The crucial role of SETDB1 in structural and functional transformation of epithelial cells during regeneration after intestinal ischemia reperfusion injury.

Su (var) 3-9, enhancer of seste, trithorax (SET)-domain bifurcated histone lysine methyltransferase (SETDB1) plays a crucial role in maintaining intestinal stem cell homeostasis; however, its physiological function in epithelial injury is largely unknown. In this study, we investigated the role of SETDB1 in epithelial regeneration using an intestinal ischemia/reperfusion injury (IRI) mouse model. Jejunum tissues were sampled after 75 min of ischemia followed by 3, 24, and 48 h of reperfusion. Morphological evaluations were performed using light microscopy and electron microscopy, and the involvement of SETDB1 in epithelial remodeling was investigated by immunohistochemistry. Expression of SETDB1 was increased following 24 h of reperfusion and localized in not only the crypt bottom but also in the transit amplifying zone and part of the villi. Changes in cell lineage, repression of cell adhesion molecule expression, and decreased histone H3 methylation status were detected in the crypts at the same time. Electron microscopy also revealed aberrant alignment of crypt nuclei and fusion of adjacent villi. Furthermore, increased SETDB1 expression and epithelial remodeling were confirmed with loss of stem cells, suggesting SETDB1 affects epithelial cell plasticity. In addition, crypt elongation and increased numbers of Ki-67 positive cells indicated active cell proliferation after IRI; however, the expression of PCNA was decreased compared to sham mouse jejunum. These morphological changes and the aberrant expression of proliferation markers were prevented by sinefungin, a histone methyltransferase inhibitor. In summary, SETDB1 plays a crucial role in changes in the epithelial structure after IRI-induced stem cell loss.

Sinefungin analogs targeting VP39 methyltransferase as potential anti-monkeypox therapeutics: a multi-step computational approach.

The increasing spread of the Monkeypox virus (MPXV) presents a significant public health challenge, emphasising the urgent requirement for effective treatments. Our study focuses on the VP39 Methyltransferase enzyme of MPXV as a critical target for therapy. By utilising virtual screening, we investigated natural compounds with structural similarities to sinefungin, a broad-acting MTase inhibitor. From an initial set of 177 compounds, we identified three promising compounds-CNP0346326, CNP0343532, and CNP008361, whose binding scores were notably close to that of sinefungin. These candidates bonded strongly to the VP39 enzyme, hinting at a notable potential to impede the virus. Our rigorous computational assays, including re-docking, extended molecular dynamics simulations, and energetics analyses, validate the robustness of these interactions. The data paint a promising picture of these natural compounds as front-runners in the ongoing race to develop MPXV therapeutics and set the stage for subsequent empirical trials to refine these discoveries into actionable medical interventions.

Anti-COVID-19 activity of some benzofused 1,2,3-triazolesulfonamide hybrids using in silico and in vitro analyses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pandemic fatal infection with no known treatment. The severity of the disease and the fast viral mutations forced the scientific community to search for potential solution. Here in the present manuscript, some benzofused1,2,3triazolesulfonamide hybrids were synthesized and evaluated for their anti- SARS-CoV-2 activity using in silico prediction then the most potent compounds were assessed using in-Vitro analysis. The in-Silico study was assessed against RNA dependent RNA polymerase, Spike protein S1, Main protease (3CLpro) and 2'-O-methyltransferase (nsp16). It was found that 4b and 4c showed high binding scores against RNA dependent RNA polymerase reached -8.40 and -8.75 â€‹kcal/mol, respectively compared to the approved antiviral (remdesivir -6.77 â€‹kcal/mol). Upon testing the binding score with SARS-CoV-2 Spike protein it was revealed that 4c exhibited the highest score (-7.22 â€‹kcal/mol) compared to the reference antibacterial drug Ceftazidime (-6.36 â€‹kcal/mol). Surprisingly, the two compounds 4b and 4c showed the highest binding scores against SARS-CoV-2 3CLpro (-8.75, -8.48 â€‹kcal/mol, respectively) and nsp16 (- 8.84 and - 8.89 â€‹kcal/mol, respectively) displaying many types of interaction with all the enzymes binding sites. The derivatives 4b and 4c were examined in vitro for their potential anti-SARS-CoV-2 and it was revealed that 4c was the most promising compound with IC50 reached 758.8108 â€‹mM and complete (100%) inhibition of the binding of SARS-CoV-2 virus to human ACE2 can be accomplished by using 0.01 â€‹mg.

