In present study, we set out to investigate the conformation dynamics of Aβ40 and Aβ42 through exploring the impact of intra-molecular interactions on conformation dynamics using equilibrium molecular dynamics simulations. Our 40 microsecond-scale simulations reveal heterogeneous conformation ensembles of Aβ40 and Aβ42 that encompass ~35% β-strand and ~60% unstructured coils. Two conformational states were identified in both alloforms: a collapsed state (CS) that resembles the structural motif of face-to-face hydrophobic clustering in amyloid fibrils, and an extended state (ES) that features the structural characteristics of anti-parallel β-sheets in amyloid oligomers. In Aβ40, the C-terminus remains unstructured and rarely interacts with other parts, thereof the hydrophobic clustering is in loose contact and the peptide assumes ES with high probability. In contrast, the C-terminus of Aβ42 adopts a β-strand structure that strongly interacts with segments E3-R5 and V18-A21. The active association leads to a more compact hydrophobic collapse and refrain the alloform from ES. Based on the structural characterization, we propose that the fibril and oligomer assembly pathways could respectively take off from CS and ES, and their aggregation propensity may be governed by the probability of visiting the corresponding conformational states at the equilibrium.

The nicotinic acetylcholine receptor (nAChR) is a key molecule involved in the propagation of signals in the central nervous system and peripheral synapses. Although numerous computational and experimental studies have been performed on this receptor, the structural dynamics of the receptor underlying the gating mechanism is still unclear. To address the mechanical fundamentals of nAChR gating, both conventional molecular dynamics (CMD) and steered rotation molecular dynamics (SRMD) simulations have been conducted on the cryo-electron microscopy (cryo-EM) structure of nAChR embedded in a dipalmitoylphosphatidylcholine (DPPC) bilayer and water molecules. A 30-ns CMD simulation revealed a collective motion amongst C-loops, M1, and M2 helices. The inward movement of C-loops accompanying the shrinking of acetylcholine (ACh) binding pockets induced an inward and upward motion of the outer beta-sheet composed of beta9 and beta10 strands, which in turn causes M1 and M2 to undergo anticlockwise motions around the pore axis. Rotational motion of the entire receptor around the pore axis and twisting motions among extracellular (EC), transmembrane (TM), and intracellular MA domains were also detected by the CMD simulation. Moreover, M2 helices undergo a local twisting motion synthesized by their bending vibration and rotation. The hinge of either twisting motion or bending vibration is located at the middle of M2, possibly the gate of the receptor. A complementary twisting-to-open motion throughout the receptor was detected by a normal mode analysis (NMA). To mimic the pulsive action of ACh binding, nonequilibrium MD simulations were performed by using the SRMD method developed in one of our laboratories. The result confirmed all the motions derived from the CMD simulation and NMA. In addition, the SRMD simulation indicated that the channel may undergo an open-close (O <--> C) motion. The present MD simulations explore the structural dynamics of the receptor under its gating process and provide a new insight into the gating mechanism of nAChR at the atomic level.

The human formyl peptide receptor 2 (FPR2) plays a crucial role in host defense and inflammation, and has been considered as a drug target for chronic inflammatory diseases. A variety of peptides with different structures and origins have been characterized as FPR2 ligands. However, the ligand-binding modes of FPR2 remain elusive, thereby limiting the development of potential drugs. Here we report the crystal structure of FPR2 bound to the potent peptide agonist WKYMVm at 2.8 Å resolution. The structure adopts an active conformation and exhibits a deep ligand-binding pocket. Combined with mutagenesis, ligand binding and signaling studies, key interactions between the agonist and FPR2 that govern ligand recognition and receptor activation are identified. Furthermore, molecular docking and functional assays reveal key factors that may define binding affinity and agonist potency of formyl peptides. These findings deepen our understanding about ligand recognition and selectivity mechanisms of the formyl peptide receptor family.

Acute myeloid leukemia is a disorder characterized by abnormal differentiation of myeloid cells and a clonal proliferation derived from primitive hematopoietic stem cells. Interventions that overcome myeloid differentiation have been shown to be a promising therapeutic strategy for acute myeloid leukemia. In this study, we demonstrate that CRISPR/Cas9-mediated knockout of dihydroorotate dehydrogenase leads to apoptosis and normal differentiation of acute myeloid leukemia cells, indicating that dihydroorotate dehydrogenase is a potential differentiation regulator and a therapeutic target in acute myeloid leukemia. By screening a library of natural products, we identified a novel dihydroorotate dehydrogenase inhibitor, isobavachalcone, derived from the traditional Chinese medicine Psoralea corylifolia Using enzymatic analysis, thermal shift assay, pull down, nuclear magnetic resonance, and isothermal titration calorimetry experiments, we demonstrate that isobavachalcone inhibits human dihydroorotate dehydrogenase directly, and triggers apoptosis and differentiation of acute myeloid leukemia cells. Oral administration of isobavachalcone suppresses subcutaneous HL60 xenograft tumor growth without obvious toxicity. Importantly, our results suggest that a combination of isobavachalcone and adriamycin prolonged survival in an intravenous HL60 leukemia model. In summary, this study demonstrates that isobavachalcone triggers apoptosis and differentiation of acute myeloid leukemia cells via pharmacological inhibition of human dihydroorotate dehydrogenase, offering a potential therapeutic strategy for acute myeloid leukemia.

