Advances in inhibitor development targeting the PWWP domain.

The PWWP domain binds to both histone and DNA of a nucleosome in a bivalent way. PWWP domain-containing proteins are involved in different biological processes, and their aberrant expression is implicated in various human diseases. Here, we discuss the recent developments and challenges in targeting the PWWP domain for therapeutic intervention.

N/C-degron pathways and inhibitor development for PROTAC applications.

Ubiquitination is a fascinating post-translational modification that has received continuous attention since its discovery. In this review, we first provide a concise overview of the E3 ubiquitin ligases, delving into classification, characteristics and mechanisms of ubiquitination. We then specifically examine the ubiquitination pathways mediated by the N/C-degrons, discussing their unique features and substrate recognition mechanisms. Finally, we offer insights into the current state of development pertaining to inhibitors that target the N/C-degron pathways, as well as the promising advances in the field of PROTAC (PROteolysis TArgeting Chimeras). Overall, this review offers a comprehensive understanding of the rapidly-evolving field of ubiquitin biology.

Structural repurposing of SGLT2 inhibitor empagliflozin for strengthening anti-heart failure activity with lower glycosuria.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been reapproved for heart failure (HF) therapy in patients with and without diabetes. However, the initial glucose-lowering indication of SGLT2i has impeded their uses in cardiovascular clinical practice. A challenge of SGLT2i then becomes how to separate their anti-HF activity from glucose-lowering side-effect. To address this issue, we conducted structural repurposing of EMPA, a representative SGLT2 inhibitor, to strengthen anti-HF activity and reduce the SGLT2-inhibitory activity according to structural basis of inhibition of SGLT2. Compared to EMPA, the optimal derivative JX01, which was produced by methylation of C2-OH of the glucose ring, exhibited weaker SGLT2-inhibitory activity (IC50 > 100 nmol/L), and lower glycosuria and glucose-lowering side-effect, better NHE1-inhibitory activity and cardioprotective effect in HF mice. Furthermore, JX01 showed good safety profiles in respect of single-dose/repeat-dose toxicity and hERG activity, and good pharmacokinetic properties in both mouse and rat species. Collectively, the present study provided a paradigm of drug repurposing to discover novel anti-HF drugs, and indirectly demonstrated that SGLT2-independent molecular mechanisms play an important role in cardioprotective effects of SGLT2 inhibitors.

Photoactivatable senolysis with single-cell resolution delays aging.

Strategies that can selectively eliminate senescent cells (SnCs), namely senolytics, have been shown to promote healthy lifespan. However, it is challenging to achieve precise, broad-spectrum and tractable senolysis. Here, we integrate multiple technologies that combine the enzyme substrate of senescence-associated ß-galactosidase (SA-ß-gal) with fluorescence tag for the precise tracking of SnCs, construction of a bioorthogonal receptor triggered by SA-ß-gal to target and anchor SnCs with single-cell resolution and incorporation of a selenium atom to generate singlet oxygen and achieve precise senolysis through controllable photodynamic therapy (PDT). We generate KSL0608-Se, a photosensitive senolytic prodrug, which is selectively activated by SA-ß-gal. In naturally-aged mice, KSL0608-Se-mediated PDT prevented upregulation of age-related SnCs markers and senescence-associated secretory phenotype factors. This treatment also countered age-induced losses in liver and renal function and inhibited the age-associated physical dysfunction in mice. We therefore provide a strategy to monitor and selectively eliminate SnCs to regulate aging.

Nicandra physalodes Extract Exerts Antiaging Effects in Multiple Models and Extends the Lifespan of Caenorhabditis elegans via DAF-16 and HSF-1.

The development of safe and effective therapeutic interventions is an important issue for delaying aging and reducing the risk of aging-related diseases. Chinese herbal medicines for the treatment of aging and other complex diseases are desired due to their multiple components and targets. Through screening for effects on lifespan of 836 Chinese herbal medicine extracts, Nicandra physalodes extract (HL0285) was found to exhibit lifespan extension activity in Caenorhabditis elegans (C. elegans). In further experiments, HL0285 improved healthspan, enhanced stress resistance, and delayed the progression of neurodegenerative diseases in C. elegans. Additionally, it ameliorated senescence in human lung fibroblasts (MRC-5 cells) and reversed liver function damage and reduced senescence marker levels in doxorubicin- (Dox-) induced aging mice. In addition, the longevity effect of HL0285 in C. elegans was dependent on the DAF-16 and HSF-1 signaling pathways, as demonstrated by the results of the mutant lifespan, gene level, and GFP level assays. In summary, we discovered that HL0285 had an antiaging effect in C. elegans, MRC-5 cells, and Dox-induced aging mice and deserves to be explored in the future studies on antiaging agents.

