Post-transcriptional capping generates coenzyme A-linked RNA.

NAD can be inserted co-transcriptionally via non-canonical initiation to form NAD-RNA. However, that mechanism is unlikely for CoA-linked RNAs due to low intracellular concentration of the required initiator nucleotide, 3'-dephospho-CoA (dpCoA). We report here that phosphopantetheine adenylyltransferase (PPAT), an enzyme of CoA biosynthetic pathway, accepts RNA transcripts as its acceptor substrate and transfers 4'-phosphopantetheine to yield CoA-RNA post-transcriptionally. Synthetic natural (RNAI) and small artificial RNAs were used to identify the features of RNA that are needed for it to serve as PPAT substrate. RNAs with 4-10 unpaired nucleotides at the 5' terminus served as PPAT substrates, but RNAs having <4 unpaired nucleotides did not undergo capping. No capping was observed when the +1A was changed to G or when 5' triphosphate was removed by RNA pyrophosphohydrolase (RppH), suggesting the enzyme recognizes pppA-RNA as an ATP analog. PPAT binding affinities were equivalent for transcripts with +1A, +1 G, or 5'OH (+1A), indicating that productive enzymatic recognition is driven more by local positioning effects than by overall binding affinity. Capping rates were independent of the number of unpaired nucleotides in the range of 4-10 nucleotides. Capping was strongly inhibited by ATP, reducing CoA-RNA production ~70% when equimolar ATP and substrate RNA were present. Dual bacterial expression of candidate RNAs with different 5' structures followed by CoA-RNA CaptureSeq revealed 12-fold enrichment of the better PPAT substrate, consistent with in vivo CoA-capping of RNA transcripts by PPAT. These results suggest post-transcriptional RNA capping as a possible mechanism for the biogenesis of CoA-RNAs in bacteria.

Dicer and PKR as Novel Regulators of Embryonic Stem Cell Fate and Antiviral Innate Immunity.

Embryonic stem cells (ESCs) represent a unique cell population in the blastocyst stage embryo. They have been intensively studied as a promising cell source for regenerative medicine. Recent studies have revealed that both human and mouse ESCs are deficient in expressing IFNs and have attenuated inflammatory responses. Apparently, the ability to express IFNs and respond to certain inflammatory cytokines is not "innate" to ESCs but rather is developmentally acquired by somatic cells during differentiation. Accumulating evidence supports a hypothesis that the attenuated innate immune response may serve as a protective mechanism allowing ESCs to avoid immunological cytotoxicity. This review describes our current understanding of the molecular basis that shapes the immune properties of ESCs. We highlight the recent findings on Dicer and dsRNA-activated protein kinase R as novel regulators of ESC fate and antiviral immunity and discuss how ESCs use alternative mechanisms to accommodate their stem cell properties.

Single protein encapsulated SN38 for tumor-targeting treatment.