Targeting the interaction between the spike protein receptor binding domain (S-RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and angiotensin-converting enzyme 2 (ACE2) is a potential therapeutic strategy for treating coronavirus disease 2019 (COVID-19). However, we still lack small-molecule drug candidates for this target due to the missing knowledge in the hot spots for the protein-protein interaction. Here, we used NanoBiT technology to identify three Ginkgolic acids from an in-house traditional Chinese medicine (TCM) library, and they interfere with the S-RBD/ACE2 interplay. Our pseudovirus assay showed that one of the compounds, Ginkgolic acid C17:1 (GA171), significantly inhibits the entry of original SARS-CoV-2 and its variants into the ACE2-overexpressed HEK293T cells. We investigated and proposed the binding sites of GA171 on S-RBD by combining molecular docking and molecular dynamics simulations. Site-directed mutagenesis and surface plasmon resonance revealed that GA171 specifically binds to the pocket near R403 and Y505, critical residues of S-RBD for S-RBD interacting with ACE2. Thus, we provide structural insights into developing new small-molecule inhibitors and vaccines against the proposed S-RBD binding site.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has forced the development of direct-acting antiviral drugs due to the coronavirus disease 2019 (COVID-19) pandemic. The main protease of SARS-CoV-2 is a crucial enzyme that breaks down polyproteins synthesized from the viral RNA, making it a validated target for the development of SARS-CoV-2 therapeutics. New chemical phenotypes are frequently discovered in natural goods. In the current study, we used a fluorogenic assay to test a variety of natural products for their ability to inhibit SARS-CoV-2 Mpro. Several compounds were discovered to inhibit Mpro at low micromolar concentrations. It was possible to crystallize robinetin together with SARS-CoV-2 Mpro, and the X-ray structure revealed covalent interaction with the protease's catalytic Cys145 site. Selected potent molecules also exhibited antiviral properties without cytotoxicity. Some of these powerful inhibitors might be utilized as lead compounds for future COVID-19 research.

Effective drugs with broad spectrum safety profile to all people are highly expected to combat COVID-19 caused by SARS-CoV-2. Here we report that nelfinavir, an FDA approved drug for the treatment of HIV infection, is effective against SARS-CoV-2 and COVID-19. Preincubation of nelfinavir could inhibit the activity of the main protease of the SARS-CoV-2 (IC50 = 8.26 μM), while its antiviral activity in Vero E6 cells against a clinical isolate of SARS-CoV-2 was determined to be 2.93 μM (EC50). In comparison with vehicle-treated animals, rhesus macaque prophylactically treated with nelfinavir had significantly lower temperature and significantly reduced virus loads in the nasal and anal swabs of the animals. At necropsy, nelfinavir-treated animals had a significant reduction of the viral replication in the lungs by nearly three orders of magnitude. A prospective clinic study with 37 enrolled treatment-naive patients at Shanghai Public Health Clinical Center, which were randomized (1:1) to nelfinavir and control groups, showed that the nelfinavir treatment could shorten the duration of viral shedding by 5.5 days (9.0 vs. 14.5 days, P = 0.055) and the duration of fever time by 3.8 days (2.8 vs. 6.6 days, P = 0.014) in mild/moderate COVID-19 patients. The antiviral efficiency and clinical benefits in rhesus macaque model and in COVID-19 patients, together with its well-established good safety profile in almost all ages and during pregnancy, indicated that nelfinavir is a highly promising medication with the potential of preventative effect for the treatment of COVID-19.

