ORPs are lipid-transport proteins belonging to the oxysterol-binding protein family. They facilitate the transfer of lipids between different intracellular membranes, such as the ER and plasma membrane. We have solved the crystal structure of the ORP8 lipid transport domain (ORD8). The ORD8 exhibited a β-barrel fold composed of anti-parallel β-strands, with three α-helices replacing β-strands on one side. This mixed alpha-beta structure was consistent with previously solved structures of ORP2 and ORP3. A large cavity (≈1860 Å3) within the barrel was identified as the lipid-binding site. Although we were not able to obtain a lipid-bound structure, we used computer simulations based on our crystal structure to dock PS and PI4P molecules into the putative lipid-binding site of the ORD8. Comparative experiments between the short ORD8ΔLid (used for crystallography) and the full-length ORD8 (lid containing) revealed the lid's importance for stable lipid binding. Fluorescence assays revealed different transport efficiencies for PS and PI4P, with the lid slowing down transport and stabilizing cargo. Coarse-grained simulations highlighted surface-exposed regions and hydrophobic interactions facilitating lipid bilayer insertion. These findings enhance our comprehension of ORD8, its structure, and lipid transport mechanisms, as well as provide a structural basis for the design of potential inhibitors.
Monkeypox, or mpox, is a disease that has recently resurfaced and spread across the globe. Despite the availability of an FDA-approved vaccine (JYNNEOS) and an effective drug (tecovirimat), concerns remain over the possible recurrence of a viral pandemic. Like any other virus, mpox virus must overcome the immune system to replicate. Viruses have evolved various strategies to overcome both innate and adaptive immunity. Poxviruses possess an unusual nuclease, poxin, which cleaves 2'-3'-cGAMP, a cyclic dinucleotide, which is an important second messenger in the cGAS-STING signaling pathway. Here, we present the crystal structure of mpox poxin. The structure reveals a conserved, predominantly β-sheet fold and highlights the high conservation of the cGAMP binding site and of the catalytic residues His17, Tyr138, and Lys142. This research suggests that poxin inhibitors could be effective against multiple poxviruses.
- MeSH
- lidé MeSH
- opičí neštovice * MeSH
- Poxviridae * MeSH
- racionální návrh léčiv MeSH
- signální transdukce MeSH
- virus opičích neštovic MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Monkeypox is a disease with pandemic potential. It is caused by the monkeypox virus (MPXV), a double-stranded DNA virus from the Poxviridae family, that replicates in the cytoplasm and must encode for its own RNA processing machinery including the capping machinery. Here, we present crystal structures of its 2'-O-RNA methyltransferase (MTase) VP39 in complex with the pan-MTase inhibitor sinefungin and a series of inhibitors that were discovered based on it. A comparison of this 2'-O-RNA MTase with enzymes from unrelated single-stranded RNA viruses (SARS-CoV-2 and Zika) reveals a conserved sinefungin binding mode, implicating that a single inhibitor could be used against unrelated viral families. Indeed, several of our inhibitors such as TO507 also inhibit the coronaviral nsp14 MTase.
- MeSH
- COVID-19 * MeSH
- infekce virem zika * MeSH
- lidé MeSH
- methyltransferasy metabolismus MeSH
- RNA virová genetika MeSH
- RNA MeSH
- SARS-CoV-2 genetika MeSH
- virové nestrukturální proteiny chemie MeSH
- virus opičích neštovic genetika metabolismus MeSH
- virus zika * genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Here, we present remarkable epoxyketone-based proteasome inhibitors with low nanomolar in vitro potency for blood-stage Plasmodium falciparum and low cytotoxicity for human cells. Our best compound has more than 2,000-fold greater selectivity for erythrocytic-stage P. falciparum over HepG2 and H460 cells, which is largely driven by the accommodation of the parasite proteasome for a D-amino acid in the P3 position and the preference for a difluorobenzyl group in the P1 position. We isolated the proteasome from P. falciparum cell extracts and determined that the best compound is 171-fold more potent at inhibiting the β5 subunit of P. falciparum proteasome when compared to the same subunit of the human constitutive proteasome. These compounds also significantly reduce parasitemia in a P. berghei mouse infection model and prolong survival of animals by an average of 6 days. The current epoxyketone inhibitors are ideal starting compounds for orally bioavailable anti-malarial drugs.
- MeSH
- antimalarika * farmakologie MeSH
- inhibitory proteasomu chemie MeSH
- lidé MeSH
- myši MeSH
- Plasmodium falciparum MeSH
- Plasmodium * MeSH
- proteasomový endopeptidasový komplex chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
SARS-CoV-2 nsp10-nsp16 complex is a 2'-O-methyltransferase (MTase) involved in viral RNA capping, enabling the virus to evade the immune system in humans. It has been considered a valuable target in the discovery of antiviral therapeutics, as the RNA cap formation is crucial for viral propagation. Through cross-screening of the inhibitors that we previously reported for SARS-CoV-2 nsp14 MTase activity against nsp10-nsp16 complex, we identified two compounds (SS148 and WZ16) that also inhibited nsp16 MTase activity. To further enable the chemical optimization of these two compounds towards more potent and selective dual nsp14/nsp16 MTase inhibitors, we determined the crystal structure of nsp10-nsp16 in complex with each of SS148 and WZ16. As expected, the structures revealed the binding of both compounds to S-adenosyl-L-methionine (SAM) binding pocket of nsp16. However, our structural data along with the biochemical mechanism of action determination revealed an RNA-dependent SAM-competitive pattern of inhibition for WZ16, clearly suggesting that binding of the RNA first may help the binding of some SAM competitive inhibitors. Both compounds also showed some degree of selectivity against human protein MTases, an indication of great potential for chemical optimization towards more potent and selective inhibitors of coronavirus MTases.
