Experimental veterinary SARS-CoV-2 vaccine cross neutralization of the Delta (B.1.617.2) variant virus in cats.

SARS-CoV-2 has exhibited varying pathogenesis in a variety of Mammalia family's including Canidae, Mustelidae, Hominidae, Cervidae, Hyaenidae, and Felidae. Novel SARS-CoV-2 variants characterized by spike protein mutations have recently resulted in clinical and epidemiological concerns, as they potentially have increased infectious rates, increased transmission, or reduced neutralization by antibodies produced via vaccination. Many variants have been identified at this time, but the variant of continuing concern has been the Delta variant (B.1.617.2), due to its increased transmissibility and infectious rate. Felines vaccinated using an experimental SARS-CoV-2 spike protein-based veterinary vaccine mounted a robust immune response to the SARS-CoV-2 spike protein. Using a reporter virus particle system and feline serum, we have verified that vaccinated felines produce antibodies that neutralize the SARS-CoV-2 Wuhan strain and variant B.1.617.2 at comparable levels.

Identification and Optimization of a Novel HIV-1 Integrase Inhibitor.

Human immunodeficiency virus-1 (HIV-1) is the causative agent of acquired immunodeficiency syndrome (AIDS). HIV-1, like all retroviruses, stably integrates its vDNA copy into host chromatin, a process allowing for permanent infection. This essential step for HIV-1 replication is catalyzed by viral integrase (IN) and aided by cellular protein LEDGF/p75. In addition, IN is also crucial for proper virion maturation as it interacts with the viral RNA genome to ensure encapsulation of ribonucleoprotein complexes within the protective capsid core. These key functions make IN an attractive target for the development of inhibitors with various mechanisms of action. We conducted a high-throughput screen (HTS) of ∼370,000 compounds using a homogeneous time-resolved fluorescence-based assay capable of capturing diverse inhibitors targeting multifunctional IN. Our approach revealed chemical scaffolds containing diketo acid moieties similar to IN strand transfer inhibitors (INSTIs) as well as novel compounds distinct from all current IN inhibitors including INSTIs and allosteric integrase inhibitors (ALLINIs). Specifically, our HTS resulted in the discovery of compound 12, with a novel IN inhibitor scaffold amenable for chemical modification. Its more potent derivative 14e similarly inhibited catalytic activities of WT and mutant INs containing archetypical INSTI- and ALLINI-derived resistant substitutions. Further SAR-based optimization resulted in compound 22 with an antiviral EC50 of ∼58 µM and a selectivity index of >8500. Thus, our studies identified a novel small-molecule scaffold for inhibiting HIV-1 IN, which provides a promising platform for future development of potent antiviral agents to complement current HIV-1 therapies.

HIV-1 integrase tetramers are the antiviral target of pyridine-based allosteric integrase inhibitors.

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a promising new class of antiretroviral agents that disrupt proper viral maturation by inducing hyper-multimerization of IN. Here we show that lead pyridine-based ALLINI KF116 exhibits striking selectivity for IN tetramers versus lower order protein oligomers. IN structural features that are essential for its functional tetramerization and HIV-1 replication are also critically important for KF116 mediated higher-order IN multimerization. Live cell imaging of single viral particles revealed that KF116 treatment during virion production compromises the tight association of IN with capsid cores during subsequent infection of target cells. We have synthesized the highly active (-)-KF116 enantiomer, which displayed EC50 of ~7 nM against wild type HIV-1 and ~10 fold higher, sub-nM activity against a clinically relevant dolutegravir resistant mutant virus suggesting potential clinical benefits for complementing dolutegravir therapy with pyridine-based ALLINIs.

