Xionghao Lin

Inhibition of HIV-1 by curcumin A, a novel curcumin analog

Despite the remarkable success of combination antiretroviral therapy at curtailing HIV progression, emergence of drug-resistant viruses, chronic low-grade inflammation, and adverse effects of combination antiretroviral therapy treatments, including metabolic disorders collectively present the impetus for development of newer and safer antiretroviral drugs. Curcumin, a phytochemical compound, was previously reported to have some in vitro anti-HIV and anti-inflammatory activities, but poor bioavailability has limited its clinical utility. To circumvent the bioavailability problem, we derivatized curcumin to sustain retro-aldol decomposition at physiological pH. The lead compound derived, curcumin A, showed increased stability, especially in murine serum where it was stable for up to 25 hours, as compared to curcumin that only had a half-life of 10 hours. Both curcumin and curcumin A showed similar inhibition of one round of HIV-1 infection in cultured lymphoblastoid (also called CEM) T cells (IC50=0.7 μM). But in primary peripheral blood mononuclear cells, curcumin A inhibited HIV-1 more potently (IC50=2 μM) compared to curcumin (IC50=12 μM). Analysis of specific steps of HIV-1 replication showed that curcumin A inhibited HIV-1 reverse transcription, but had no effect on HIV-1 long terminal repeat basal or Tat-induced transcription, or NF-κB-driven transcription at low concentrations that affected reverse transcription. Finally, we showed curcumin A induced expression of HO-1 and decreased cell cycle progression of T cells. Our findings thus indicate that altering the core structure of curcumin could yield more stable compounds with potent antiretroviral and anti-inflammatory activities.

Increased iron export by ferroportin induces restriction of HIV-1 infection in sickle cell disease

The low incidence of HIV-1 infection in patients with sickle cell disease (SCD) and inhibition of HIV-1 replication in vitro under the conditions of low intracellular iron or heme treatment suggests a potential restriction of HIV-1 infection in SCD. We investigated HIV-1 ex vivo infection of SCD peripheral blood mononuclear cells (PBMCs) and found that HIV-1 replication was inhibited at the level of reverse transcription (RT) and transcription. We observed increased expression of heme and iron-regulated genes, previously shown to inhibit HIV-1, including ferroportin, IKBα, HO-1, p21, and SAM domain and HD domain-containing protein 1 (SAMHD1). HIV-1 inhibition was less pronounced in hepcidin-treated SCD PBMCs and more pronounced in the iron or iron chelators treated, suggesting a key role of iron metabolism. In SCD PBMCs, labile iron levels were reduced and protein levels of ferroportin, HIF-1α, IKBα, and HO-1 were increased. Hemin treatment induced ferroportin expression and inhibited HIV-1 in THP-1 cells, mimicking the HIV-1 inhibition in SCD PBMCs, especially as hepcidin similarly prevented HIV-1 inhibition. In THP-1 cells with knocked down ferroportin, IKBα, or HO-1 genes but not HIF-1α or p21, HIV-1 was not inhibited by hemin. Activity of SAMHD1-regulatory CDK2 was decreased, and SAMHD1 phosphorylation was reduced in SCD PBMCs and hemin-treated THP-1 cells, suggesting SAMHD1-mediated HIV-1 restriction in SCD. Our findings point to ferroportin as a trigger of HIV-1 restriction in SCD settings, linking reduced intracellular iron levels to the inhibition of CDK2 activity, reduction of SAMHD1 phosphorylation, increased IKBα expression, and inhibition of HIV-1 RT and transcription.

Activation of HIV-1 with Nanoparticle-Packaged Small-Molecule Protein Phosphatase-1-Targeting Compound

Complete eradication of HIV-1 infection is impeded by the existence of latent HIV-1 reservoirs in which the integrated HIV-1 provirus is transcriptionally inactive. Activation of HIV-1 transcription requires the viral Tat protein and host cell factors, including protein phosphatase-1 (PP1). We previously developed a library of small compounds that targeted PP1 and identified a compound, SMAPP1, which induced HIV-1 transcription. However, this compound has a limited bioavailability in vivo and may not be able to reach HIV-1-infected cells and induce HIV-1 transcription in patients. We packaged SMAPP1 in polymeric polyethylene glycol polymethyl methacrylate nanoparticles and analyzed its release and the effect on HIV-1 transcription in a cell culture. SMAPP1 was efficiently packaged in the nanoparticles and released during a 120-hr period. Treatment of the HIV-1-infected cells with the SMAPP1-loaded nanoparticles induced HIV-1 transcription. Thus, nanoparticles loaded with HIV-1-targeting compounds might be useful for future anti-HIV-1 therapeutics.