BACKGROUND: The alkaloid camptothecin analog SN38 is a potent antineoplastic agent, but cannot be used directly for clinical application due to its poor water solubility. Currently, the prodrug approach on SN38 has resulted in 3 FDA-approved cancer therapeutics, irinotecan, ONIVYDE, and Trodelvy. However, only 2-8% of irinotecan can be transformed enzymatically in vivo into the active metabolite SN38, which severely limits the drug's efficacy. While numerous drug delivery systems have been attempted to achieve effective SN38 delivery, none have produced drug products with antitumor efficacy better than irinotecan in clinical trials. Therefore, novel approaches are urgently needed for effectively delivering SN38 to cancer cells with better efficacy and lower toxicity. METHODS: Based on the unique properties of human serum albumin (HSA), we have developed a novel single protein encapsulation (SPE) technology to formulate cancer therapeutics for improving their pharmacokinetics (PK) and antitumor efficacy and reducing their side effects. Previous application of SPE technology to doxorubicin (DOX) formulation has led to a promising drug candidate SPEDOX-6 (FDA IND #, 152154), which will undergo a human phase I clinical trial. Using the same SPE platform on SN38, we have now produced two SPESN38 complexes, SPESN38-5 and SPESN38-8. We conducted their pharmacological evaluations with respect to maximum tolerated dose, PK, and in vivo efficacy against colorectal cancer (CRC) and soft tissue sarcoma (STS) in mouse models. RESULTS: The lyophilized SPESN38 complexes can dissolve in aqueous media to form clear and stable solutions. Maximum tolerated dose (MTD) of SPESN38-5 is 250 mg/kg by oral route (PO) and 55 mg/kg by intravenous route (IV) in CD-1 mice. SPESN38-8 has the MTD of 45 mg/kg by IV in the same mouse model. PK of SPESN38-5 by PO at 250 mg/kg gave mouse plasma AUC0-∞ of 0.05 and 4.5 nmol × h/mL for SN38 and SN38 glucuronidate (SN38G), respectively, with a surprisingly high molar ratio of SN38G:SN38 = 90:1. However, PK of SPESN38-5 by IV at 55 mg/kg yielded much higher mouse plasma AUC0-∞ of 19 and 28 nmol × h/mL for SN38 and SN38G, producing a much lower molar ratio of SN38G:SN38 = 1.5:1. Antitumor efficacy of SPESN38-5 and irinotecan (control) was evaluated against HCT-116 CRC xenograft tumors. The data indicates that SPESN38-5 by IV at 55 mg/kg is more effective in suppressing HCT-116 tumor growth with lower systemic toxicity compared to irinotecan at 50 mg/kg. Additionally, SPESN38-8 and DOX (control) by IV were evaluated in the SK-LMS-1 STS mouse model. The results show that SPESN38-8 at 33 mg/kg is highly effective for inhibiting SK-LMS-1 tumor growth with low toxicity, in contrast to DOX's insensitivity to SK-LMS-1 with high toxicity. CONCLUSION: SPESN38 complexes provide a water soluble SN38 formulation. SPESN38-5 and SPESN38-8 demonstrate better PK values, lower toxicity, and superior antitumor efficacy in mouse models, compared with irinotecan and DOX.

Dicer represses the interferon response and the double-stranded RNA-activated protein kinase pathway in mouse embryonic stem cells.

Recent studies have demonstrated that embryonic stem cells (ESCs) are deficient in expressing type I interferons (IFN), the cytokines that play key roles in antiviral responses. However, the underlying molecular mechanisms and biological implications of this finding are poorly understood. In this study, we developed a synthetic RNA-based assay that can simultaneously assess multiple forms of antiviral responses. Dicer is an enzyme essential for RNA interference (RNAi), which is used as a major antiviral mechanism in invertebrates. RNAi activity is detected in wild-type ESCs but is abolished in Dicer knockout ESCs (D-/-ESCs) as expected. Surprisingly, D-/-ESCs have gained the ability to express IFN, which is otherwise deficient in wild-type ESCs. Furthermore, D-/-ESCs have constitutively active double-stranded RNA (dsRNA)-activated protein kinase (PKR), an enzyme that is also involved in antiviral response. D-/-ESCs show increased sensitivity to the cytotoxicity resulting from RNA transfection. The effects of dsRNA can be partly replicated with a synthetic B2RNA corresponding to the retrotransposon B2 short interspersed nuclear element. B2RNA has secondary structure features of dsRNA and accumulates in D-/-ESCs, suggesting that B2RNA could be a cellular RNA that activates PKR and contributes to the decreased cell proliferation and viability of D-/-ESCs. Treatment of D-/-ESCs with a PKR inhibitor and IFNß-neutralizing antibodies increased cell proliferation rate and cell viability. Based on these findings, we propose that, in ESCs, Dicer acts as a repressor of antiviral responses and plays a key role in the maintenance of proliferation, viability, and pluripotency of ESCs.

Cloning, expression and purification of the low-complexity region of RanBP9 protein.