HIV-1 inserts its genetic code into human genomes, turning healthy cells into virus factories. To do this, the virus uses an enzyme called integrase. Front-line treatments against HIV-1 called "integrase strand-transfer inhibitors" stop this enzyme from working. These inhibitors have helped to revolutionize the treatment of HIV/AIDS by protecting the cells from new infections. But, the emergence of drug resistance remains a serious problem. As the virus evolves, it changes the shape of its integrase protein, substantially reducing the effectiveness of the current therapies. One way to overcome this problem is to develop other therapies that can kill the drug resistant viruses by targeting different parts of the integrase protein. It should be much harder for the virus to evolve the right combination of changes to escape two or more treatments at once. A promising class of new compounds are "allosteric integrase inhibitors". These chemical compounds target a part of the integrase enzyme that the other treatments do not yet reach. Rather than stopping the integrase enzyme from inserting the viral code into the human genome, the new inhibitors make integrase proteins clump together and prevent the formation of infectious viruses. At the moment, these compounds are still experimental. Before they are ready for use in people, researchers need to better understand how they work, and there are several open questions to answer. Integrase proteins work in groups of four and it is not clear how the new compounds make the integrases form large clumps, or what this does to the virus. Understanding this should allow scientists to develop improved versions of the drugs. To answer these questions, Koneru et al. first examined two of the new compounds. A combination of molecular analysis and computer modelling revealed how they work. The compounds link many separate groups of four integrases with each other to form larger and larger clumps, essentially a snowball effect. Live images of infected cells showed that the clumps of integrase get stuck outside of the virus's protective casing. This leaves them exposed, allowing the cell to destroy the integrase enzymes. Koneru et al. also made a new compound, called (-)-KF116. Not only was this compound able to tackle normal HIV-1, it could block viruses resistant to the other type of integrase treatment. In fact, in laboratory tests, it was 10 times more powerful against these resistant viruses. Together, these findings help to explain how allosteric integrase inhibitors work, taking scientists a step closer to bringing them into the clinic. In the future, new versions of the compounds, like (-)-KF116, could help to tackle drug resistance in HIV-1.

Resistance to pyridine-based inhibitor KF116 reveals an unexpected role of integrase in HIV-1 Gag-Pol polyprotein proteolytic processing.

The pyridine-based multimerization selective HIV-1 integrase (IN) inhibitors (MINIs) are a distinct subclass of allosteric IN inhibitors. MINIs potently inhibit HIV-1 replication during virion maturation by inducing hyper- or aberrant IN multimerization but are largely ineffective during the early steps of viral replication. Here, we investigated the mechanism for the evolution of a triple IN substitution (T124N/V165I/T174I) that emerges in cell culture with a representative MINI, KF116. We show that HIV-1 NL4-3(IN T124N/V165I/T174I) confers marked (>2000-fold) resistance to KF116. Two IN substitutions (T124N/T174I) directly weaken inhibitor binding at the dimer interface of the catalytic core domain but at the same time markedly impair HIV-1 replication capacity. Unexpectedly, T124N/T174I IN substitutions inhibited proteolytic processing of HIV-1 polyproteins Gag and Gag-Pol, resulting in immature virions. Strikingly, the addition of the third IN substitution (V165I) restored polyprotein processing, virus particle maturation, and significant levels of replication capacity. These results reveal an unanticipated role of IN for polyprotein proteolytic processing during virion morphogenesis. The complex evolutionary pathway for the emergence of resistant viruses, which includes the need for the compensatory V165I IN substitution, highlights a relatively high genetic barrier exerted by MINI KF116. Additionally, we have solved the X-ray structure of the drug-resistant catalytic core domain protein, which provides means for rational development of second-generation MINIs.

Computational and synthetic approaches for developing Lavendustin B derivatives as allosteric inhibitors of HIV-1 integrase.

Through structure-based virtual screening and subsequent activity assays of selected natural products, Lavendustin B was previously identified as an inhibitor of HIV-1 integrase (IN) interaction with its cognate cellular cofactor, lens epithelium-derived growth factor (LEDGF/p75). In order to improve the inhibitory potency we have employed in silico-based approaches. Particularly, a series of new analogues was designed and docked into the LEDGF/p75 binding pocket of HIV-1 IN. To identify promising leads we used the Molecular Mechanics energies combined with the Generalized Born and Surface Area continuum solvation (MM-GBSA) method, molecular dynamics simulations and analysis of hydrogen bond occupancies. On the basis of these studies, six analogues of Lavendustine B, containing the benzylamino-hydroxybenzoic scaffold, were selected for synthesis and structure activity-relationship (SAR) studies. Our results demonstrated a good correlation between computational and experimental data, and all six analogues displayed an improved potency for inhibiting IN binding to LEDGF/p75 in vitro to respect to the parent compound Lavendustin B. Additionally, these analogs show to inhibit weakly LEDGF/p75-independent IN catalytic activity suggesting a multimodal allosteric mechanism of action. Nevertheless, for the synthesized compounds similar profiles for HIV-1 inhibition and cytoxicity were highlighted. Taken together, our studies elucidated the mode of action of Lavendustin B analogs and provided a path for their further development as a new promising class of HIV-1 integrase inhibitors.

