Compounds producing an effective combinatorial regimen for disruption of HIV‐1 latency

Abstract Highly active antiretroviral therapy (HAART) has improved the outlook for the HIV epidemic, but does not provide a cure. The proposed “shock‐and‐kill” strategy is directed at inducing latent HIV reservoirs, which may then be purged via boosted immune response or targeting infected cells. We describe five novel compounds that are capable of reversing HIV latency without affecting the general T‐cell activation state. The new compounds exhibit synergy for reactivation of latent provirus with other latency‐reversing agents (LRAs), in particular ingenol‐3‐angelate/PEP005. One compound, designated PH02, was efficient at reactivating viral transcription in several cell lines bearing reporter HIV‐1 at different integration sites. Furthermore, it was capable of reversing latency in resting CD4+ T lymphocytes from latently infected aviremic patient cells on HAART, while producing minimal cellular toxicity. The combination of PH02 and PEP005 produces a strong synergistic effect for reactivation, as demonstrated through a quantitative viral outgrowth assay (qVOA), on CD4+ T lymphocytes from HIV‐1‐infected individuals. We propose that the PH02/PEP005 combination may represent an effective novel treatment for abrogating persistent HIV‐1 infection.

Thank you for the submission of your manuscript to EMBO Molecular Medicine. We have now heard back from the three referees whom we asked to evaluate your manuscript.
As you will see from the comments below, the three referees are enthusiastic about the study, even though recommendations are made to provide more mechanistic insights into the mode of action, that I would like to encourage you to address, at least in part.
We would welcome the submission of a revised version within three months for further consideration. Please note that EMBO Molecular Medicine strongly supports a single round of revision and that, as acceptance or rejection of the manuscript will depend on another round of review, your responses should be as complete as possible.
EMBO Molecular Medicine has a "scooping protection" policy, whereby similar findings that are published by others during review or revision are not a criterion for rejection. Should you decide to submit a revised version, I do ask that you get in touch after three months if you have not completed it, to update us on the status.
Please also contact us as soon as possible if similar work is published elsewhere. If other work is published we may not be able to extend the revision period beyond three months.
Please read below for important editorial formatting and consult our author's guidelines for proper formatting of your revised article for EMBO Molecular Medicine.
I look forward to receiving your revised manuscript. ***** Reviewer's comments ***** Referee #1 (Comments on Novelty/Model System): The work is interesting and correctly performed. The authors first screened about 180,000 compounds to identify molecules reactivating a latent HIV provirus in a model cell line. They selected 5 compounds that were further characterized, either alone or in combination with known molecules. One compound, termed PH02, reactivates viral replication in CD4+ T cells from HIV-1 infected individuals under successful antiretroviral treatment. The compound works synergistically with previously characterized compounds, including PEP005. The novelty resides in the identification of PH02 and other compounds, and their synergistic activity against the viral reservoir.
Referee #2 (Comments on Novelty/Model System): The manuscript by Hashemi et al. deals with the issue of identifying compounds that, in combination, could lead latent HIV-1 proviruses out of their latent state. In particular, the authors have identified 5 compounds by high throughput screening (HTS) that reverse latent HIV-1 infection without causing a generalized T cell activation. These novel compounds synergize with previously identified latency reversing agents (LRA), particularly with Ingenol in both cell lines and primary resting CD4+ T cells isolated from pts. receiving cART showing synergy by the standard Q-VOA in the case of the combination between PH02 and PEP005.
The paper is very clearly written and represents and impressive thorough analysis of candidate novel LRA up to their validation in resting CD4 T cells of patients under suppressive cART.
Referee #2 (Remarks): impressive work! it would be impossible to reduce it to a short report. The main limitation is the concept of "shock and kill" as a whole; however, within this hypotethical model this study has been really well conducted.
Referee #3 (Comments on Novelty/Model System): Phenotypic screen for novel latency reactivating agents is wel performed and well described.