Recombinant expression and purification of proteins is key for biochemical and biophysical investigations. Although this has become a routine and standard procedure for many proteins, intrinsically disordered ones and those with low complexity sequences pose difficulties. Proteins containing low complexity regions (LCRs) are increasingly becoming significant for their roles in both normal and pathological processes. Here, we report cloning, expression and purification of N-terminal LCR of RanBP9 protein (Nt-RanBP9). RanBP9 is a scaffolding protein present in both cytoplasm and nucleus that is implicated in many cellular processes. Nt-RanBP9 is a poorly understood region of the protein perhaps due to difficulties posed by the LCR. Indeed, conventional methods presented difficulties in Nt-RanBP9 cloning due to its high GC content resulting in insignificant protein expression. These led us to use a different approach of cloning by expressing the protein as a fusion construct containing mCherry or mEGFP using in vivo DNA recombination methods. Our results indicate that expression of mEGFP-tagged Nt-RanBP9 followed by thrombin cleavage of the tag was the most effective method to obtain the protein with >90% purity and good yields. We report and discuss the challenges in obtaining the N-terminal region of RanBP9, a protein with functional implications in multiple biological processes and neurodegenerative diseases.

Efficient one-pot enzymatic synthesis of dephospho coenzyme A.

Dephospho coenzyme A (depCoA) is the last intermediate for CoA biosynthesis, and it can be used as a transcription initiator to prepare CoA-linked RNA by in vitro transcription. However, commercially available depCoA is expensive. We hereby describe a simple and efficient enzymatic synthesis of depCoA in a single-step from commercially available and inexpensive oxidized pantethine (Ox-Pan) and ATP. A plasmid (pCoaDAa) was constructed to co-express and co-purify two enzymes pantothenate kinase (PanK/coaA) and phosphopantetheine adenylyltransferase (PPAT/coaD). Starting from Ox-Pan and ATP, two different synthetic routes of one-pot reaction catalyzed by PanK and PPAT, followed by a simple column purification step, afforded depCoA and its oxidized dimer (Ox-depCoA) with high yields and purity. The simplicity and low cost of our method should make depCoA easily accessible to a broad scientific community, and promote research on CoA-related areas in biology and biomedicine.

Attenuated Innate Immunity in Embryonic Stem Cells and Its Implications in Developmental Biology and Regenerative Medicine.

Embryonic stem cells (ESCs) represent a promising cell source for regenerative medicine. Intensive research over the past 2 decades has led to the feasibility of using ESC-differentiated cells (ESC-DCs) in regenerative medicine. However, increasing evidence indicates that ESC-DCs generated by current differentiation methods may not have equivalent cellular functions to their in vivo counterparts. Recent studies have revealed that both human and mouse ESCs as well as some types of ESC-DCs lack or have attenuated innate immune responses to a wide range of infectious agents. These findings raise important concerns for their therapeutic applications since ESC-DCs, when implanted to a wound site of a patient, where they would likely be exposed to pathogens and inflammatory cytokines. Understanding whether an attenuated immune response is beneficial or harmful to the interaction between host and grafted cells becomes an important issue for ESC-based therapy. A substantial amount of recent evidence has demonstrated that the lack of innate antiviral responses is a common feature to ESCs and other types of pluripotent cells. This has led to the hypothesis that mammals may have adapted different antiviral mechanisms at different stages of organismal development. The underdeveloped innate immunity represents a unique and uncharacterized property of ESCs that may have important implications in developmental biology, immunology, and in regenerative medicine.

Antiviral responses in mouse embryonic stem cells: differential development of cellular mechanisms in type I interferon production and response.

We have recently reported that mouse embryonic stem cells (mESCs) are deficient in expressing type I interferons (IFNs) in response to viral infection and synthetic viral RNA analogs (Wang, R., Wang, J., Paul, A. M., Acharya, D., Bai, F., Huang, F., and Guo, Y. L. (2013) J. Biol. Chem. 288, 15926-15936). Here, we report that mESCs are able to respond to type I IFNs, express IFN-stimulated genes, and mediate the antiviral effect of type I IFNs against La Crosse virus and chikungunya virus. The major signaling components in the IFN pathway are expressed in mESCs. Therefore, the basic molecular mechanisms that mediate the effects of type I IFNs are functional in mESCs; however, these mechanisms may not yet be fully developed as mESCs express lower levels of IFN-stimulated genes and display weaker antiviral activity in response to type I IFNs when compared with fibroblasts. Further analysis demonstrated that type I IFNs do not affect the stem cell state of mESCs. We conclude that mESCs are deficient in type I IFN expression, but they can respond to and mediate the cellular effects of type I IFNs. These findings represent unique and uncharacterized properties of mESCs and are important for understanding innate immunity development and ESC physiology.

