Myricetin allosterically inhibits Dengue NS2B-NS3 protease as studied by NMR and MD simulations

Dengue NS2B-NS3 protease existing in equilibrium between the active and inactive forms is essential for virus replication, thus representing a key drug target. Here Myricetin, a plant flavonoid, was characterized to non-competitively inhibit Dengue protease. Further NMR study identified the protease residues perturbed by binding to Myricetin, which were utilized to construct the Myricetin-protease complexes. Strikingly, in the active form Myricetin binds a new allosteric site (AS2) far away from the active site pocket and allosteric site (AS1) for binding Curcumin, while in the inactive form it binds both AS1 and AS2. To decipher the mechanism for the allosteric inhibition by Myricetin, we conducted molecular dynamics (MD) simulations on different forms of Dengue NS2B-NS3 protease. Unexpectedly, the binding of Myricetin to AS2 is sufficient to disrupt the active conformation by displacing the characteristic NS2B C-terminal b- hairpin from the active site pocket. By contrast, the binding of Myricetin to AS1 and AS2 results in locking the inactive conformation. Therefore Myricetin represents the first small molecule which allosterically inhibits Dengue protease by both disrupting the active conformation and locking the inactive conformation. The results enforce the notion that a global allosteric network exists in Dengue NS2B-NS3 protease, which is susceptible to allosteric inhibition by small molecules such as Myricetin and Curcumin. As Myricetin has been extensively used as a food additive, it might be directly utilized to fight the Dengue infections and as a promising starting for further design of potent allosteric inhibitors.

Human RIPK3 maintains MLKL in an inactive conformation prior to cell death by necroptosis

The ancestral origins of the lytic cell death mode, necroptosis, lie in host defense. However, the dysregulation of necroptosis in inflammatory diseases has led to widespread interest in targeting the pathway therapeutically. This mode of cell death is executed by the terminal effector, the MLKL pseudokinase, which is licensed to kill following phosphorylation by its upstream regulator, RIPK3 kinase. The precise molecular details underlying MLKL activation are still emerging and, intriguingly, appear to mechanistically-diverge between species. Here, we report the structure of the human RIPK3 kinase domain alone and in complex with the MLKL pseudokinase. These structures reveal how human RIPK3 structurally differs from its mouse counterpart, and how human RIPK3 maintains MLKL in an inactive conformation prior to induction of necroptosis. Residues within the RIPK3:MLKL C-lobe interface are crucial to complex assembly and necroptotic signaling in human cells, thereby rationalizing the strict species specificity governing RIPK3 activation of MLKL.

Differences in the dynamics of the tandem-SH2 modules of the Syk and ZAP-70 tyrosine kinases

The catalytic activity of Syk-family tyrosine kinases is regulated by a tandem-SH2 module (tSH2 module). In the autoinhibited state, this module adopts a conformation which stabilizes an inactive conformation of the kinase domain. The binding of the tSH2 module to doubly-phosphorylated tyrosine-containing motifs necessitates a conformational change, thereby relieving kinase inhibition and promoting activation. We determined the crystal structure of the isolated tSH2 module of Syk and find, in contrast to ZAP-70, that its conformation more closely resembles that of the peptide-bound state, rather than the autoinhibited state. Hydrogen-deuterium exchange by mass spectrometry, as well as molecular dynamics simulations, reveal that the dynamics of the tSH2 modules of Syk and ZAP-70 differ, with most of these differences occurring in the C-terminal SH2 domain. Our data suggest that the conformational landscapes of the tSH2 modules in Syk and ZAP-70 have been tuned differently, such that the auto-inhibited conformation of the Syk tSH2 module is less stable. This feature of Syk likely contributes to its ability to more readily escape autoinhibition when compared to ZAP-70, consistent with tighter control of downstream signaling pathways in T cells.

Permeant fluorescent probes visualize the activation of SARM1 and uncover an anti-neurodegenerative drug candidate

SARM1 regulates axonal degeneration through its NAD-metabolizing activity and is a drug target for neurodegenerative disorders. We designed and synthesized fluorescent conjugates of styryl derivative with pyridine to serve as substrates of SARM1, which exhibited large red-shifts after conversion. With the conjugates, SARM1 activation was visualized in live cells following elevation of endogenous NMN or treatment with a cell-permeant NMN-analog. In neurons, imaging documented mouse SARM1 activation preceded vincristine-induced axonal degeneration by hours. Library screening identified a derivative of nisoldipine as a covalent inhibitor of SARM1 that reacted with the cysteines, especially Cys311 in its ARM domain and blocked its NMN-activation, protecting axons from degeneration. The Cryo-EM structure showed that SARM1 was locked into an inactive conformation by the inhibitor, uncovering a potential neuroprotective mechanism of dihydropyridines.

Permeant Fluorescent Probes Visualize the Activation of SARM1 and Uncover an Anti-neurodegenerative Drug Candidate

SARM1 regulates axonal degeneration through its NAD-metabolizing activity and is a drug target for neurodegenerative disorders. We designed and synthesized fluorescent conjugates of styryl derivative with pyridine to serve as substrates of SARM1, which exhibited large red-shifts after conversion. With the conjugates, SARM1 activation was visualized in live cells following elevation of endogenous NMN or treatment with a cell-permeant NMN-analog. In neurons, imaging documented SARM1 activation preceded vincristine-induced axonal degeneration by hours. Library screening identified a derivative of nisoldipine as a covalent inhibitor of SARM1 that reacted with Cys311 in its Armadillo-domain and blocked its NMN-activation, protecting axons from degeneration. CryoEM showed that SARM1 was locked into an inactive conformation by the inhibitor, uncovering an unsuspected neuroprotective mechanism of dihydropyridines.