N-Acylamino Saccharin as an Emerging Cysteine-Directed Covalent Warhead and Its Application in the Identification of Novel FBPase Inhibitors toward Glucose Reduction.

With a resurgence of covalent drugs, there is an urgent need for the identification of new moieties capable of cysteine bond formation. Herein, we report on the N-acylamino saccharin moieties capable of novel covalent reactions with cysteine. Their utility as alternative electrophilic warheads was demonstrated through the covalent modification of fructose-1,6-bisphosphatase (FBPase), a promising target associated with cancer and type 2 diabetes. The cocrystal structure of title compound W8 bound with FBPase unexpectedly revealed that the N-acylamino saccharin moiety worked as an electrophile warhead that covalently modified the noncatalytic C128 site in FBPase while releasing saccharin, suggesting a previously undiscovered covalent reaction mechanism of saccharin derivatives with cysteine. Treatment of title compound W8 displayed potent inhibition of glucose production in vitro and in vivo. This newly discovered reactive warhead supplements the current repertoire of cysteine covalent modifiers while avoiding some of the limitations generally associated with established moieties.

[Relationship between carbon dioxide combining power and the short-term prognosis in acute ischemic stroke patients after thrombolysis].

OBJECTIVE: To investigate the effect of venous blood carbon dioxide binding capacity (CO2-CP) on the short-term prognosis of patients with acute ischemic stroke (AIS) after thrombolytic therapy. METHODS: A total of 86 AIS inpatients who received thrombolytic therapy in the emergency department of Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University from April 2019 to May 2021 were analyzed retrospectively. According to the venous blood CO2-CP levels at admission, the patients were divided into two groups: low CO2-CP group (CO2-CP < 23 mmol/L, n = 52) and high CO2-CP group (CO2-CP ≥ 23 mmol/L, n = 34). The CO2-CP levels and changes between the two groups before and after thrombolytic therapy were compared. The National Institutes of Health Stroke scale (NIHSS) score was used to evaluate the improvement rate of patients after thrombolytic therapy [NIHSS score at admission-NIHSS score at discharge)/NIHSS score at admission ×100%] and in-hospital death was also recorded. The correlation between CO2-CP levels and prognosis of patients with AIS during emergency visit was analyzed, the receiver operator characteristic curve (ROC curve) was drawn and the area under the ROC curve (AUC) was calculated to evaluate the predictive value of CO2-CP in the prognosis of AIS patients. RESULTS: The CO2-CP levels of low CO2-CP group and high CO2-CP group after thrombolytic therapy were significantly higher than those before treatment (mmol/L: 23.08±2.34 vs. 20.46±1.51, 25.24±2.16 vs. 23.94±1.07, both P < 0.05). The differences of CO2-CP before and after treatment in low CO2-CP group were significantly higher than those in high CO2-CP group (mmol/L: 2.62±0.83 vs. 1.30±1.09, P < 0.05). The improvement rate of CO2-CP levels in the high CO2-CP group (NIHSS improvement rate > 45%) was significantly higher than that in the low CO2-CP group [85.29% (29/34) vs. 23.08% (12/52)], while the mortality in the low CO2-CP group was significantly higher than that in the high CO2-CP group [11.54% (6/52) vs. 0% (0/34), P < 0.05]. The AUC of CO2-CP for the prognosis of patients with AIS thrombolysis was 0.820, the 95% confidence interval (95%CI) was 0.727-0.924, P = 0.000 1. CONCLUSION: AIS patients with CO2-CP levels less than 23 mmol/L have a poor short-term prognosis, which has certain predictive and clinical reference value for choosing thrombolytic time in emergency stroke patients.

Discovery of novel dual RAGE/SERT inhibitors for the potential treatment of the comorbidity of Alzheimer's disease and depression.