A series of peptidomimetic compounds containing benzothiazolyl ketone and [2.2.1] azabicyclic ring was designed, synthesized and evaluated in the hope of obtaining potent oral 3CLpro inhibitors with improved pharmacokinetic properties. Among the target compounds, 11b had the best enzymatic potency (IC50 = 0.110 μM) and 11e had the best microsomal stability (t1/2 > 120 min) and good enzyme activity (IC50 = 0.868 μM). Therefore, compounds 11b and 11e were chosen for further evaluation of pharmacokinetics in ICR mice. The results exhibited that the AUC(0-t) of 11e was 5143 h*ng/mL following single-dose oral administration of 20 mg/kg, and the F was 67.98%. Further structural modification was made to obtain compounds 11g-11j based on 11e. Among them, 11j exhibited the best enzyme inhibition activity against SARS-CoV-2 3CLpro (IC50 = 1.646 μM), the AUC(0-t) was 32473 h*ng/mL (20 mg/kg, po), and the F was 48.1%. In addition, 11j displayed significant anti-SARS-CoV-2 activity (EC50 = 0.18 μM) and low cytotoxicity (CC50 > 50 μM) in Vero E6 cells. All of the above results suggested that compound 11j was a promising lead compound in the development of oral 3CLpro inhibitors and deserved further research.

The signal transduction of acetylated histone can be processed through a recognition module, bromodomain. Several inhibitors targeting BRD4, one of the bromodomain members, are in clinical trials as anticancer drugs. Hereby, we report our efforts on discovery and optimization of a new series of 2-thiazolidinones as BRD4 inhibitors along our previous study. In this work, guided by crystal structure analysis, we reversed the sulfonamide group and identified a new binding mode. A structure-activity relationship study on this new series led to several potent BRD4 inhibitors with IC50 of about 0.05-0.1 μM in FP binding assay and GI50 of 0.1-0.3 μM in cell based assays. To complete the lead-like assessment of this series, we further checked its effects on BRD4 downstream protein c-Myc, investigated its selectivity among five different bromodomain proteins, as well as the metabolic stability test, and reinforced the utility of 2-thiazolidinone scaffold as BET bromodomain inhibitors in novel anticancer drug development.

The c-Met kinase has emerged as an attractive target for developing antitumor agents because of its close relationship with the development of many human cancers, poor clinical outcomes and even drug resistance. A series of novel c-Met kinase inhibitors have been identified with multiple workflow in this work, including virtual screening, X-ray crystallography, biological evaluation and structural optimization. The experimentally determined crystal structure of the best hit compound HL-11 in c-Met kinase domain was highly consistent with the computational prediction. Comparison of the hit compounds with different c-Met kinase inhibitory activity by molecular dynamics simulations suggested the key protein-ligand interactions for structural optimization. Based on these, structural optimization produced compound 11e with better c-Met kinase inhibitory activity and improved anti-proliferative activity. These experimental findings proved the reliability and efficiency of our in silico methods. This strategy will facilitate further lead discovery and optimization for novel c-Met kinase inhibitors.

Previous researches suggested that polysaccharides from brown algae had anti-virus activity. We hypothesized that nature polysaccharide from marine plants might have the effect on anti-SARS-CoV-2 activity. By high throughput screening to target 3CLpro enzyme using polysaccharides library, we discover a crude polysaccharide 375 from seaweed Ecklonia kurome blocked 3CLpro enzymatic activity and shows good anti-SARS-CoV-2 infection activity in cell. Further, we show that homogeneous polysaccharide 37502 from the 375 may bind to 3CLpro well and disturb spike protein binding to ACE2 receptor. The structure characterization uncovers that 37502 is alginate. These results imply that the bioactivities of 375 on SARS-CoV-2 may target multiple key molecules implicated in the virus infection and replication. The above results suggest that 375 may be a potential drug candidate against SARS-CoV-2.