Mouse embryonic stem cells are deficient in type I interferon expression in response to viral infections and double-stranded RNA.

Embryonic stem cells (ESCs) are considered to be a promising cell source for regenerative medicine because of their unlimited capacity for self-renewal and differentiation. However, little is known about the innate immunity in ESCs and ESC-derived cells. We investigated the responses of mouse (m)ESCs to three types of live viruses as follows: La Crosse virus, West Nile virus, and Sendai virus. Our results demonstrated mESCs were susceptible to viral infection, but they were unable to express type I interferons (IFNα and IFNß, IFNα/ß), which differ from fibroblasts (10T1/2 cells) that robustly express IFNα/ß upon viral infections. The failure of mESCs to express IFNα/ß was further demonstrated by treatment with polyIC, a synthetic viral dsRNA analog that strongly induced IFNα/ß in 10T1/2 cells. Although polyIC transiently inhibited the transcription of pluripotency markers, the stem cell morphology was not significantly affected. However, polyIC can induce dsRNA-activated protein kinase in mESCs, and this activation resulted in a strong inhibition of cell proliferation. We conclude that the cytosolic receptor dsRNA-activated protein kinase is functional, but the mechanisms that mediate type I IFN expression are deficient in mESCs. This conclusion is further supported by the findings that the major viral RNA receptors are either expressed at very low levels (TLR3 and MDA5) or may not be active (retinoic acid-inducible gene I) in mESCs.

Delivery of antiviral small interfering RNA with gold nanoparticles inhibits dengue virus infection in vitro.

Dengue virus (DENV) infection in humans can cause flu-like illness, life-threatening haemorrhagic fever or even death. There is no specific anti-DENV therapeutic or approved vaccine currently available, partially due to the possibility of antibody-dependent enhancement reaction. Small interfering RNAs (siRNAs) that target specific viral genes are considered a promising therapeutic alternative against DENV infection. However, in vivo, siRNAs are vulnerable to degradation by serum nucleases and rapid renal excretion due to their small size and anionic character. To enhance siRNA delivery and stability, we complexed anti-DENV siRNAs with biocompatible gold nanoparticles (AuNPs) and tested them in vitro. We found that cationic AuNP-siRNA complexes could enter Vero cells and significantly reduce DENV serotype 2 (DENV-2) replication and infectious virion release under both pre- and post-infection conditions. In addition, RNase-treated AuNP-siRNA complexes could still inhibit DENV-2 replication, suggesting that AuNPs maintained siRNA stability. Collectively, these results demonstrated that AuNPs were able to efficiently deliver siRNAs and control infection in vitro, indicating a novel anti-DENV strategy.

Topological deep learning based deep mutational scanning.

High-throughput deep mutational scanning (DMS) experiments have significantly impacted protein engineering, drug discovery, immunology, cancer biology, and evolutionary biology by enabling the systematic understanding of protein functions. However, the mutational space associated with proteins is astronomically large, making it overwhelming for current experimental capabilities. Therefore, alternative methods for DMS are imperative. We propose a topological deep learning (TDL) paradigm to facilitate in silico DMS. We utilize a new topological data analysis (TDA) technique based on the persistent spectral theory, also known as persistent Laplacian, to capture both topological invariants and the homotopic shape evolution of data. To validate our TDL-DMS model, we use SARS-CoV-2 datasets and show excellent accuracy and reliability for binding interface mutations. This finding is significant for SARS-CoV-2 variant forecasting and designing effective antibodies and vaccines. Our proposed model is expected to have a significant impact on drug discovery, vaccine design, precision medicine, and protein engineering.

An effective live-attenuated Zika vaccine candidate with a modified 5' untranslated region.