Depression is identified as one of the most common psychiatric symptoms in Alzheimer's disease (AD). The comorbidity of AD and depression increases the burden of clinical treatment and care in elderly patients. In order to find new treatment options, we first proposed the dual RAGE/SERT inhibitors by fusing the key pharmacophore of vilazodone and azeliragon for the potential treatment of AD with comorbid depression. After a series of structural modifications, 34 dual-target directed ligands were designed and synthesized, and their RAGE and SERT inhibitory activities were systematically evaluated. Among them, compound 12 showed good dual-target bioactivities against RAGE (IC50 = 8.26 ± 1.12 µM) and SERT (IC50 = 31.09 ± 5.15 nM) in vitro, better safety profile than azeliragon, good liver microsomal stability, weak CYP inhibition, and acceptable pharmacokinetic properties. Moreover, 12 ameliorated Aß25-35-induced neurotoxicity in SH-SY5Y cells and alleviated the depressive symptom in tail suspension test. In brief, these results indicated that 12 is a prospective prototype for the potential treatment of AD with comorbid depression.

Anti-aging effects of chlorpropamide depend on mitochondrial complex-II and the production of mitochondrial reactive oxygen species.

Sulfonylureas are widely used oral anti-diabetic drugs. However, its long-term usage effects on patients' lifespan remain controversial, with no reports of influence on animal longevity. Hence, the anti-aging effects of chlorpropamide along with glimepiride, glibenclamide, and tolbutamide were studied with special emphasis on the interaction of chlorpropamide with mitochondrial ATP-sensitive K+ (mitoK-ATP) channels and mitochondrial complex II. Chlorpropamide delayed aging in Caenorhabditis elegans, human lung fibroblast MRC-5 cells and reduced doxorubicin-induced senescence in both MRC-5 cells and mice. In addition, the mitochondrial membrane potential and ATP levels were significantly increased in chlorpropamide-treated worms, which is consistent with the function of its reported targets, mitoK-ATP channels. Increased levels of mitochondrial reactive oxygen species (mtROS) were observed in chlorpropamide-treated worms. Moreover, the lifespan extension by chlorpropamide required complex II and increased mtROS levels, indicating that chlorpropamide acts on complex II directly or indirectly via mitoK-ATP to increase the production of mtROS as a pro-longevity signal. This study provides mechanistic insight into the anti-aging effects of sulfonylureas in C. elegans.

Identification of Guaifenesin-Andrographolide as a Novel Combinatorial Drug Therapy for Epilepsy Using Network Virtual Screening and Experimental Validation.

Combinatorial drug therapy has attracted substantial attention as an emerging strategy for the treatment of diseases with complex pathological mechanisms. We previously developed a potentially universal computational screening approach for combination drugs and used this approach to successfully identify some beneficial combinations for the treatment of heart failure. Herein, this screening approach was used to identify novel combination drugs for the treatment of epilepsy in an approved drug library. The combination of guaifenesin-andrographolide was first discovered as a promising therapy with synergistic anticonvulsant activities in maximal electroshock (MES)- and subcutaneous pentylenetetrazol (sc-PTZ)-induced epilepsy models in vivo. The studies of network analysis, fluorescence imaging, and N-methyl-d-aspartate (NMDA)-induced cytotoxicity further revealed that guaifenesin-andrographolide might synergistically affect NMDA receptors and then alleviate the pathogenesis of epilepsy. Therefore, we report that the combination of guaifenesin-andrographolide exerts effects against epilepsy through a novel synergistic mechanism and is thus a potential treatment for epilepsy, providing a promising mechanism for the design of novel combinatorial drug treatments against epilepsy.

Structure-Guided Discovery of the Novel Covalent Allosteric Site and Covalent Inhibitors of Fructose-1,6-Bisphosphate Aldolase to Overcome the Azole Resistance of Candidiasis.