FB2001 is a drug candidate that targets the main protease of SARS-CoV-2 via covalently binding to cysteine 145. In this study, we evaluated the inhibitory activities of FB2001 against several SARS-CoV-2 variants in vitro and in vivo (in mice), and we also evaluated the histopathological analysis and immunostaining of FB2001 on lung and brain which have been rarely reported. The results showed that FB2001 exhibited potent antiviral efficacy against several current SARS-CoV-2 variants in Vero E6 cells, namely, B.1.1.7 (Alpha): EC50 = 0.39 ± 0.01 μM, EC90 = 0.75 ± 0.01 μM; B.1.351 (Beta): EC50 = 0.28 ± 0.11 μM, EC90 = 0.57 ± 0.21 μM; B.1.617.2 (Delta): EC50 = 0.27 ± 0.05 μM, EC90 = 0.81 ± 0.20 μM; B.1.1.529 (Omicron): EC50 = 0.26 ± 0.06 μM and EC50 = 0.042 ± 0.007 μM (in the presence of a P-glycoprotein inhibitor). FB2001 remained potent against SARS-CoV-2 replication in the presence of high concentrations of human serum, which indicating that human serum had no significant effect on the in vitro inhibitory activity. Additionally, this inhibitor exhibited an additive effect against SARS-CoV-2 when combined with Remdesivir. Furthermore, FB2001 significantly reduced the SARS-CoV-2 copy numbers and titers in the lungs and brains in vivo, and alleviated the pathological symptoms. In addition, FB2001 could alleviated local bleeding, erythrocyte overflow, edema, and inflammatory cell infiltration in brain tissue, and inhibitors reducing viral titers and improving inflammation in the brain have been rarely reported. A physiologically based pharmacokinetic model was established and verified to predict the FB2001 concentration in human lungs. When FB2001 was administered at 200 mg twice a day for 5 days, the observed Ctrough ss in plasma and predicted Ctrough ss of lung total concentration were 0.163 and 2.5 μg/mL, which were approximately 9 and 132-fold higher than the EC50 of 0.019 μg/mL (0.042 μM) against Omicron variant. Taken together, our study suggests that FB2001 is a promising therapeutic agent in COVID-19 treatment and can be combined with remdesivir to achieve improved clinical outcomes. Owing to its good safety and tolerability in healthy human (NCT05197179 and NCT04766931), FB2001 has been approved for Phase II/III clinical trial (NCT05445934).

The 3C-like protease (3CLpro) is essential for the replication and transcription of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), making it a promising target for the treatment of corona virus disease 2019 (COVID-19). In this study, a series of 2,3,5-substituted [1,2,4]-thiadiazole analogs were discovered to be able to inhibit 3CLpro as non-peptidomimetic covalent binders at submicromolar levels, with IC50 values ranging from 0.118 to 0.582 μM. Interestingly, these compounds were also shown to inhibit PLpro with the same level of IC50 values, but had negligible effect on proteases such as chymotrypsin, cathepsin B, and cathepsin L. Subsequently, the antiviral abilities of these compounds were evaluated in cell-based assays, and compound 6g showed potent antiviral activity with an EC50 value of 7.249 μM. It was proposed that these compounds covalently bind to the catalytic cysteine 145 via a ring-opening metathesis reaction mechanism. To understand this covalent-binding reaction, we chose compound 6a, one of the identified hit compounds, as a representative to investigate the reaction mechanism in detail by combing several computational predictions and experimental validation. The process of ring-opening metathesis was theoretically studied using quantum chemistry calculations according to the transition state theory. Our study revealed that the 2,3,5-substituted [1,2,4]-thiadiazole group could covalently modify the catalytic cysteine in the binding pocket of 3CLpro as a potential warhead. Moreover, 6a was a known GPCR modulator, and our study is also a successful computational method-based drug-repurposing study.

The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (Mpro) and papain like protease (PLpro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851Mpro inhibitors and 205 PLpro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both Mpro and PLpro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of Mpro inhibitors and over 20% of PLpro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 Mpro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.

Heterodimerization of the metabotropic glutamate receptors (mGlus) has shown importance in the functional modulation of the receptors and offers potential drug targets for treating central nervous system diseases. However, due to a lack of molecular details of the mGlu heterodimers, understanding of the mechanisms underlying mGlu heterodimerization and activation is limited. Here we report twelve cryo-electron microscopy (cryo-EM) structures of the mGlu2-mGlu3 and mGlu2-mGlu4 heterodimers in different conformational states, including inactive, intermediate inactive, intermediate active and fully active conformations. These structures provide a full picture of conformational rearrangement of mGlu2-mGlu3 upon activation. The Venus flytrap domains undergo a sequential conformational change, while the transmembrane domains exhibit a substantial rearrangement from an inactive, symmetric dimer with diverse dimerization patterns to an active, asymmetric dimer in a conserved dimerization mode. Combined with functional data, these structures reveal that stability of the inactive conformations of the subunits and the subunit-G protein interaction pattern are determinants of asymmetric signal transduction of the heterodimers. Furthermore, a novel binding site for two mGlu4 positive allosteric modulators was observed in the asymmetric dimer interfaces of the mGlu2-mGlu4 heterodimer and mGlu4 homodimer, and may serve as a drug recognition site. These findings greatly extend our knowledge about signal transduction of the mGlus.

In this paper, a series of peptidomimetic SARS-CoV-2 3CL protease inhibitors with new P2 and P4 positions were synthesized and evaluated. Among these compounds, 1a and 2b exhibited obvious 3CLpro inhibitory activities with IC50 of 18.06 nM and 22.42 nM, respectively. 1a and 2b also showed excellent antiviral activities against SARS-CoV-2 in vitro with EC50 of 313.0 nM and 170.2 nM, respectively, the antiviral activities of 1a and 2b were 2- and 4-fold better than that of nirmatrelvir, respectively. In vitro studies revealed that these two compounds had no significant cytotoxicity. Further metabolic stability tests and pharmacokinetic studies showed that the metabolic stability of 1a and 2b in liver microsomes was significantly improved, and 2b had similar pharmacokinetic parameters to that of nirmatrelvir in mice.