- MeSH
- COVID-19 * farmakoterapie MeSH
- lidé MeSH
- methyltransferasy chemie MeSH
- RNA virová metabolismus MeSH
- SARS-CoV-2 * MeSH
- virové nestrukturální proteiny chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cyclic dinucleotides (CDNs) are second messengers that activate stimulator of interferon genes (STING). The cGAS-STING pathway plays a promising role in cancer immunotherapy. Here, we describe the synthesis of CDNs containing 7-substituted 7-deazapurine moiety. We used mouse cyclic GMP-AMP synthase and bacterial dinucleotide synthases for the enzymatic synthesis of CDNs. Alternatively, 7-(het)aryl 7-deazapurine CDNs were prepared by Suzuki-Miyaura cross-couplings. New CDNs were tested in biochemical and cell-based assays for their affinity to human STING. Eight CDNs showed better activity than 2'3'-cGAMP, the natural ligand of STING. The effect on cytokine and chemokine induction was also evaluated. The best activities were observed for CDNs bearing large aromatic substituents that point above the CDN molecule. We solved four X-ray structures of complexes of new CDNs with human STING. We observed π-π stacking interactions between the aromatic substituents and Tyr240 that are involved in the stabilization of CDN-STING complexes.
- MeSH
- cytokiny MeSH
- interferony MeSH
- lidé MeSH
- ligandy MeSH
- membránové proteiny * metabolismus MeSH
- myši MeSH
- nukleotidy cyklické * chemie MeSH
- nukleotidyltransferasy MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Positive-sense single-stranded RNA (+RNA) viruses have proven to be important pathogens that are able to threaten and deeply damage modern societies, as illustrated by the ongoing COVID-19 pandemic. Therefore, compounds active against most or many +RNA viruses are urgently needed. Here, we present PR673, a helquat-like compound that is able to inhibit the replication of SARS-CoV-2 and tick-borne encephalitis virus in cell culture. Using in vitro polymerase assays, we demonstrate that PR673 inhibits RNA synthesis by viral RNA-dependent RNA polymerases (RdRps). Our results illustrate that the development of broad-spectrum non-nucleoside inhibitors of RdRps is feasible.
- MeSH
- COVID-19 * MeSH
- lidé MeSH
- pandemie MeSH
- RNA-dependentní RNA-polymerasa MeSH
- SARS-CoV-2 MeSH
- viry klíšťové encefalitidy * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Covalent DNA interstrand crosslinks are toxic DNA damage lesions that block the replication machinery that can cause a genomic instability. Ubiquitous abasic DNA sites are particularly susceptible to spontaneous cross-linking with a base from the opposite DNA strand. Detection of a crosslink induces the DNA helicase ubiquitination that recruits NEIL3, a DNA glycosylase responsible for the lesion removal. NEIL3 utilizes several zinc finger domains indispensable for its catalytic NEI domain repairing activity. They recruit NEIL3 to the repair site and bind the single-stranded DNA. However, the molecular mechanism underlying their roles in the repair process is unknown. Here, we report the structure of the tandem zinc-finger GRF domain of NEIL3 and reveal the molecular details of its interaction with DNA. Our biochemical data indicate the preferential binding of the GRF domain to the replication fork. In addition, we obtained a structure for the catalytic NEI domain in complex with the DNA reaction intermediate that allowed us to construct and validate a model for the interplay between the NEI and GRF domains in the recognition of an interstrand cross-link. Our results suggest a mechanism for recognition of the DNA replication X-structure by NEIL3, a key step in the interstrand cross-link repair.
Coronaviral methyltransferases (MTases), nsp10/16 and nsp14, catalyze the last two steps of viral RNA-cap creation that takes place in cytoplasm. This cap is essential for the stability of viral RNA and, most importantly, for the evasion of innate immune system. Non-capped RNA is recognized by innate immunity which leads to its degradation and the activation of antiviral immunity. As a result, both coronaviral MTases are in the center of scientific scrutiny. Recently, X-ray and cryo-EM structures of both enzymes were solved even in complex with other parts of the viral replication complex. High-throughput screening as well as structure-guided inhibitor design have led to the discovery of their potent inhibitors. Here, we critically summarize the tremendous advancement of the coronaviral MTase field since the beginning of COVID pandemic.
- MeSH
- aminokyseliny chemie MeSH
- antivirové látky chemie farmakologie MeSH
- Coronavirus účinky léků enzymologie genetika MeSH
- lidé MeSH
- methyltransferasy antagonisté a inhibitory chemie metabolismus MeSH
- metylace MeSH
- molekulární konformace MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- objevování léků MeSH
- RNA virová chemie genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
