC50 = 0.37 µM against PTP1B and Papp = 1.5 × 10-6 cm/s). We believe that this strategy can be widely utilized to modify potent lead compounds with low cell-permeability.

Discovery of 2-ethoxy-4-(methoxymethyl)benzamide derivatives as potent and selective PTP1B inhibitors.

Protein tyrosine phosphatase 1B (PTP1B), a key negative regulator of insulin signaling, is considered as a promising and validated therapeutic target for type 2 diabetes mellitus (T2DM) and obesity. Upon careful study, a series of 2-ethoxy-4-(methoxymethyl)benzamide and 2-ethoxy-5-(methoxymethyl)benzamide analogs designed by the "bioisosteric principle" were discovered, wherein their PTP1B inhibitory potency, type of PTP1B inhibition, selectivity and membrane permeability were evaluated. Among them, compound 10m exhibited high inhibitory activity (IC50 = 0.07 µM), significant selectivity (32-fold) over T-cell PTPase (TCPTP) as well as good membrane permeability (Papp = 2.41 × 10-6 cm/s). Further studies on cell viability and cellular activity revealed that compound 10m could enhance insulin-stimulated glucose uptake with no significant cytotoxicity.

Anti-diabetic potential of Pueraria lobata root extract through promoting insulin signaling by PTP1B inhibition.

Type 2 diabetes mellitus is a fast-growing epidemic affecting people globally. We initiated the project by searching the possible target of the Pueraria lobata root extract (P. lobata). We conducted the IC50 assays of P. lobata on the four diabetes-related proteins: PTP1B, TCPTP, SHP-2 and DPP-4. Results indicated that P. lobata exhibited high PTP1B inhibitory activity with IC50 of 0.043 mg/ml. Treated insulin-resistant HepG2 cells with 0.0115 mg/ml of P. lobata increased the glucose uptake by two times compared with the negative control. Further, we performed OGTT test on the diabetic C57BL/6 male mice. 20% decreased blood glucose (AUC) was obtained with a dose of 1 g/kg P. lobata compared with the negative control. Herein, we were able to demonstrate the antidiabetic effects of P. lobata might be related to the inhibition of PTP1B and therefore, bettering the insulin signaling pathway.

Surrogating and redirection of pyrazolo[1,5-a]pyrimidin-7(4H)-one core, a novel class of potent and selective DPP-4 inhibitors.

The initial focus on characterizing novel pyrazolo[1,5-a]pyrimidin-7(4H)-one derivatives as DPP-4 inhibitors, led to a potent and selective inhibitor compound b2. This ligand exhibits potent in vitro DPP-4 inhibitory activity (IC50: 80 nM), while maintaining other key cellular parameters such as high selectivity, low cytotoxicity and good cell viability. Subsequent optimization of b2 based on docking analysis and structure-based drug design knowledge resulted in d1. Compound d1 has nearly 2-fold increase of inhibitory activity (IC50: 49 nM) and over 1000-fold selectivity against DPP-8 and DPP-9. Further in vivo IPGTT assays showed that compound b2 effectively reduce glucose excursion by 34% at the dose of 10 mg/kg in diabetic mice. Herein we report the optimization and design of a potent and highly selective series of pyrazolo[1,5-a]pyrimidin-7(4H)-one DPP-4 inhibitors.

Y-shaped bis-arylethenesulfonic acid esters: Potential potent and membrane permeable protein tyrosine phosphatase 1B inhibitors.

Known PTP1B inhibitors with bis-anionic moieties exhibit potent inhibitory activity, good selectivity, however, they are incapable of penetrating cellular membranes. Based upon our finding of a new pharmacophoric group in inhibition of PTP1B and the structural characteristics of the binding pocket of PTP1B, a series of bis-arylethenesulfonic acid ester derivatives were designed and synthesized. These novel molecules, particularly Y-shaped bis-arylethenesulfonic acid ester derivatives, exhibited high PTP1B inhibitory activity, moderate selectivity, and great potential in penetrating cellular membranes (compound 7p, CLogP=9.73, Papp=9.6×10-6cm/s; IC50=140, 1290 and 920nM on PTP1B, TCPTP and SHP2, respectively). Docking simulations suggested that these Y-shaped inhibitors might interact with multiple secondary binding sites in addition to the catalytic site of PTP1B.

Synthesis, biological evaluation and molecular docking analysis of 2-phenyl-benzofuran-3-carboxamide derivatives as potential inhibitors of Staphylococcus aureus Sortase A.