Fructose-1,6-bisphosphate aldolase (FBA) represents an attractive new antifungal target. Here, we employed a structure-based optimization strategy to discover a novel covalent binding site (C292 site) and the first-in-class covalent allosteric inhibitors of FBA from Candida albicans (CaFBA). Site-directed mutagenesis, liquid chromatography-mass spectrometry, and the crystallographic structures of APO-CaFBA, CaFBA-G3P, and C157S-2a4 revealed that S268 is an essential pharmacophore for the catalytic activity of CaFBA, and L288 is an allosteric regulation switch for CaFBA. Furthermore, most of the CaFBA covalent inhibitors exhibited good inhibitory activity against azole-resistant C. albicans, and compound 2a11 can inhibit the growth of azole-resistant strains 103 with the MIC80 of 1 µg/mL. Collectively, this work identifies a new covalent allosteric site of CaFBA and discovers the first generation of covalent inhibitors for fungal FBA with potent inhibitory activity against resistant fungi, establishing a structural foundation and providing a promising strategy for the design of potent antifungal drugs.

Rapid Repurposing of Novel Combination Drugs for the Treatment of Heart Failure via a Computationally Guided Network Screening Approach.

Combination drugs, characterized by high efficacy and few side effects, have received extensive attention from pharmaceutical companies and researchers for the treatment of complex diseases such as heart failure (HF). Traditional combination drug discovery depends on large-scale high-throughput experimental approaches that are time-consuming and costly. Herein we developed a novel, rapid, and potentially universal computer-guided combination drug-network-screening approach based on a set of databases and web services that are easy for individuals to obtain and operate, and we discovered for the first time that the menthol-allethrin combination screened by this approach exhibited a significant synergistic cardioprotective effect in vitro. Further mechanistic studies indicated that allethrin and menthol could synergistically block calcium channels. Allethrin bound to the central cavity of the voltage-dependent L-type calcium channel subunit alpha-1S (CACNA1S) lead to a conformational change in an allosteric site of CACNA1S, thereby enhancing the binding of menthol to this allosteric site. In summary, we reported a potentially universal computational approach to combination drug screening that has been used to discover a new combination of menthol-allethrin against HF in vitro, providing a new synergistic mechanism and prospective agent for HF treatment.

The novel therapeutic strategy of vilazodone-donepezil chimeras as potent triple-target ligands for the potential treatment of Alzheimer's disease with comorbid depression.

Depression is one of the most frequent comorbid psychiatric symptoms of Alzheimer's disease (AD), and no efficacious drugs have been approved specifically for this purpose thus far. Herein, we proposed a novel therapeutic strategy that merged the key pharmacophores of the antidepressant vilazodone (5-HT1A receptor partial agonist and serotonin transporter inhibitor) and the anti-AD drug donepezil (acetylcholinesterase inhibitor) together to develop a series of multi-target-directed ligands for potential therapy of the comorbidity of AD and depression. Accordingly, 55 vilazodone-donepezil chimeric derivatives were designed and synthesized, and their triple-target activities against acetylcholinesterase, 5-HT1A receptor, and serotonin transporter were systematically evaluated. Among them, compound 5 displayed strong triple-target bioactivities in vitro, low hERG potassium channel inhibition and acceptable brain distribution. Importantly, oral intake of 5 mg/kg of the compound 5 dihydrochloride significantly alleviated the depressive symptoms and ameliorated cognitive dysfunction in mouse models. In brief, these results highlight vilazodone-donepezil chimeras as a prospective therapeutic approach for the treatment of the comorbidity of AD and depression.

Biological evaluation and SAR analysis of novel covalent inhibitors against fructose-1,6-bisphosphatase.

Fructose-1,6-bisphosphatase (FBPase) is an attractive target for affecting the GNG pathway. In our previous study, the C128 site of FBPase has been identified as a new allosteric site, where several nitrovinyl compounds can bind to inhibit FBPase activity. Herein, a series of nitrostyrene derivatives were further synthesized, and their inhibitory activities against FBPase were investigated in vitro. Most of the prepared nitrostyrene compounds exhibit potent FBPase inhibition (IC50 < 10 µM). Specifically, when the substituents of F, Cl, OCH3, CF3, OH, COOH, or 2-nitrovinyl were installed at the R2 (meta-) position of the benzene ring, the FBPase inhibitory activities of the resulting compounds increased 4.5-55 folds compared to those compounds with the same groups at the R1 (para-) position. In addition, the preferred substituents at the R3 position were Cl or Br, thus compound HS36 exhibited the most potent inhibitory activity (IC50 = 0.15 µM). The molecular docking and site-directed mutation suggest that C128 and N125 are essential for the binding of HS36 and FBPase, which is consistent with the C128-N125-S123 allosteric inhibition mechanism. The reaction enthalpy calculations show that the order of the reactions of compounds with thiol groups at the R3 position is Cl > H > CH3. CoMSIA analysis is consistent with our proposed binding mode. The effect of compounds HS12 and HS36 on glucose production in primary mouse hepatocytes were further evaluated, showing that the inhibition was 71% and 41% at 100 µM, respectively.