Amino Acid Metabolism and Transport Mechanisms as Potential Antifungal Targets.

Discovering new drugs for treatment of invasive fungal infections is an enduring challenge. There are only three major classes of antifungal agents, and no new class has been introduced into clinical practice in more than a decade. However, recent advances in our understanding of the fungal life cycle, functional genomics, proteomics, and gene mapping have enabled the identification of new drug targets to treat these potentially deadly infections. In this paper, we examine amino acid transport mechanisms and metabolism as potential drug targets to treat invasive fungal infections, including pathogenic yeasts, such as species of Candida and Cryptococcus, as well as molds, such as Aspergillus fumigatus. We also explore the mechanisms by which amino acids may be exploited to identify novel drug targets and review potential hurdles to bringing this approach into clinical practice.

Cycloalkane analogues of sinefungin as EHMT1/2 inhibitors.

A series of cycloalkyl substituted analogues of the natural product sinefungin lacking the amino-acid moiety was designed and synthesized. Two stereoisomers (6-R and 6-S) were separated and their bioactivities examined against EHMT1/2. Of which, compound 14d showed an inhibitory activity against EHMT1/2 (88.9%, IC50=21.8µM for EHMT1 and 77.6%, IC50=39.6µM for EHMT2, respectively) similar to that of sinefungin (100.0%, IC50=28.4µM for EHMT1 and 79.5%, IC50=30.1µM for EHMT2, respectively). Further studies against other methyltransferases such as PRMT1 showed no activity except that 12d displayed about 20% inhibition.

Crystal structure of Rv2258c from Mycobacterium tuberculosis H37Rv, an S-adenosyl-l-methionine-dependent methyltransferase.

The Mycobacterium tuberculosis Rv2258c protein is an S-adenosyl-L-methionine (SAM)-dependent methyltransferase (MTase). Here, we have determined its crystal structure in three forms: a ligand-unbound form, a binary complex with sinefungin (SFG), and a binary complex with S-adenosyl-L-homocysteine (SAH). The monomer structure of Rv2258c consists of two domains which are linked by a long α-helix. The N-terminal domain is essential for dimerization and the C-terminal domain has the Class I MTase fold. Rv2258c forms a homodimer in the crystal, with the N-terminal domains facing each other. It also exists as a homodimer in solution. A DALI structural similarity search with Rv2258c reveals that the overall structure of Rv2258c is very similar to small-molecule SAM-dependent MTases. Rv2258c interacts with the bound SFG (or SAH) in an extended conformation maintained by a network of hydrogen bonds and stacking interactions. Rv2258c has a relatively large hydrophobic cavity for binding of the methyl-accepting substrate, suggesting that bulky nonpolar molecules with aromatic rings might be targeted for methylation by Rv2258c in M. tuberculosis. However, the ligand-binding specificity and the biological role of Rv2258c remain to be elucidated due to high variability of the amino acid residues defining the substrate-binding site.

A convergent synthesis of carbocyclic sinefungin and its C5 epimer.

A convergent synthesis of carbocyclic sinefungin 2 and its C5 epimer 3 is described. The key features in our synthesis include the use of commercial available L-methionine and readily available (1R, 4S)-4-hydroxy-2-cyclopentenyl acetate as starting materials, cross-metathesis coupling, enzymatic kinetic resolution and Staudinger reduction. The current synthesis is flexible and therefore provides convenient access to the synthesis of various carbocyclic SIN analogues for biological evaluation.

Structure and function of the NS5 methyltransferase domain from Usutu virus.