Zika virus (ZIKV) is a mosquito-transmitted flavivirus that has caused devastating congenital Zika syndrome (CZS), including microcephaly, congenital malformation, and fetal demise in human newborns in recent epidemics. ZIKV infection can also cause Guillain-Barré syndrome (GBS) and meningoencephalitis in adults. Despite intensive research in recent years, there are no approved vaccines or antiviral therapeutics against CZS and adult Zika diseases. In this report, we developed a novel live-attenuated ZIKV strain (named Z7) by inserting 50 RNA nucleotides (nt) into the 5' untranslated region (UTR) of a pre-epidemic ZIKV Cambodian strain, FSS13025. We used this particular ZIKV strain as it is attenuated in neurovirulence, immune antagonism, and mosquito infectivity compared with the American epidemic isolates. Our data demonstrate that Z7 replicates efficiently and produces high titers without causing apparent cytopathic effects (CPE) in Vero cells or losing the insert sequence, even after ten passages. Significantly, Z7 induces robust humoral and cellular immune responses that completely prevent viremia after a challenge with a high dose of an American epidemic ZIKV strain PRVABC59 infection in type I interferon (IFN) receptor A deficient (Ifnar1-/-) mice. Moreover, adoptive transfer of plasma collected from Z7 immunized mice protects Ifnar1-/- mice from ZIKV (strain PRVABC59) infection. These results suggest that modifying the ZIKV 5' UTR is a novel strategy to develop live-attenuated vaccine candidates for ZIKV and potentially for other flaviviruses.

Establishment of FAP-overexpressing Cells for FAP-targeted Theranostics.

OBJECTIVE: Fibroblast activation protein (FAP) has been widely studied and exploited for its clinical applications. One of the difficulties in interpreting reports of FAP-targeted theranostics is due to the lack of accurate controls, making the results less specific and less confirmative. This study aimed to establish a pair of cell lines, in which one highly expresses FAP (HT1080-hFAP) and the other has no detectable FAP (HT1080-vec) as control, to accurately evaluate the specificity of the FAP-targeted theranostics in vitro and in vivo. METHODS: The cell lines of the experimental group (HT1080-hFAP) and no-load group (HT1080-vec) were obtained by molecular construction of the recombinant plasmid pIRES-hFAP. The expression of hFAP in HT1080 cells was detected by PCR, Western blotting and flow cytometry. CCK-8, Matrigel transwell invasion assay, scratch test, flow cytometry and immunofluorescence were used to verify the physiological function of FAP. The activities of human dipeptidyl peptidase (DPP) and human endopeptidase (EP) were detected by ELISA in HT1080-hFAP cells. PET imaging was performed in bilateral tumor-bearing nude mice models to evaluate the specificity of FAP. RESULTS: RT-PCR and Western blotting demonstrated the mRNA and protein expression of hFAP in HT1080-hFAP cells but not in HT1080-vec cells. Flow cytometry confirmed that nearly 95% of the HT1080-hFAP cells were FAP positive. The engineered hFAP on HT1080 cells had its ability to retain enzymatic activities and a variety of biological functions, including internalization, proliferation-, migration-, and invasion-promoting activities. The HT1080-hFAP xenografted tumors in nude mice bound and took up 68GA-FAPI-04 with superior selectivity. High image contrast and tumor-organ ratio were obtained by PET imaging. The HT1080-hFAP tumor retained the radiotracer for at least 60 min. CONCLUSION: This pair of HT1080 cell lines was successfully established, making it feasible for accurate evaluation and visualization of therapeutic and diagnostic agents targeting the hFAP.

Protein-encapsulated doxorubicin reduces cardiotoxicity in hiPSC-cardiomyocytes and cardiac spheroids while maintaining anticancer efficacy.

The chemotherapeutic doxorubicin (DOX) detrimentally impacts the heart during cancer treatment. This necessitates development of non-cardiotoxic delivery systems that retain DOX anticancer efficacy. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), cardiac fibroblasts (hiPSC-CFs), multi-lineage cardiac spheroids (hiPSC-CSs), patient-specific hiPSCs, and multiple human cancer cell lines to compare the anticancer efficacy and reduced cardiotoxicity of single protein encapsulated DOX (SPEDOX-6), to standard unformulated (UF) DOX. Cell viability assays and immunostaining in human cancer cells, hiPSC-ECs, and hiPSC-CFs revealed robust uptake of SPEDOX-6 and efficacy in killing these proliferative cell types. In contrast, hiPSC-CMs and hiPSC-CSs exhibited substantially lower cytotoxicity during SPEDOX-6 treatment compared with UF DOX. SPEDOX-6-treated hiPSC-CMs and hiPSC-CSs maintained their functionality, as indicated by sarcomere contractility assessment, calcium imaging, multielectrode arrays, and RNA sequencing. This study demonstrates the potential of SPEDOX-6 to alleviate cardiotoxic side effects associated with UF DOX, while maintaining its anticancer potency.