Development of disulfide-derived fructose-1,6-bisphosphatase (FBPase) covalent inhibitors for the treatment of type 2 diabetes.

Fructose-1,6-bisphosphatase (FBPase), as a key rate-limiting enzyme in the gluconeogenesis (GNG) pathway, represents a practical therapeutic strategy for type 2 diabetes (T2D). Our previous work first identified cysteine residue 128 (C128) was an important allosteric site in the structure of FBPase, while pharmacologically targeting C128 attenuated the catalytic ability of FBPase. Herein, ten approved cysteine covalent drugs were selected for exploring FBPase inhibitory activities, and the alcohol deterrent disulfiram displayed superior inhibitory efficacy among those drugs. Based on the structure of lead compound disulfiram, 58 disulfide-derived compounds were designed and synthesized for investigating FBPase inhibitory activities. Optimal compound 3a exhibited significant FBPase inhibition and glucose-lowering efficacy in vitro and in vivo. Furthermore, 3a covalently modified the C128 site, and then regulated the N125-S124-S123 allosteric pathway of FBPase in mechanism. In summary, 3a has the potential to be a novel FBPase inhibitor for T2D therapy.

Identification of the New Covalent Allosteric Binding Site of Fructose-1,6-bisphosphatase with Disulfiram Derivatives toward Glucose Reduction.

Fructose 1,6-bisphosphatase (FBPase) has attracted substantial interest as a target associated with cancer and type 2 diabetes. Herein, we found that disulfiram and its derivatives can potently inhibit FBPase by covalently binding to a new C128 allosteric site distinct from the original C128 site in APO FBPase. Further identification of the allosteric inhibition mechanism reveals that the covalent binding of a fragment of 214 will result in the movement of C128 and the dissociation of helix H4 (123-128), which in turn allows S123 to more easily form new hydrogen bonds with K71 and D74 in helix H3 (69-72), thereby inhibiting FBPase activity. Notably, both disulfiram and 212 might moderately reduce blood glucose output in vivo. Therefore, our current findings not only identify a new covalent allosteric site of FBPase but also establish a structural foundation and provide a promising way for the design of covalent allosteric drugs for glucose reduction.

Cov_FB3D: A De Novo Covalent Drug Design Protocol Integrating the BA-SAMP Strategy and Machine-Learning-Based Synthetic Tractability Evaluation.

De novo drug design actively seeks to use sets of chemical rules for the fast and efficient identification of structurally new chemotypes with the desired set of biological properties. Fragment-based de novo design tools have been successfully applied in the discovery of noncovalent inhibitors. Nevertheless, these tools are rarely applied in the field of covalent inhibitor design. Herein, we present a new protocol, called Cov_FB3D, which involves the in silico assembly of potential novel covalent inhibitors by identifying the active fragments in the covalently binding site of the target protein. In this protocol, we propose a BA-SAMP strategy, which combines the noncovalent moiety score with the X-Score as the molecular mechanism (MM) level, and the covalent candidate score with the PM7 as the QM level. The synthetic accessibility of each suggested compound could be further evaluated with machine-learning-based synthetic complexity evaluation (SCScore). An in-depth test of this protocol against the crystal structures of 15 covalent complexes consisting of BTK inhibitors, KRAS inhibitors, EGFR inhibitors, EphB1 inhibitors, MAGL inhibitors, and MAPK inhibitors revealed that most of these inhibitors could be de novo reproduced from the fragments by Cov_FB3D. The binding modes of most generated reference poses could accurately reproduce the known binding mode of most of the reference covalent adduct in the binding site (RMSD ≤ 2 Å). In particular, most of these inhibitors were ranked in the top 2%, using the BA-SAMP strategy. Notably, the novel human ALDOA inhibitor (T1) with potent inhibitory activity (0.34 ± 0.03 µM) and greater synthetic accessibility was successfully de novo designed by this protocol. The positive results confirm the abilities of Cov_FB3D protocol.