In silico analysis of antihypertensive and hepatotoxicity potential of the n-butanol fraction of the methanol extract of of cantaloupe (Cucumis melo var. cantalupensis)

Latar belakang: Hipertensi merupakan faktor risiko utama penyakit kardiovaskular. Penduduk Indonesia cenderung mengkonsumsi herbal dalam terapi hipertensi dalam mempertahankan kadar tekanan darah seperti buah blewah (Cucumis melo var. cantalupensis). Namun mekanisme kerja buah blewah dalam menurunkan tekanan darah, dan potensi toksisitasnya jika dikonsumsi dalam jangka panjang masih belum jelas. Tujuan studi ini adalah untuk menganalisis mekanisme antihipertensi dari buah blewah dan potensi toksiknya melalui pendekatan in silico. Metode: Bubuk blewah kering dimaserasi menggunakan metanol absolut, difraksinasi menggunakan n-butanol. Uji fitokimia dilakukan dengan metode LC-MS, kemudian senyawa bioaktif ditelusuri hingga SMILESnya di PubChem. Analisis QSAR untuk analisis potensi antihipertensi dilakukan dengan PASS server. Kelas toksisitas dan potensi hepatotoksisitas dianalisis menggunakan ProTox-II, dilanjutkan dengan analisis networking menggunakan STITCH dan STRINGdb. Hasil: Setidaknya terdapat 434 jenis senyawa yang terdapat pada fraksi n-butanol dari ekstrak metanol buah blewah (FBEMB). Berdasarkan analisis STITCH dan STRINGdb, FBEMB dapat bekerja dalam menurunkan tekanan darah melalui mekanisme aksi seperti senyawa amlodipine, yang menstabilkan saluran kalsium tipe-L yang terisi tegangan dalam konformasi tidak aktifnya. Dengan demikian, mencegah kontraksi myocyte yang bergantung pada kalsium dan vasokonstriksi. FBEMB mungkin berpotensi hepatotoksik melalui mekanisme kerja senyawa seperti itrakonazol yang menghambat enzim sitokrom P450 yang mempengaruhi gangguan pada sintesis ergosterol, dan efavirenz yang memiliki efek neurotoksik. Penghambatan sitokrom P450 dapat menyebabkan toksisitas obat dan kerusakan hati. Kesimpulan: FBEMB dapat bekerja dalam menurunkan tekanan darah melalui mekanisme penstabilan saluran kalsium tipe-L yang terisi tegangan dalam konformasi tidak aktifnya. Kata kunci: in silico, antihipertensi, hepatotoksisitas, blewah Abstract Background: Hypertension is a major cardiovascular disease risk factor. Indonesian people tend to consume herbal medicine to maintain hypertension therapy, i.e cantaloupe (Cucumis melo var. cantalupensis). However, the mechanism of action of cantaloupe in lowering blood pressure and toxicity potential for long term consumption is unclear. The study aimed to analyze the antihypertensive mechanism of cantaloupe and its toxic potential through the in silico. Methods: The dried cantaloupe powder was macerated using absolute methanol, then fractionated using n-butanol. The phytochemical test was done by LC-MS method, then the bioactive compounds were traced to their SMILES in the PubChem. The QSAR analysis of the antihypertensive potential was done using the PASS server. The toxicity class and hepatotoxicity potential were analyzed using ProTox-II, followed by networking analysis using STITCH and STRINGdb. Results: At least 434 types of compounds were shown in the n-butanol fraction of the methanol extract of cantaloupe (BFMEC). Based on the networking analysis, BFMEC may work in lowering blood pressure through the action mechanism of the amlodipine compound-like, which stabilizes voltage-gated L-type calcium channels in an inactive conformation, thus, prevents calcium-dependent myocyte contraction and vasoconstriction. BFMEC presumably has hepatotoxic through the action mechanism of itraconazole compound-like inhibited cytochrome P450-dependent enzymes, affecting the impairment of ergosterol synthesis, and efavirenz which has neurotoxic effects. The inhibition of cytochrome P450 may cause drug toxicity and liver damage. Conclusion: BFMEC may work in lowering blood pressure through the action mechanism which stabilizes voltage-gated L-type calcium channels in an inactive conformation. Keywords: in silico, antihypertensive, hepatotoxicity, cantaloupe

Structural basis for SARM1 inhibition and activation under energetic stress

SARM1, an executor of axonal degeneration, displays NADase activity that depletes the key cellular metabolite, NAD+, in response to nerve injury. The basis of SARM1 inhibition and its activation under stress conditions are still unknown. Here, we present cryo-EM maps of SARM1 at 2.9 and 2.7 Å resolutions. These indicate that SARM1 homo-octamer avoids premature activation by assuming a packed conformation, with ordered inner and peripheral rings, that prevents dimerization and activation of the catalytic domains. This inactive conformation is stabilized by binding of SARM1’s own substrate NAD+ in an allosteric location, away from the catalytic sites. This model was validated by mutagenesis of the allosteric site, which led to constitutively active SARM1. We propose that the reduction of cellular NAD+ concentration contributes to the disassembly of SARM1's peripheral ring, which allows formation of active NADase domain dimers, thereby further depleting NAD+ to cause an energetic catastrophe and cell death.

An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike

The SARS-CoV-2 virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryogenic electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.