Phenyl-1-Pyridin-2yl-Ethanone-Based Iron Chelators Increase IκB-α Expression, Modulate CDK2 and CDK9 Activities, and Inhibit HIV-1 Transcription

ABSTRACT HIV-1 transcription is activated by the Tat protein, which recruits CDK9/cyclin T1 to the HIV-1 promoter. CDK9 is phosphorylated by CDK2, which facilitates formation of the high-molecular-weight positive transcription elongation factor b (P-TEFb) complex. We previously showed that chelation of intracellular iron inhibits CDK2 and CDK9 activities and suppresses HIV-1 transcription, but the mechanism of the inhibition was not understood. In the present study, we tested a set of novel iron chelators for the ability to inhibit HIV-1 transcription and elucidated their mechanism of action. Novel phenyl-1-pyridin-2yl-ethanone (PPY)-based iron chelators were synthesized and examined for their effects on cellular iron, HIV-1 inhibition, and cytotoxicity. Activities of CDK2 and CDK9, expression of CDK9-dependent and CDK2-inhibitory mRNAs, NF-κB expression, and HIV-1- and NF-κB-dependent transcription were determined. PPY-based iron chelators significantly inhibited HIV-1, with minimal cytotoxicity, in cultured and primary cells chronically or acutely infected with HIV-1 subtype B, but they had less of an effect on HIV-1 subtype C. Iron chelators upregulated the expression of IκB-α, with increased accumulation of cytoplasmic NF-κB. The iron chelators inhibited CDK2 activity and reduced the amount of CDK9/cyclin T1 in the large P-TEFb complex. Iron chelators reduced HIV-1 Gag and Env mRNA synthesis but had no effect on HIV-1 reverse transcription. In addition, iron chelators moderately inhibited basal HIV-1 transcription, equally affecting HIV-1 and Sp1- or NF-κB-driven transcription. By virtue of their involvement in targeting several key steps in HIV-1 transcription, these novel iron chelators have the potential for the development of new therapeutics for the treatment of HIV-1 infection.

Inhibition of HIV-1 infection in humanized mice and metabolic stability of protein phosphatase-1-targeting small molecule 1E7-03.

We recently identified the protein phosphatase-1 - targeting compound, 1E7-03 which inhibited HIV-1 in vitro. Here, we investigated the effect of 1E7-03 on HIV-1 infection in vivo by analyzing its metabolic stability and antiviral activity of 1E7-03 and its metabolites in HIV-1 infected NSG-humanized mice. 1E7-03 was degraded in serum and formed two major degradation products, DP1 and DP3, which bound protein phosphatase-1 in vitro. However, their anti-viral activities were significantly reduced due to inefficient cell permeability. In cultured cells, 1E7-03 reduced expression of several protein phosphatase-1 regulatory subunits including Sds22 as determined by a label free quantitative proteomics analysis. In HIV-1-infected humanized mice, 1E7-03 significantly reduced plasma HIV-1 RNA levels, similar to the previously described HIV-1 transcription inhibitor F07#13. We synthesized a DP1 analog, DP1-07 with a truncated side chain, which showed improved cell permeability and longer pharmacokinetic retention in mice. But DP1-07 was less efficient than 1E7-03 as a HIV-1 inhibitor both in vitro and in vivo, indicating that the full side chain of 1E7-03 was essential for its anti-HIV activity. Analysis of 1E7-03 stability in plasma and liver microsomes showed that the compound was stable in human, primate and ferret plasma but not in rodent plasma. However, 1E7-03 was not stable in human liver microsomes. Our findings suggest that 1E7-03 is a good candidate for future development of HIV-1 transcription inhibitors. Further structural modification and advanced formulations are needed to improve its metabolic stability and enhance its antiviral activity in non-human primate animals and humans.