Single Protein Encapsulated SN38 for Tumor-Targeting Treatment.

Background: The alkaloid camptothecin analog SN38 is a potent antineoplastic agent, but cannot be used directly for clinical application due to its poor water solubility. Currently, the prodrug approach on SN38 has resulted in 3 FDA-approved cancer therapeutics, irinotecan, ONIVYDE, and Trodelvy. However, only 2-8% of irinotecan can be transformed enzymatically in vivo into the active metabolite SN38, which severely limits the drug's efficacy. While numerous drug delivery systems have been attempted to achieve effective SN38 delivery, none have produced drug products with antitumor efficacy better than irinotecan in clinical trials. Therefore, novel approaches are urgently needed for effectively delivering SN38 to cancer cells with better efficacy and lower toxicity. Methods: Based on the unique properties of human serum albumin (HSA), we have developed a novel single protein encapsulation (SPE) technology to formulate cancer therapeutics for improving their pharmacokinetics (PK) and antitumor efficacy and reducing their side effects. Previous application of SPE technology to doxorubicin (DOX) formulation has led to a promising drug candidate SPEDOX-6 (FDA IND #, 152154), which will undergo a human phase I clinical trial. Using the same SPE platform on SN38, we have now produced two SPESN38 complexes, SPESN38-5 and SPESN38-8. We conducted their pharmacological evaluations with respect to maximum tolerated dose, PK, and in vivo efficacy against colorectal cancer (CRC) and soft tissue sarcoma (STS) in mouse models. Results: The lyophilized SPESN38 complexes can dissolve in aqueous media to form clear and stable solutions. Maximum tolerated dose (MTD) of SPESN38-5 is 250 mg/kg by oral route (PO) and 55 mg/kg by intravenous route (IV) in CD-1 mice. SPESN38-8 has the MTD of 45 mg/kg by IV in the same mouse model. PK of SPESN38-5 by PO at 250 mg/kg gave mouse plasma AUC0-∞ of 0.0548 and 4.5007 (nmol × h/mL) for SN38 and SN38 glucuronidate (SN38G), respectively, with a surprisingly high molar ratio of SN38G:SN38 = 82:1. However, PK of SPESN38-5 by IV at 55 mg/kg yielded much higher mouse plasma AUC0-∞ of 18.80 and 27.78 nmol × h/mL for SN38 and SN38G, producing a much lower molar ratio of SN38G:SN38 = 1.48:1. Antitumor efficacy of SPESN38-5 and irinotecan (control) was evaluated against HCT-116 CRC xenograft tumors. The data indicates that SPESN38-5 by IV at 55 mg/kg is more effective in suppressing HCT-116 tumor growth with lower systemic toxicity compared to irinotecan at 50 mg/kg. Additionally, SPESN38-8 and DOX (control) by IV were evaluated in the SK-LMS-1 STS mouse model. The results show that SPESN38-8 at 33 mg/kg is highly effective for inhibiting SK-LMS-1 tumor growth with low toxicity, in contrast to DOX's insensitivity to SK-LMS-1 with high toxicity. Conclusion: SPESN38 complexes provide a water soluble SN38 formulation. SPESN38-5 and SPESN38-8 demonstrate better PK values, lower toxicity, and superior antitumor efficacy in mouse models, compared with irinotecan and DOX.

Synthesis of photolabile transcription initiators and preparation of photocleavable functional RNA by transcription.

Two new photolabile adenosine-containing transcription initiators with terminal thiol and amino functionalities are chemically synthesized. Transcription in the presence of the transcription initiators under the T7 phi2.5 promoter produces 5' thiol- and amino-functionalized RNA conjugated by a photocleavable (PC) linker. Further RNA functionalization with biotin may be achieved through acyl transfer reactions from either biotinyl AMP to the RNA thiol group or biotin NHS to the RNA amino group. Photocleavage of the PC linker displays relatively fast kinetics with a half-life of 4-5 min. The availability of these transcription initiators makes new photolabile RNA accessible for affinity purification of RNA, in vitro selection of functional RNAs, and functional RNA caging.