Usutu virus (USUV), is a mosquito-borne flavivirus currently spreading outside the African continent producing substantial avian mortality. In contrast, infected humans could exhibit mild neurological symptoms or remain asymptomatic. As in other flaviviruses, the capped USUV genome encodes three structural and seven non-structural (NS) proteins. Among the NS proteins, NS5 plays crucial roles in virus replication, harbouring the capping and methyltransferase (MTase) activities in its N-terminal domain and the RNA-dependent RNA polymerase (RdRP) activity at the C-terminus. In this work, we present the first structural and functional characterization of the USUV MTase domain. The first structure of the USUV MTase has been determined in complex with its natural ligands (S-adenosyl-L-methionine [SAM]) and S-adenosyl-L-homocysteine [SAH]) at 2.2 Å resolution, showing a molecular dimer in the crystal asymmetric unit. One molecule is bound to the methyl donor SAM while the second is bound to the reaction by-product SAH. Both molecules are almost identical and also show a high structural similarity to the MTase domains of other flaviviruses. The structure of the USUV MTase bound to the inhibitor sinefungin at 1.8 Å resolution is also described. Careful comparisons of the interactions in the SAM-binding cavity prompt us to hypothesize about the strength and weakness of the structure-based design of antivirals directed to the SAM/SAH binding site that could be effective to deal with this threat.

The S-adenosylmethionine analog sinefungin inhibits the trimethylguanosine synthase TGS1 to promote telomerase activity and telomere lengthening.

Mutations in many genes that control the expression, the function, or the stability of telomerase cause telomere biology disorders (TBDs), such as dyskeratosis congenita, pulmonary fibrosis, and aplastic anemia. Mutations in a subset of the genes associated with TBDs cause reductions of the telomerase RNA moiety hTR, thus limiting telomerase activity. We have recently found that loss of the trimethylguanosine synthase TGS1 increases both hTR abundance and telomerase activity and leads to telomere elongation. Here, we show that treatment with the S-adenosylmethionine analog sinefungin inhibits TGS1 activity, increases the hTR levels, and promotes telomere lengthening in different cell types. Our results hold promise for restoring telomere length in stem and progenitor cells from TBD patients with reduced hTR levels.

In silico structure modelling of SARS-CoV-2 Nsp13 helicase and Nsp14 and repurposing of FDA approved antiviral drugs as dual inhibitors.

The high mortality rate from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in humans and the lack of effective therapeutic regime for its treatment necessitates the identification of new antivirals. SARS-CoV-2 relies on non-structural proteins such as Nsp13 helicase and nsp14 which are the key components of the replication-transcription complex (RTC) to complete its infectious life cycle. Therefore, targeting these essential viral proteins with small molecules will most likely to halt the disease pathogenesis. The lack of experimental structures of these proteins deters the process of structure-based identification of their specific inhibitors. In the present study, the in silico models of SARS-CoV-2 nsp13 helicase and nsp14 protein were elucidated using a comparative homology modelling approach. These in silico model structures were validated using various parameters such as Ramachandran plot, Verify 3D score, ERRAT score, knowledge-based energy and Z-score. The in silico models were further used for virtual screening of the Food and Drug Administration (FDA) approved antiviral drugs. Simeprevir (SMV), Paritaprevir (PTV) and Grazoprevir (GZR) were the common leads identified which show higher binding affinity to both nsp13 helicase and nsp14 as compared to the control inhibitors and therefore, they might be potential dual-target inhibitors. The leads also establish a network of hydrogen bonds and hydrophobic interactions with the key residues lining the active site pockets. The present findings suggest that these FDA approved antiviral drugs can be subjected to repurposing against SARS-CoV-2 infection after verifying the in silico results through in vitro and in vivo studies.

A high-throughput screen for the identification of compounds that inhibit nematode gene expression by targeting spliced leader trans-splicing.

Infections with parasitic nematodes are among the most significant of the neglected tropical diseases affecting about a billion people living mainly in tropical regions with low economic activity. The most effective current strategy to control nematode infections involves large scale treatment programs with anthelmintic drugs. This strategy is at risk from the emergence of drug resistant parasites. Parasitic nematodes also affect livestock, which are treated with the same limited group of anthelmintic drugs. Livestock parasites resistant to single drugs, and even multi-drug resistant parasites, are appearing in many areas. There is therefore a pressing need for new anthelmintic drugs. Here we use the nematode Caenorhabditis elegans as a model for parasitic nematodes and demonstrate that sinefungin, a competitive inhibitor of methyltransferases, causes a delay in development and reduced fecundity, and inhibits spliced leader trans-splicing. Spliced leader trans-splicing is an essential step in gene expression that does not occur in the hosts of parasitic nematodes, and is therefore a potential target for new anthelmintic drugs. We have exploited the ability of sinefungin to inhibit spliced leader trans-splicing to adapt a green fluorescent protein based reporter gene assay that monitors spliced leader trans-splicing for high-throughput screening for new anthelmintic compounds. We have established a protocol for robust high-throughput screening, combining mechanical dispensing of living C. elegans into 384- or 1536- well plates with addition of compounds using an acoustic liquid dispenser, and the detection of the inhibition of SL trans-splicing using a microplate reader. We have tested this protocol in a first pilot screen and envisage that this assay will be a valuable tool in the search for new anthelmintic drugs.