In Gram-positive bacteria, Sortase A (Srt A) is a critical cysteine transpeptidase that is responsible for recognizing and assembling surface virulence proteins through the recognition of a LPXTG (leucine, proline, X, threonine, and glycine, where X is any amino acid) signal. Mutants lacking genes for Srt A attenuate infections without affecting microbial viability. Here a series of 2-phenyl-benzofuran-3-carboxamide derivatives were synthesized and identified as potent Srt A inhibitors. Activity assays revealed that multiple compounds exhibited excellent inhibitory activity against Srt A compared with known Sortase A inhibitor pHMB (IC50=130µM). Structural activity relationships (SARs) demonstrated that the amide group at 3-position was essential for inhibitory activity. Replacement of the hydroxyl group at the 2-phenyl position of benzofuran with other substitutions such as a methoxyl, halogen or nitro group reduced the enzyme inhibitory activity in most cases. The compound Ia-22 was found to be the most potent inhibitor against the enzyme with an IC50 value of 30.8µM. Molecular docking studies showed Ia-22 shared similar binding pattern with substrate LPXTG in the binding pocket of Srt A (PDB: 2KID) including i-butyl stretching, L-shape pattern kinking, and H-bond interaction with Srt A functional site residues Cys184, Trp194 and Arg197.

Synthesis, biological evaluation and molecular docking of 2-phenyl-benzo[d]oxazole-7-carboxamide derivatives as potential Staphylococcus aureus Sortase A inhibitors.

A series of novel 2-phenyl-benzo[d]oxazole-7-carboxamide derivatives were designed, synthesized and evaluated for their in vitro inhibitory activities against Staphylococcus aureus Sortase A with known Sortase A inhibitor pHMB as positive compound (IC50=130µM). Most compounds exhibited excellent inhibitory activity (IC50=19.8-184.2µM). Structure-activity relationship studies demonstrated that substitution at 7-position and 2-position of benzoxazole had great influence on the activities. Specifically, the substituent at 7-position is indispensable for inhibitory activity. The molecular docking studies revealed the i-butyl amide group went towards the ß6/ß7 loop-ß8 substructure of the protein and the benzoxazole core lied in a hydrophobic pocket composed of Ala118, Val166, Val168, Val169 and Ile182, shaping the whole molecule into a L-shape mode to be recognized by Sortase A.

Design, synthesis, and biological evaluation of 2-substituted ethenesulfonic acid ester derivatives as selective PTP1B inhibitors.

Fifteen 2-substituted ethenesulfonic acid ester derivatives were designed, synthesized, and evaluated for the inhibitory activities against protein tyrosine phosphatase 1B (PTP1B) and T-Cell protein tyrosine phosphatase (TCPTP). The structural activity relationship (SAR) of these compounds are discussed to clarify the impact of the linker and the optimized tail on the inhibitory activity of PTP1B and selectivity over TCPTP. Most of the compounds exhibit excellent inhibitory activities against PTP1B with IC50 values of 1.5-8.9 µM. SAR analysis reveal that the substituents at the hydrophobic tail significantly alter the inhibitory activity against PTP1 B and selectivity over TCPTP, e.g. compound 5d showed excellent inhibitory activity to PTP1B with IC50 = 7.8 µM, and -6-fold selectivity over TCPTP. Combined with our previous findings, we confirm that the linker length and the substituted hydrophobic tail have decisive influence on the PTP1B inhibitory activity and selectivity.

Identification of 2-substituted ethenesulfonic acid ester derivatives as novel, potent and selective inhibitors of protein tyrosine phosphatase 1B.

Sixteen 2-substituted ethenesulfonic acid ester derivatives were designed, synthesized and evaluated for the inhibitory activity against tyrosine phosphatase 1B (PTP1B) and T-Cell protein tyrosine phosphatase (TCPTP). The structural activity relationship (SAR) of these compounds demonstrated that the hydrophilic head, aromatic center and the hydrophobic tail affected the inhibitory activities against PTP1B and the selectivity over TCPTP. Most of the compounds exhibited excellent inhibitory activity against PTP1B with IC50 value of 1.0 µM - 31.2 µM. SAR analysis revealed that the hydrophilic head was indispensable in the maintain of inhibitory activity against PTP1B, the aromatic center significantly altered the selectivity of PTP1B over TCPTP, and the hydrophobic tail significantly altered the inhibitory activity against PTP1B.

Synthesis, biological activity evaluation and mechanism analysis of new ganglioside GM3 derivatives as potential agents for nervous functional recovery.