Protein Phosphatase 1–Targeting Small-Molecule C31 Inhibits Ebola Virus Replication

Background Ebola virus (EBOV) infection causes severe hemorrhagic fever. EBOV transcription is controlled by host protein phosphatase 1 (PP1), which dephosphorylates VP30 protein. We previously developed 1E7-03, a compound targeting a noncatalytic site of PP1 that induced VP30 phosphorylation and inhibited EBOV transcription. Here, we attempted to further improve 1E7-03, which was not stable in murine serum. Results High-throughput screening with EBOV-green fluorescent protein was conducted on 72 1E7-03 analogs and identified 6 best inhibitory and the least toxic compounds. A parallel in silico screening of compounds from the ZINC database by docking to PP1 identified the best-binding compound C31, which was also present among the top 6 compounds found in the viral screen. C31 showed the best EBOV inhibitory activity among the top 6 compounds and also inhibited EBOV minigenome. C31 bound to the PP1 C-terminal groove in vitro and increased VP30 phosphorylation in cultured cells. C31 demonstrated improved stability in mouse plasma and cell permeability, compared with 1E7-03. It was also detected for 24 hours after injection in mice. Conclusion C31 represents a novel PP1-targeting EBOV inhibitor with improved pharmacological properties that can be further evaluated for future antifiloviral therapy.

Proteomic Profile of Tears for the Diagnosis of Uveitis Associated with Juvenile Idiopathic Arthritis: Setting Targets to Achieve Results

The paper presents epidemiologic and pathophysiological aspects of the problem statement for early recognition of Uveitis (Uv) associated with Juvenile Idiopathic Arthritis (JIA) in terms of the proteomic profile of tears as well as the results of an attempt to solve this problem by means of the Tandem Mass-Spectrometry (TMS). The solution of this problem is of the highest relevance due to revolutionary changes in treatment strategies after introducing highly effective biologics. Content analysis of literature reviews reveals the following: 1. the incidence of JIA-Uv in the Northwest Federal District of Russian Federation averages 0.5-0.7 per 100 000 of children with the prevalence being ten-fold higher than incidence, 2. without Methotrexate treatment 4-7 years after the diagnosis of JIA-Uv cataract is revealed in 35-40% of children and in 5% – glaucoma as well, 3. even with Methotrexate in 28-40% of children the complications of JIA-Uv inevitably will be revealed with blurred vision in 10-36% of children, 4. timely diagnosis of JIA-Uv and adequate treatment reduce the risk of complications by 4% per year, 5. current medical care system reveals in one third of children already the complications of JIA-Uv. Revelation in tears of the motif mode for protein interaction network, triggering mobilization/inhibition of cells which moderate Uv would contradict the traditional point of view on existing natural anatomic and physiologic barriers, isolating the intraocular space, but however seems to be possible since JIA is a systemic disease and Uv leads to damage of the blood-retinal barriers. To reveal protein biomarkers of JIA-Uv tears of 31 children aged 2-17 years were studied: 17 – chronic JIA-Uv, 4 – JIA without Uv, 4 – idiopathic Uv, 3 – systemic vasculitis, 3 – healthy children. We used the current clinical guidelines and standards to diagnose the pathology and TMS with hierarchical clustering methodology for protein identification: nano C18 column attached to Shimadzu nano LC coupled in-line to LTQ Orbitrap XL tandem mass spectrometer, data-dependent 4-event scan method, a survey FT-MS parent scan followed by sequential data dependent FT-MS/MS scans on the three most abundant peptide ions. Proteins were identified from the mass spectra results with Proteome Discoverer 1.2 software for protein database search using the International Protein Index (IPI) and Human Protein Database. Quantification was conducted using SIEVE 2.0 after normalization to albumin keeping in mind the validity of proportional change of its concentration after stimulation of lacri-mation. Data from SIEVE were exported to IPA (Ingenuity Pathway Analysis) for filtering. The extracellular proteins selected in Ingenuity were further analyzed for disease relation and networks formation. TMS revealed more than 3000 proteins in tears and 300 of them have been considered to be the first row candidates to be biomarkers of JIA-Uv. The top two proteins, lactoferrin and lipocalin were upregulated over ten-fold in children with Uv. Pathway analysis placed these proteins into the inflammation-related IL-1 and TNF-α related networks which also included proteins involved in the development of endothelial dysfunction, inflammation and retinopathy. In addition, IL-23, which was previously linked to Uveitis, was found to be upregulated. Taken together, our proof-of-principle study presents a novel and yet untested approach for detection of early biomarkers of Uveitis and identified several candidate proteins.