We present an unconventional approach to antiviral drug discovery, which is used to identify potent small molecules against rabies virus. First, we conceptualized viral capsid assembly as occurring via a hostcatalyzed biochemical pathway, in contrast to the classical view of capsid formation by self-assembly. This suggested opportunities for antiviral intervention by targeting previously unappreciated catalytic host proteins, which were pursued. Second, we hypothesized these host proteins to be components of heterogeneous, labile, and dynamic multi-subunit assembly machines, not easily isolated by specific target protein-focused methods. This suggested the need to identify active compounds before knowing the precise protein target. A cell-free translation-based small molecule screen was established to recreate the hypothesized interactions involving newly synthesized capsid proteins as host assembly machine substrates. Hits from the screen were validated by efficacy against infectious rabies virus in mammalian cell culture. Used as affinity ligands, advanced analogs were shown to bind a set of proteins that effectively reconstituted drug sensitivity in the cell-free screen and included a small but discrete subfraction of cellular ATP-binding cassette family E1 (ABCE1), a host protein previously found essential for HIV capsid formation. Taken together, these studies advance an alternate view of capsid formation (as a host-catalyzed biochemical pathway), a different paradigm for drug discovery (whole pathway screening without knowledge of the target), and suggest the existence of labile assembly machines that can be rendered accessible as next-generation drug targets by the means described.assembly intermediate | viral-host interaction | whole pathway screen | drug discovery paradigm | protein heterogeneity

Five different variants of L7/L12 containing single cysteine substitutions, two in the N-terminal (NTD) and three in the C-terminal domain (CTD), were produced, modified with [125I]N-[4-(p-azidosalicylamido)butyl]-3-(2'-pyridyldithio) propionamide ([125I]APDP), a sulfhydryl-specific, heterobifunctional, cleavable photo-cross-linking reagent, and reconstituted into ribosomes. These were irradiated, the total proteins were extracted and reductively cleaved, and the cross-linked proteins were identified. The effect of zero-length disulfide cross-linking on binding and activity was also determined. The same sites in L7/L12 were used to attach a rhodamine dye. The formation of ground-state rhodamine dimers caused the appearance of a new absorption band at 518 nm that was used to estimate the extent of interaction of the probes in the free protein and in complexes with L10. The three sites in the CTD, but not the N-terminal sites, cross-linked to L2 and L5 and to 30S proteins S2, S3, S7, S14, and S18 in a manner influenced by elongation factors. Binding to the ribosome and, therefore, function were blocked by zero-length cross-linking within the NTD, but not the CTD. Binding also disrupted rhodamine dimers in the NTD. No rhodamine dimers formed in the CTD.

The Escherichia coli ribosomal protein, L7/L12, is the most extensively investigated representative of the small, four-copy, dimeric acidic proteins that are found in large ribosomal subunits of all organisms and exist as a conserved quaternary structural element in which two dimers are integrated into the ribosome through binding to a common anchoring protein (1-3). One or both of the L7/L12 dimers form a conspicuous morphological feature on the ribosome known in E. coli as the L7/L12 stalk (4). The proteins can be simply and selectively removed from and reconstituted into the ribosome (5), a property that greatly facilitates experiments of the type reported here in which genetically and biochemically modified proteins replace the wild type.The L7/L12 polypeptide is composed of two distinct organized structural domains linked by a flexible hinge (6, 7), as summarized in Fig. 1. The elongated, helical N-terminal domain, residues 1-33, is responsible for dimer interaction (8), and the globular C-terminal domain, residues 53-120, is necessary for factor binding (9 -11). The C-terminal domains can be cross-linked to each other in different orientations yet retain full functional activity in supporting polypeptide synthesis (12). Flexibility of L7/L12 has been demonstrated in solution by fluorescence techniques (13) and in the ribosome by proton NMR (14, 15), by electron microscopy (16), and with fluorescence probes attached to the C-terminal domains (13,17,18).Immunoelectron microscopy with monoclonal antibodies (19) directly showed the presence of the C-terminal domain at the tip of the stalk and the N-terminal domain at the base of the stalk. This was consistent with earlier demonstrations that the N-terminal domain was responsible for binding of the fulllength L7/L12 to L10 and to the ribosome (9, 10). It was shown that one dimer per particle was sufficient to form a visible stalk (20), despite earlier studies with polyclonal antibodies that had suggested that both dimers were present in the stalk (21). Different studies indicated that the presence of one L7/L12 dimer on the body of the 50 S particle in an extended conformation directed toward the central protuberance (22, 23). Additional evidence that a C-terminal domain can occupy a location not only extended across the body of the ribosome but also near the base of the stalk came from cross-linking between a predetermined location in the C-terminal domain, 25) and also from hinge deletion studies (26,27). A different heterobifunctional reagent, APDP 1 showed a cross-link between Cys-89 and L11 and also with L10 in a lesser extent near the EF-G binding site near the base of the stalk (28). The site-specific cross-linking experiments led to the proposal of a possible "bent" conformation for one of the dimers in which the C-terminal domain could lie on the body of the subunit near the N-terminal domain at the base of the stalk. We asked the question whether there was a preferred surface of the C-terminal domain that made these contacts and designed cysteine prob...