Perspectives on SARS-CoV-2 Main Protease Inhibitors.

The main protease (Mpro) plays a crucial role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication and is highly conserved, rendering it one of the most attractive therapeutic targets for SARS-CoV-2 inhibition. Currently, although two drug candidates targeting SARS-CoV-2 Mpro designed by Pfizer are under clinical trials, no SARS-CoV-2 medication is approved due to the long period of drug development. Here, we collect a comprehensive list of 817 available SARS-CoV-2 and SARS-CoV Mpro inhibitors from the literature or databases and analyze their molecular mechanisms of action. The structure-activity relationships (SARs) among each series of inhibitors are discussed. Additionally, we broadly examine available antiviral activity, ADMET (absorption, distribution, metabolism, excretion, and toxicity), and animal tests of these inhibitors. We comment on their druggability or drawbacks that prevent them from becoming drugs. This Perspective sheds light on the future development of Mpro inhibitors for SARS-CoV-2 and future coronavirus diseases.

Rational design of targeted cancer therapeutics through the multiconjugation of folate and cleavable siRNA to RAFT-synthesized (HPMA-s-APMA) copolymers.

A well-defined N-(2-hydroxypropyl)methacrylamide-s-N-(3-aminopropyl)methacrylamide (HPMA-s-APMA) copolymer, synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, was utilized for the rational design of multiconjugates containing both a gene therapeutic, small interfering RNA (siRNA), and a cancer cell targeting moiety, folate. The copolymer contains a biocompatible poly(HPMA) portion (91 mol %) and a primary amine, APMA, portion (9 mol %). A fraction (20 mol %) of the APMA repeats were converted to activated thiols utilizing the amine- and sulfhydryl-reactive molecule N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP). 5'-Thiolated sense strand RNAs were then coupled to the polymer through a disulfide exchange with pendant pyridyldithio moieties, giving an 89 +/- 4% degree of conjugation. The unmodified APMA units (80 mol %) were subsequently coupled to amine reactive folates with 81 +/- 1% efficiency. This yielded a multiconjugate copolymer with 91 mol % HPMA, 2 mol % RNA, and 6 mol % folate. siRNA formation was achieved by annealing antisense strands to the conjugated RNA sense strands. Subsequent siRNA cleavage under intracellular conditions demonstrated the potential utility of this carrier in gene delivery. The multiconjugate copolymer and siRNA release were characterized by UV-vis spectroscopy and polyacrylamide gel electrophoresis.

Tailored design of Au nanoparticle-siRNA carriers utilizing reversible addition-fragmentation chain transfer polymers.

The facile synthesis of polymer-stabilized Au nanoparticles (AuNPs) capable of forming neutral, sterically stable complexes with small interfering RNA (siRNA) is reported. The amine-containing cationic block of poly(N-2-hydroxypropyl methacrylamide(70)-block-N-[3-(dimethylamino)propyl] methacrylamide(24)) [P(HPMA(70)-b-DMAPMA(24))] was utilized to promote the in situ reduction of Au(3+) to AuNPs and subsequently bind small interfering RNA, while the nonimmunogenic, hydrophilic block provided steric stabilization. The ratio of [DMAPMA](0)/[Au(3+)](0) utilized in the reduction reaction was found to be critical to the production of polymer-stabilized AuNPs capable of complexing siRNA. Significant protection ( approximately 100 times) against nucleases was demonstrated by enzymatic tests, while gene down-regulation experiments indicated successful delivery of siRNA to cancerous cells.

Synthesis of symmetrical thiol-adenosine conjugate and 5' thiol-RNA preparation by efficient one-step transcription.

A symmetrical transcription initiator containing two adenosines and conjugated thiol functionality (ThioAMP dimer) is chemically synthesized. Transcription in the presence of ThioAMP dimer under the T7 Φ2.5 promoter yields 5' thiol-labeled RNA (5' HS-RNA) with up to 90% labeling efficiency, depending on the concentration ratio of ThioAMP dimer to ATP. The resulting 5' HS-RNA may be used directly or after thiopropyl Separose 6B affinity column purification. Biotinylation of 5' HS-RNA and formation of gold nanoparticle-RNA nanoplexes are demonstrated.