The persistent pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants accentuates the great demand for developing effective therapeutic agents. Here, we report the development of an orally bioavailable SARS-CoV-2 3C-like protease (3CLpro) inhibitor, namely simnotrelvir, and its preclinical evaluation, which lay the foundation for clinical trials studies as well as the conditional approval of simnotrelvir in combination with ritonavir for the treatment of COVID-19. The structure-based optimization of boceprevir, an approved HCV protease inhibitor, leads to identification of simnotrelvir that covalently inhibits SARS-CoV-2 3CLpro with an enthalpy-driven thermodynamic binding signature. Multiple enzymatic assays reveal that simnotrelvir is a potent pan-CoV 3CLpro inhibitor but has high selectivity. It effectively blocks replications of SARS-CoV-2 variants in cell-based assays and exhibits good pharmacokinetic and safety profiles in male and female rats and monkeys, leading to robust oral efficacy in a male mouse model of SARS-CoV-2 Delta infection in which it not only significantly reduces lung viral loads but also eliminates the virus from brains. The discovery of simnotrelvir thereby highlights the utility of structure-based development of marked protease inhibitors for providing a small molecule therapeutic effectively combatting human coronaviruses.

Previously, from the tannic sumac plant (Rhus coriaria), we developed the Rutan 25 mg oral drug tablets with antiviral activity against influenza A and B viruses, adenoviruses, paramyxoviruses, herpes virus, and cytomegalovirus. Here, our re-purposing study demonstrated that Rutan at 25, 50, and 100 mg/kg provided a very effective and safe treatment for COVID-19 infection, simultaneously inhibiting two vital enzyme systems of the SARS-CoV-2 virus: 3C-like proteinase (3CLpro) and RNA-dependent RNA polymerase (RdRp). There was no drug accumulation in experimental animals' organs and tissues. A clinical study demonstrated a statistically significant decrease in the C-reactive protein and a reduction of the viremia period. In patients receiving Rutan 25 mg (children) and 100 mg (adults), the frequency of post-COVID-19 manifestations was significantly less than in the control groups not treated with Rutan tablets. Rutan, having antiviral activity, can provide safe treatment and prevention of COVID-19 in adults and children.

Bacterial porins are water-filled β-barrel channels that allow translocation of solutes across the outer membrane. They feature a constriction zone, contributed by the plunging of extracellular loop 3 (L3) into the channel lumen. Porins are generally in the open state, but undergo gating in response to external voltages. To date the underlying mechanism is unclear. Here we report results from molecular dynamics simulations on the two porins of Providenica stuartii, Omp-Pst1 and Omp-Pst2, which display distinct voltage sensitivities. Voltage gating was observed in Omp-Pst2, where the binding of cations in-between L3 and the barrel wall results in exposing a conserved aromatic residue in the channel lumen, thereby halting ion permeation. Comparison of Omp-Pst1 and Omp-Pst2 structures and trajectories suggests that their sensitivity to voltage is encoded in the hydrogen-bonding network anchoring L3 onto the barrel wall, as we observed that it is the strength of this network that governs the probability of cations binding behind L3. That Omp-Pst2 gating is observed only when ions flow against the electrostatic potential gradient of the channel furthermore suggests a possible role for this porin in the regulation of charge distribution across the outer membrane and bacterial homeostasis.

Fatty acid binding protein 4 (FABP4) is a potential drug target for diabetes and atherosclerosis. For discovering new chemical entities as FABP4 inhibitors, structure-based virtual screening (VS) was performed, bioassay demonstrated that 16 of 251 tested compounds are FABP4 inhibitors, among which compound m1 are more active than endogenous ligand linoleic acid (LA). Based on the structure of m1, new derivatives were designed and prepared, leading to the discovery of two more potent inhibitors, compounds 9 and 10. To further explore the binding mechanisms of these new inhibitors, we determined the X-ray structures of the complexes of FABP4-9 and FABP4-10, which revealed similar binding conformations of the two compounds. Residue Ser53 and Arg126 formed direct hydrogen bonding with the ligands. We also found that 10 could significantly reduce the levels of lipolysis on mouse 3T3-L1 adipocytes. Taken together, in silico, in vitro and crystallographic data provide useful hints for future development of novel inhibitors against FABP4.