Antiviral compounds displaying remarkable features have been identified by an unconventional drug screen and advanced through animal validation. Efficacy is observed against the six viral families causing most human respiratory viral disease, irrespective of strain, including both influenza (FLUV) and SARS-CoV-2, with cell culture EC50 at or below 100 nM. Survival benefit is demonstrated in pigs against another member of family Coronaviridae, porcine epidemic diarrhea virus (PEDV), and shown equally effective in mild and severe disease. Respiratory syncytial virus (RSV) titer is reduced by drug treatment in cotton rats. A substantial barrier to viral resistance is demonstrated for FLUV. Drug resin affinity chromatography (DRAC) reveals a novel drug target: a multi-protein complex (MPC) formed transiently, in an energy-dependent fashion, and composed of host proteins implicated in both viral lifecycles and manipulation of innate immunity. The protein composition of this host MPC is modified upon viral infection, with increase or decrease of some proteins and appearance or complete loss of others. Valosin-containing protein, also known as Transitional endoplasmatic reticulum ATPase (VCP/p97), is present in the target MPC of uninfected cells and significantly increased in both FLUV and CoV infection. SQSTM1/p62, a key regulator of the autophagy pathway of innate immunity whose dysfunction is implicated in cytokine storm, is i) found in the target MPC from uninfected cells, ii) diminished in DRAC eluates by infection, and iii) restored by drug treatment of infected cells. 14-3-3 is one of likely several proteins that comprise the drug-binding site. Advanced compounds with improved pharmacokinetic (PK) properties and lung exposure are approaching criteria for a Target Product Profile. We propose these novel drug targets to comprise a previously unappreciated molecular basis for homeostasis that is modified by viruses to allow exploitation for viral propagation and is restored by treatment with the therapeutic compounds presented. This discovery has transformative implications for treatment of respiratory viral-related disease, applicable to everything from seasonal FLUV to COVID-19 and future novel respiratory viruses, due to the pan-family nature of drug activity and barrier to resistance development.

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Two structurally-unrelated small molecule chemotypes, represented by compounds PAV-617 and PAV-951, with antiviral activity in cell culture against monkeypox virus (MPXV) and human immunodeficiency virus (HIV) respectively, were studied for anti-cancer efficacy. Each exhibited apparent pan-cancer cytotoxicity, reasonable pharmacokinetics, and non-toxicity in mice at active concentrations. Anti-tumor properties of each compound were validated in mouse xenografts against A549 human lung cancer. The targets of these compounds are unconventional: each binds to a different transient, energy-dependent multi-protein complex containing the protein KAP-1(TRIM28), an allosteric modulator known to broadly regulate mechanisms underlying viral and nonviral disease states including cancer. Treatment with these compounds alters the target multi-protein complexes in a manner consistent with allosteric modulation as their mechanism of action. These compounds appear to remove a block, crucial for cancer survival and progression, on the homeostatic linkage of uncontrolled cellular proliferation to apoptosis. These compounds may provide starting points for development of next-generation non-toxic, cancer therapeutics.

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Rheumatoid arthritis is a chronic crippling disease, where protein-based tumor necrosis factor-alpha (TNF-α) inhibitors show significant relief, but with potentially fatal side effects. A need for a safe, oral, cost-effective small molecule or phyto-pharmaceutical is warranted. BV-9238 is an Ayurvedic poly-herbal formulation containing specialized standardized extracts of Withania somnifera, Boswellia serrata, Zingiber officinale and Curcuma longa. The anti-inflammatory and anti-arthritic effects of BV-9238 were evaluated for inhibition of TNF-α and nitric oxide (NO) production, in lipopolysaccharide-stimulated, RAW 264.7, mouse macrophage cell line. BV-9238 reduced TNF-α and NO production, without any cytotoxic effects. Subsequently, the formulation was tested in adjuvant-induced arthritis (AIA) and carrageenan-induced paw edema (CPE) rat animal models. AIA was induced in rats by injecting Freund's complete adjuvant intra-dermally in the paw, and BV-9238 and controls were administered orally for 21 days. Arthritic scores in AIA study and inflamed paw volume in CPE study were significantly reduced upon treatment with BV-9238. These results suggest that the anti-inflammatory and anti-arthritic effects of BV-9238 are due to its inhibition of TNF-α, and NO, and this formulation shows promise as an alternate therapy for inflammatory disorders where TNF-α and NO play important roles.