Allosteric HIV-1 integrase inhibitors promote aberrant protein multimerization by directly mediating inter-subunit interactions: Structural and thermodynamic modeling studies.

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) bind at the dimer interface of the IN catalytic core domain (CCD), and potently inhibit HIV-1 by promoting aberrant, higher-order IN multimerization. Little is known about the structural organization of the inhibitor-induced IN multimers and important questions regarding how ALLINIs promote aberrant IN multimerization remain to be answered. On the basis of physical chemistry principles and from our analysis of experimental information, we propose that inhibitor-induced multimerization is mediated by ALLINIs directly promoting inter-subunit interactions between the CCD dimer and a C-terminal domain (CTD) of another IN dimer. Guided by this hypothesis, we have built atomic models of inter-subunit interfaces in IN multimers by incorporating information from hydrogen-deuterium exchange (HDX) measurements to drive protein-protein docking. We have also developed a novel free energy simulation method to estimate the effects of ALLINI binding on the association of the CCD and CTD. Using this structural and thermodynamic modeling approach, we show that multimer inter-subunit interface models can account for several experimental observations about ALLINI-induced multimerization, including large differences in the potencies of various ALLINIs, the mechanisms of resistance mutations, and the crucial role of solvent exposed R-groups in the high potency of certain ALLINIs. Our study predicts that CTD residues Tyr226, Trp235 and Lys266 are involved in the aberrant multimer interfaces. The key finding of the study is that it suggests the possibility of ALLINIs facilitating inter-subunit interactions between an external CTD and the CCD-CCD dimer interface.

The Competitive Interplay between Allosteric HIV-1 Integrase Inhibitor BI/D and LEDGF/p75 during the Early Stage of HIV-1 Replication Adversely Affects Inhibitor Potency.

Allosteric HIV-1 integrase inhibitors (ALLINIs) have recently emerged as a promising class of antiretroviral agents and are currently in clinical trials. In infected cells, ALLINIs potently inhibit viral replication by impairing virus particle maturation but surprisingly exhibit a reduced EC50 for inhibiting HIV-1 integration in target cells. To better understand the reduced antiviral activity of ALLINIs during the early stage of HIV-1 replication, we investigated the competitive interplay between a potent representative ALLINI, BI/D, and LEDGF/p75 with HIV-1 integrase. While the principal binding sites of BI/D and LEDGF/p75 overlap at the integrase catalytic core domain dimer interface, we show that the inhibitor and the cellular cofactor induce markedly different multimerization patterns of full-length integrase. LEDGF/p75 stabilizes an integrase tetramer through the additional interactions with the integrase N-terminal domain, whereas BI/D induces protein-protein interactions in C-terminal segments that lead to aberrant, higher-order integrase multimerization. We demonstrate that LEDGF/p75 binds HIV-1 integrase with significantly higher affinity than BI/D and that the cellular protein is able to reverse the inhibitor induced aberrant, higher-order integrase multimerization in a dose-dependent manner in vitro. Consistent with these observations, alterations of the cellular levels of LEDGF/p75 markedly affected BI/D EC50 values during the early steps of HIV-1 replication. Furthermore, genome-wide sequencing of HIV-1 integration sites in infected cells demonstrate that LEDGF/p75-dependent integration site selection is adversely affected by BI/D treatment. Taken together, our studies elucidate structural and mechanistic details of the interplay between LEDGF/p75 and BI/D during the early stage of HIV-1 replication.