Ribosome-inactivating proteins (RIPs) including ricin, Shiga toxin, and trichosanthin, are RNA N-glycosidases that depurinate a specific adenine residue (A-4324 in rat 28S ribosomal RNA, rRNA) in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. RIPs are grouped into three types according to the number of subunits and the organization of the precursor sequences. RIPs are two-domain proteins, with the active site located in the cleft between the N- and C-terminal domains. It has been found that the basic surface residues of the RIPs promote rapid and specific targeting to the ribosome and a number of RIPs have been shown to interact with the C-terminal regions of the P proteins of the ribosome. At present, the structural basis for the interaction of trichosanthin and ricin-A chain toward P2 peptide is known. This review surveys the structural features of the representative RIPs and discusses how they approach and interact with the ribosome.

Ricin is a type 2 ribosome-inactivating protein (RIP), containing a catalytic A chain and a lectin-like B chain. It inhibits protein synthesis by depurinating the N-glycosidic bond at α-sarcin/ricin loop (SRL) of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation center of the ribosome. Here, we present the 1.6 Å crystal structure of Ricin A chain (RTA) complexed to the C-terminal peptide of the ribosomal stalk protein P2, which plays a crucial role in specific recognition of elongation factors and recruitment of eukaryote-specific RIPs to the ribosomes. Our structure reveals that the C-terminal GFGLFD motif of P2 peptide is inserted into a hydrophobic pocket of RTA, while the interaction assays demonstrate the structurally untraced SDDDM motif of P2 peptide contributes to the interaction with RTA. This interaction mode of RTA and P protein is in contrast to that with trichosanthin (TCS), Shiga-toxin (Stx) and the active form of maize RIP (MOD), implying the flexibility of the P2 peptide-RIP interaction, for the latter to gain access to ribosome.

Trichosanthin (TCS) is an RNA N-glycosidase that depurinates adenine-4324 in the conserved α-sarcin/ricin loop (α-SRL) of rat 28 S ribosomal RNA (rRNA). TCS has only one chain, and is classified as type 1 ribosome-inactivating protein (RIP). Our structural studies revealed that TCS consists of two domains, with five conserved catalytic residues Tyr70, Tyr111, Glu160, Arg163 and Phe192 at the active cleft formed between them. We also found that the structural requirements of TCS to interact with the ribosomal stalk protein P2 C-terminal tail. The structural analyses suggest TCS attacks ribosomes by first binding to the C-terminal domain of ribosomal P protein. TCS exhibits a broad spectrum of biological and pharmacological activities including anti-tumor, anti-virus, and immune regulatory activities. This review summarizes an updated knowledge in the structural and functional studies and the mechanism of its multiple pharmacological effects.

To improve the pharmacological properties of maize ribosome-inactivating protein (maize RIP) for targeting HIV-infected cells, the previously engineered TAT-fused active form of maize RIP (MOD) was further engineered for cysteine-directed PEGylation. In this work, two potential antigenic sites, namely Lys-78 and Lys-264, were identified. They were mutated to cysteine residue and conjugated with PEG_5k or PEG_20k. The resultant PEG derivatives of MOD variants were examined for ribosome-inactivating activity, circulating half-life and immunogenicity. Our results showed that MOD-PEG conjugates had two- to five-fold lower biological activity compared to the wild-type. Mutation of the two sites respectively did not decrease the anti-MOD IgG and IgE level in mice, but the conjugation of PEG did dramatically reduce the antigenicity. Furthermore, pharmacokinetics studies demonstrated that attachment of PEG_20k prolonged the plasma half-life by five-fold for MOD-K78C and 17-fold for MOD-K264C, respectively. The site-specific mutation together with PEGylation therefore generated MOD derivatives with improved pharmacological properties.

The RNA-dependent RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pivotal antiviral target due to its essential role in viral replication and high conservation. Non-nucleoside analog inhibitors (NNIs) are a promising class of antiviral compounds that target SARS-CoV-2 RdRp, potentially circumventing resistance profiles associated with nucleoside analogues. Although multiple NNIs against SARS-CoV-2 RdRp have been reported, the knowledge regarding the modes of action remains fragmented, and the lack of a standardized cell-free assay has hindered cross-comparison of various NNIs. Notably, whether clinically approved drugs or the NNIs targeting hepatitis C virus (HCV) RdRp (NS5B) can be used to inhibit SARS-CoV-2 RdRp remains largely unexplored, and the chemical and mechanistic diversity of the NNIs are poorly defined. Here, we conducted a high-throughput screening of an FDA-approved drug library using a fluorescence-based RNA polymerase assay to identify novel NNIs targeting SARS-CoV-2 RdRp. Twenty-nine primary hits, along with sixteen previously reported SARS-CoV-2 NNIs and ten HCV RdRp NNIs, were systematically evaluated using a gel-based RdRp inhibition assay, resulting in seventeen positive compounds which include five novel inhibitors (verteporfin, benzbromarone, epigallocatechin gallate, mitoxantrone, and trifarotene) and two NNIs targeting HCV NS5B (JTK-109 and BILB-1941). Parallel dose-response analysis enabled direct efficacy comparison of the NNIs, showing suramin, C646, and baicalein as the most potent inhibitors. Gel mobility shift assay further revealed varied kinetics of the NNIs in disrupting the RdRp-RNA interaction, reflecting their diverse inhibitory mechanisms. Together, our findings expand the repertoire of NNIs against SARS-CoV-2 RdRp, establish a unified framework for comparative evaluation of RdRp inhibitors, and provide insights into the differential impacts of the NNIs on RdRp-RNA complex formation, thereby supporting future development of allosteric antiviral therapeutics.