Discovery of novel allosteric site and covalent inhibitors of FBPase with potent hypoglycemic effects.

Fructose-1,6-bisphosphatase (FBPase) is an essential enzyme of GNG pathway. Significant advances demonstrate the FBPase plays a critical role in treatment of diabetes. Numerous FBPase inhibitors were developed by targeting AMP site, nevertheless, none of these inhibitors has exhibited suitable potency and druggability. Herein, a new allosteric site (C128) on FBPase was discovered, and several nitrostyrene compounds exhibiting potent FBPase inhibitions were found covalently bind to C128 site on FBPase. Mutagenesis suggest that C128 is the only cysteine that can influence FBPase inhibition, the N125-S124-S123 pathway was most likely involved in allosteric signaling transmission between C128 and active site. However, these nitrostyrenes may bind with multiple cysteine besides C128 in FBPase. To improve pocket selectivity, a series of novel compounds (14a-14n) were re-designed rationally by integrating fragment-based covalent virtual screening and machine-learning-based synthetic complexity evaluation. As expected, the mass spectrometry validated that the proportion of title compounds binding to the C128 in FBPase was significantly higher than that of nitrostyrenes. Notably, under physiological and pathological conditions, the treatment of compounds 14b, 14c, 14i or 14n led to potent inhibition of glucose production, as well as decreased triglyceride and total cholesterol levels in mouse primary hepatocytes. We highlight a novel paradigm that molecular targeting C128 site on FBPase can have potent hypoglycemic effect.

In silico screening of a novel scaffold for fructose-1,6-bisphosatase (FBPase) inhibitors.

Fructose-1, 6-bisphosphatase (FBPase) has been regarded as an attractive drug target to control blood glucose against Type 2 diabetes (T2D). In this study, by using the strategy of pharmacophore-based virtual screening, a novel scaffold inhibitor targeted the AMP allosteric site of human liver FBPase were screened, their inhibitory activities were further tested. The experimental results showed that compound H27 exhibited high inhibitory activities with the IC50 value of 5.3 µM. Therefore, compound H27 was chosen as the probe molecule, it's possible binding conformation targeted into FBPase was identified by using DOX2.0 strategy. The importance of key residues (T27, T31, K112 and R140) in allosteric site of FBPase for the binding inhibitors were validated by mutation experiments. The agreement between theory and experiment suggest that the interactional information of FBPase and inhibitors (H27) were reliable. On basis of these rational interactional information, the compound H29 was further designed to exhibit more potential FBPase inhibition (IC50 = 2.5 µM).

New insight into the binding modes of TNP-AMP to human liver fructose-1,6-bisphosphatase.

Human liver fructose-1,6-bisphosphatase (FBPase) contains two binding sites, a substrate fructose-1,6-bisphosphate (FBP) active site and an adenosine monophosphate (AMP) allosteric site. The FBP active site works by stabilizing the FBPase, and the allosteric site impairs the activity of FBPase through its binding of a nonsubstrate molecule. The fluorescent AMP analogue, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) has been used as a fluorescent probe as it is able to competitively inhibit AMP binding to the AMP allosteric site and, therefore, could be used for exploring the binding modes of inhibitors targeted on the allosteric site. In this study, we have re-examined the binding modes of TNP-AMP to FBPase. However, our present enzyme kinetic assays show that AMP and FBP both can reduce the fluorescence from the bound TNP-AMP through competition for FBPase, suggesting that TNP-AMP binds not only to the AMP allosteric site but also to the FBP active site. Mutagenesis assays of K274L (located in the FBP active site) show that the residue K274 is very important for TNP-AMP to bind to the active site of FBPase. The results further prove that TNP-AMP is able to bind individually to the both sites. Our present study provides a new insight into the binding mechanism of TNP-AMP to the FBPase. The TNP-AMP fluorescent probe can be used to exam the binding site of an inhibitor (the active site or the allosteric site) using FBPase saturated by AMP and FBP, respectively, or the K247L mutant FBPase.