Neuronal regenerative ability is vital for the treatment of neurodegenerative diseases and neuronal injuries. Recent studies have revealed that Ganglioside GM3 and its derivatives may possess potential neuroprotective and neurite growth-promoting activities. Herein, six GM3 derivatives were synthesized and evaluated their potential neuroprotective effects and neurite outgrowth-promoting activities on a cellular model of Parkinson's disease and primary nerve cells. Amongst these derivatives, derivatives N-14 and 2C-12 demonstrated neuroprotective effects in the MPP + model in SH-SY5Y cells. 2C-12 combined with NGF (nerve growth factor) induced effecially neurite growth in primary nerve cells. Further action mechanism revealed that derivative 2C-12 exerts neuroprotective effects by regulating the Wnt signaling pathway, specifically involving the Wnt7b gene. Overall, this study establishes a foundation for further exploration and development of GM3 derivatives with neurotherapeutic potential.

Discovery of Sortase A covalent inhibitors with benzofuranene cyanide structures as potential antibacterial agents against Staphylococcus aureus.

Sortase A (SrtA) is a cysteine transpeptidase of most gram-positive bacteria that is responsible for the anchoring of many surface protein virulence factors to the cell wall. SrtA ablation has demonstrated to alleviate the infection without affecting the viability of bacteria. Herein, a series of benzofuran cyanide derivatives were synthesized and evaluated. Several compounds exhibited excellent inhibitory activity against SrtA with IC50 values from 3.3 µM to 21.8 µM compared with the known SrtA inhibitor pHMB (IC50 = 130 µM). Ⅲ-1, Ⅲ-15, Ⅲ-34 and V-1 showed potent inhibitory effects on biofilm formation with IC50 values from 2.1 µM to 54.2 µM. Invasion assays showed the four compounds caused a decrease of 4%-24.0% in the uptake of the S. aureus strain by 293T cells. Further assay showed that compound Ⅲ-15 decreased the amount of cell wall-associated protein A by 26.5%. Structure-activity relationship and docking studies demonstrated that the acrylonitrile moiety of the compounds played an important role in enhancing the activity. When the double bond of acrylonitrile changed to single bond, the activity was decreased significantly. This indicates that acrylonitrile, which is a Michael receptor, can inhibit the activity of SrtA by covalent binding effectively to the thiol group of Cys184.

Design, synthesis and neurite outgrowth activity of novel ganglioside GM1 derivatives by remodeling of the fatty acid moiety.

Ganglioside GM1 is a glycosphingolipid found on mammalian cell membranes, and it is involved in ischemic encephalopathy, spinal cord injury and neurodegenerative diseases. Fatty acids, as a structure module of GM1, have been reported to affect its physiological function and neurite growth activity. Due to the limitation of preparation methods, the function of GM1 derivatives containing different fatty acids in nerve cells has not been systematically studied. To discover novel GM1 derivatives as nerve growth-promoting agents, we developed an efficient SA_SCDase enzymatic synthetic system of GM1 derivatives, yielding twenty GM1 derivatives with unsaturated fatty acid chains in high total yields (16-67%). Subsequently, the neurite outgrowth activities of GM1 derivatives were assessed on Neuro2a Cells. Among all the GM1 derivatives, GM1 (d18:1/C16:1) induced demonstrably neurite outgrowth activity. The subsequent RNA-sequencing (RNA-seq) and Western blot analysis was then performed and indicated that the mechanism of nerve cells growth involved cholesterol biosynthesis regulation by up-regulating SREBP2 expression or ERBB4 phosphorylation to activate the PI3K-mTOR pathway.

Plasmalogens Eliminate Aging-Associated Synaptic Defects and Microglia-Mediated Neuroinflammation in Mice.

Neurodegeneration is a pathological condition in which nervous system or neuron losses its structure, function, or both leading to progressive neural degeneration. Growing evidence strongly suggests that reduction of plasmalogens (Pls), one of the key brain lipids, might be associated with multiple neurodegenerative diseases, including Alzheimer's disease (AD). Plasmalogens are abundant members of ether-phospholipids. Approximately 1 in 5 phospholipids are plasmalogens in human tissue where they are particularly enriched in brain, heart and immune cells. In this study, we employed a scheme of 2-months Pls intragastric administration to aged female C57BL/6J mice, starting at the age of 16 months old. Noticeably, the aged Pls-fed mice exhibited a better cognitive performance, thicker and glossier body hair in appearance than that of aged control mice. The transmission electron microscopic (TEM) data showed that 2-months Pls supplementations surprisingly alleviate age-associated hippocampal synaptic loss and also promote synaptogenesis and synaptic vesicles formation in aged murine brain. Further RNA-sequencing, immunoblotting and immunofluorescence analyses confirmed that plasmalogens remarkably enhanced both the synaptic plasticity and neurogenesis in aged murine hippocampus. In addition, we have demonstrated that Pls treatment inhibited the age-related microglia activation and attenuated the neuroinflammation in the murine brain. These findings suggest for the first time that Pls administration might be a potential intervention strategy for halting neurodegeneration and promoting neuroregeneration.