Novel HDAC Inhibitors Disrupt HIV-1 Latency Danielle Zhou, Austin Niu, Rashmi Ramani, Dr. George Kyei

Department of Molecular Oncology, Washington University School of Medicine

Abstract

Introduction

Results

Implications

References

Funding Sources: Department of Molecular Oncology Summer Undergraduate Research Fellowship

The latent nature of HIV-1 residing in CD4+ T-cells remains a significant barrier to the eradication of the virus. Development of ART (Antiretroviral Therapy) has allowed HIV suppression, but there is no sterilizing cure. The development of LRAs (Latency Reversing Agents) enables elimination of the latent reservoir by reactivating the provirus, allowing immune cytopathic effects to clear out the infection without globally reactivating the cell. Previous studies found that one type of LRA, Histone Deacetylase (HDAC) inhibitors, is capable of inducing activation of the provirus in various models. Further testing revealed that the specific drug compound MC2625, a Class 1 HDAC specific inhibitor, strongly implicated reactivation in latent HIV-1. In this study, we performed an MTT assay and Western Blot, and generated a dose response curve to analyze the properties of this compound and its ability to reactivate HIV-1. Using the J-Lat 10.6 model of latency, we found that the compound, while able to potently reactivate HIV-1, was somewhat toxic at concentrations that generated an effect.

In the dose response curve, a plateau was reached around 700 nM, showing that the drug The MTT assay revealed levels of toxicity at 2000+ nM, which means that the drug may be slightly toxic at these high concentrations. The Western Blot confirmed the production of viral proteins.

The results have identified MC2625 as a potentially significant LRA compound for the reactivation of HIV-1. .

1. Xing, Sifei, and Robert F. Siliciano. "Targeting HIV Latency: Pharmacologic Strategies toward Eradication." Drug Discovery Today 18, no. 0 (2013): 541-51. Accessed January 21, 2015. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3672351/.

Future Research MC2625 can be further tested to synergize with different compounds in order to more effectively reduce the size of latent HIV-1 reservoir. Different cell lines and models of latency testing can be implemented to for validation. In the long term, successful candidates may eventually reach Phase I clinical trials. The findings from this research aim to reduce the size of latent HIV reservoir, possibly enough so that patients can be taken off of expensive ART regimens. The implications of this project will hopefully improve the health and well-being of HIV positive individuals. HDACs are a class of enzymes that de-acetylate

histones, resulting in local gene repression. This makes them a vital key to the repression of HIV-1. The Class 1 HDAC MC2625 has shown implications of reactivation, but due to the limited reach of previous research, not much is currently known about the compound.

Figure  1:  Dose  Response  Curve  in  J-­‐Lat  10.6  Model  A

B

Figure  2:  Toxicity  Assay  Using  MTT  A

B

Figure  3:  Western  Blot  

Methods The J-Lat 10.6 cell line was used to model a dose response curve in triplicate, measuring % GFP positive cells against drug concentration, until a plateau was reached. An MTT assay profile was developed to measure the toxicity level of increasing drug concentrations, and analyzed by FACS (Fluorescent Activated Cell Sorting). A BCA assay was performed in preparation of a Western Blot analysis, which measures the protein band probing for Actin and Gag within DMSO, TNF-alpha, SAHA, and MC2625 to confirm validity of results.

DMSO TNF-α SAHA MC2625 DMSO TNF-α SAHA MC2625

Anti-P24 Anti-Actin

300 nM 1200 nM 300 nM 1200 nM

25 KD

25 KD