Molecular dissection of an inhibitor targeting the HIV integrase dependent preintegration complex nuclear import

Abstract

Human immunodeficiency virus (HIV) continues to be a major contributor to morbidity and mortality worldwide, particularly in developing nations where high cost and logistical issues severely limit the use of current HIV therapeutics. This, combined HIV's high propensity to develop resistance, means that new antiviral agents against novel targets are still urgently required. We previously identified novel anti-HIV agents directed against the nuclear import of the HIV integrase (IN) protein, which plays critical roles in the HIV lifecycle inside the cell nucleus, as well as in transporting the HIV preintegration complex (PIC) into the nucleus. Here we investigate the structure activity relationship of a series of these compounds for the first time, including a newly identified anti-IN compound, budesonide, showing that the extent of binding to the IN core domain correlates directly with the ability of the compound to inhibit IN nuclear transport in a permeabilised cell system. Importantly, compounds that inhibited the nuclear transport of IN were found to significantly decrease HIV viral replication, even in a dividing cell system. Significantly, budesonide or its analogue flunisolide, were able to effect a significant reduction in the presence of specific nuclear forms of the HIV DNA (2-LTR circles), suggesting that the inhibitors work though blocking IN, and potentially PIC, nuclear import. The work presented here represents a platform for further development of these specific inhibitors of HIV replication with therapeutic and prophylactic potential.

Ivermectin as a Broad-Spectrum Host-Directed Antiviral: The Real Deal?

“…Of several compounds subsequently confirmed to be active against IMPα/β1 and possess antiviral activity as a consequence [7,14,29,36], ivermectin has been the best characterized in this regard, and shown to have broad-spectrum activities, a number of which are summarized in Table 1. It was initially shown to inhibit nuclear import not only of IN, but also of simian virus SV40 large tumour antigen (T-ag) and other IMPα/β1-dependent (but not IMPβ1-dependent) cargoes, consistent with the idea that IMPα (not IN) is the direct target [34,35]. Subsequent work has confirmed this, with ivermectin's ability to inhibit the nuclear accumulation of various different host, including NF-kB p65 [37] and viral proteins demonstrated in transfected and infected cell systems (see Table 1) [14,34].…”

“…We identified ivermectin in 2011 in a proof-of-principle, high-throughput screen using recombinantly-expressed proteins and a 1200 compound library for inhibitors of HIV-1 Integrase (IN) recognition by IMPα/β1 [34]; specific inhibitors targeting IMPα/β1 directly (such as ivermectin) and not IN (such as budenoside) were identified using a nested counterscreen strategy [34,35]. Of several compounds subsequently confirmed to be active against IMPα/β1 and possess antiviral activity as a consequence [7,14,29,36], ivermectin has been the best characterized in this regard, and shown to have broad-spectrum activities, a number of which are summarized in Table 1.…”

Multifaceted HIV integrase functionalities and therapeutic strategies for their inhibition

“…IN has been proposed to play additional roles in the HIV-1 lifecycle, including virus particle uncoating after cell entry (246) and PIC nuclear import (247,248 (268) or KPNA2/KPNB1 (269) in vitro displayed comparatively weak antiviral activities of less than 50% inhibition at 100 M and 50% inhibition at ϳ50 -100 M, respectively. To establish IN-KPN interactions as bona fide antiviral targets, it will be important to show that resistance to compounds with 10 -100-fold greater potencies maps to the IN region of HIV-1 pol.…”

Novel Flavivirus Antiviral That Targets the Host Nuclear Transport Importin α/β1 Heterodimer

“…This study is the first to document GW5074′s ability to inhibit flavivirus infection through limiting NS5 nuclear targeting; this adds to a growing body of literature documenting the viability of targeting viral protein nuclear import as a means to inhibit a range of viruses [8,12,13,14,15,16,20,21,25,31,32,33]. It is clear from the present study that inhibitors preventing nuclear import of key viral proteins such as DENV NS5, either by targeting IMPs directly [12,13,20,21,25] as here in the case of GW5074 or the viral component of the host–pathogen interface in the case of 4-HPR [14,31], can be efficacious in limiting flavivirus infection. Figure 6 highlights the different mechanisms of GW5074 and the known anti-DENV agent 4-HPR [14] in blocking NS5–IMPα/β1 interaction, and thereby preventing NS5 nuclear translocation and its subsequent impact on the antiviral response/host mRNA splicing, etc.…”