Referee #3 (Remarks):
Authors report on a phenotypic screen for novel latency reactivating agents. The screen is performed well and the hits are interesting. The paper is very well written. Unfortunately, although some epigenetic characteristics are tested, the true mechanism of action of the novel compounds is not elucidated. I find this a prerequisite for a paper in EMBO Molecular Medicine. (Remarks): impressive work! it would be impossible to reduce it to a short report. The main limitation is the concept of "shock and kill" as a whole; however, within this hypotethical model this study has been really well conducted.
Referee #3: Phenotypic screen for novel latency reactivating agents is well performed and well described.
(Remarks): Authors report on a phenotypic screen for novel latency reactivating agents. The screen is performed well and the hits are interesting. The paper is very well written. Unfortunately, although some epigenetic characteristics are tested, the true mechanism of action of the novel compounds is not elucidated. I find this a prerequisite for a paper in Embo Molecular Medicine.
We thank all three referees for their encouraging comments; several of the referees commented that this was "impressive work" and overall the screen was well performed. All of the referees have commented that the writing was good, and therefore we have made only minor corrections to spelling and grammar in most of the text throughout the revisions. Referee #2 alludes to "limitations of shock and kill", but without mentioning specifics in their comments. We, like others working in this field, recognize the steep incline that potential shock and kill therapies face for effective treatment, and consequently we have noted this in the Introduction in the paragraph ending "Therefore, it is likely that more advanced combination strategies must be used to produce efficient provirus induction for eradication of cells that produce replication-competent viruses using the shock and kill strategy." Referee #3, notes that we have not described a mechanism for reactivation of HIV latency by thecompounds. We believe it important to point out that this study represents the first analysis of completely synthetic compound libraries where novel compounds were identified that can reactivate HIV latency in cell lines, as well as infected PBMCs from patients, and importantly can also cause induction of replication competent virus from latently infected samples from patients. One previous study describes a screen using a similar number of compounds (~200,000), but this study failed to identify compound s capable of reactivating expression of replication competent virus from patient samples (Micheva-Viteva et al., 2011). Furthermore, we note that most comparable previous screens were performed with much smaller "known drug" libraries or libraries of natural products where mechanistic activity had already been identified or at least suspected.
We agree that it will be important to understand the biochemical effect(s) of the compounds we have identified, and we have spent considerable effort over the past several months towards this goal. Unfortunately none of the most obvious possible mechanisms that could reactivate HIV from latency seem to be affected. We provide additional evidence in the revised manuscript showing that PH02 does not affect global histone acetylation and also does not affect expression of key transcription factors regulating activation from the HIV enhancer. In additional experiments, not described in the manuscript, we have determined, unfortunately, that none of the PH compounds inhibit growth or other obvious phenotypes in yeast. From these efforts we believe that the compounds must affect previously uncharacterized mechanisms for maintenance of viral latency. Identification of mechanism of action for compounds identified in synthetic small molecule libraries is never trivial, and will require detailed analysis of responses using proteomics and genomics, which seems beyond the scope of the present manuscript. Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed reports from the referees that were asked to re-assess it. As you will see the reviewers are now globally supportive and I am pleased to inform you that we will be able to accept your manuscript pending final amendments.
***** Reviewer's comments ***** Referee #1 (Remarks for Author): The manuscript has been improved and the authors have addressed my concerns Referee #3 (Remarks for Author): The lack of MOA dampens my enthusiasm but the work deserves publication so the work can be shared and continued. 1.a. How was the sample size chosen to ensure adequate power to detect a pre--specified effect size? 1.b. For animal studies, include a statement about sample size estimate even if no statistical methods were used.
2. Describe inclusion/exclusion criteria if samples or animals were excluded from the analysis. Were the criteria pre-established?
3. Were any steps taken to minimize the effects of subjective bias when allocating animals/samples to treatment (e.g. randomization procedure)? If yes, please describe.
For animal studies, include a statement about randomization even if no randomization was used.
4.a. Were any steps taken to minimize the effects of subjective bias during group allocation or/and when assessing results (e.g. blinding of the investigator)? If yes please describe. In the pink boxes below, please ensure that the answers to the following questions are reported in the manuscript itself. Every question should be answered. If the question is not relevant to your research, please write NA (non applicable). We encourage you to include a specific subsection in the methods section for statistics, reagents, animal models and human subjects.

definitions of statistical methods and measures:
a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).
Please fill out these boxes ê (Do not worry if you cannot see all your text once you press return) a specification of the experimental system investigated (eg cell line, species name).

B--Statistics and general methods
the assay(s) and method(s) used to carry out the reported observations and measurements an explicit mention of the biological and chemical entity(ies) that are being measured. an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.

Data
the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner. figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way. graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range; Each figure caption should contain the following information, for each panel where they are relevant:

Captions
The data shown in figures should satisfy the following conditions: Source Data should be included to report the data underlying graphs. Please follow the guidelines set out in the author ship guidelines on Data Presentation.

YOU MUST COMPLETE ALL CELLS WITH A PINK BACKGROUND ê
The sample sizes were chosen to obtain a 95% confidence interval of statistical significance between and within the groups of samples.

NA
Yes, for every figure with statistical tests, there were sufficient numbers of replicates to perform these tests.
Yes, a ratio paired t--test and a one--way analysis of variance (ANOVA) were used when appropriate.
Yes, the error bars were reported as standard errors from the standard deviation.