Design, synthesis and in vitro anti-Zika virus evaluation of novel Sinefungin derivatives.

We report herein the design and synthesis of a series of novel Sinefungin (SIN) derivatives, based on the structures of SIN and its analogue EPZ004777. Our results reveal that target compounds 1ad-af, 1ba-bb and 1bf-bh show better activity (IC50 = 4.56-20.16 µM) than EPZ004777 (IC50 = 35.19 µM). Surprisingly, SIN was founded to be not as active (IC50 > 50 µM) as we and other research groups predicted. Interestingly, the intermediates 9a-b and 11b display potent anti-ZIKV potency (IC50 = 6.33-29.98 µM), and compound 9a also exhibits acceptable cytotoxicity (CC50 > 200 µM), suggesting their promising potential to be leads for further development.

Synthesis and Assays of Inhibitors of Methyltransferases.

Epigenetic regulation requires site-specific modification of the genome and is involved in multiple physiological processes and disease etiology. Methyltransferases, which catalyze the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to various substrates, are critical components of the epigenetic machinery. This group of enzymes can methylate diverse substrates including DNA, RNA, proteins, and small-molecule metabolites. Their dysregulation has also been implicated in multiple disease states such as cancer, neurological, and cardiovascular disorders. Developing potent and selective small-molecule inhibitors of methyltransferases is valuable not only for therapeutic intervention but also for investigating the roles of these enzymes in disease progression. In this chapter, we will discuss the strategies of designing and synthesizing methyltransferases inhibitors based on the SAM scaffold. Following the section of inhibitor design, we will briefly review representative assays that are available to evaluate the potency of these inhibitors along with a detailed description of the most commonly used radiometric assay.

Analogues of the Natural Product Sinefungin as Inhibitors of EHMT1 and EHMT2.

A series of analogues of the natural product sinefungin lacking the amino acid moiety was synthesized and probed for their ability to inhibit EHMT1 and EHMT2. This study led to inhibitors 3b and 4d of methyltransferase activity of EHMT1 and EHMT2 and it demonstrates that such analogues constitute an interesting scaffold to develop selective methyltransferase inhibitors. Surprisingly, the inhibition was not competitive toward AdoMet.

Yeast-based assays for the high-throughput screening of inhibitors of coronavirus RNA cap guanine-N7-methyltransferase.

The 5'-cap structure is a distinct feature of eukaryotic mRNAs and is important for RNA stability and protein translation by providing a molecular signature for the distinction of self or non-self mRNA. Eukaryotic viruses generally modify the 5'-end of their RNAs to mimic the cellular mRNA structure, thereby facilitating viral replication in host cells. However, the molecular organization and biochemical mechanisms of the viral capping apparatus typically differ from its cellular counterpart, which makes viral capping enzymes attractive targets for drug discovery. Our previous work showed that SARS coronavirus (SARS-CoV) non-structural protein 14 represents a structurally novel and unique guanine-N7-methyltransferase (N7-MTase) that is able to functionally complement yeast cellular N7-MTase. In the present study, we developed a yeast-based system for identifying and screening inhibitors against coronavirus N7-MTase using both 96-well and 384-well microtiter plates. The MTase inhibitors previously identified by in vitro biochemical assays were tested, and some, such as sinefungin, effectively suppressed N7-MTase in the yeast system. However, other compounds, such as ATA and AdoHcy, did not exert an inhibitory effect within a cellular context. These results validated the yeast assay system for inhibitor screening yet also demonstrated the difference between cell-based and in vitro biochemical assays. The yeast system was applied to the screening of 3000 natural product extracts, and three were observed to more potently inhibit the activity of coronavirus than human N7-MTase.