Quantum Mechanical-Based Stability Evaluation of Crystal Structures for HIV-Targeted Drug Cabotegravir

The long-acting parenteral formulation of the HIV integrase inhibitor cabotegravir (GSK744) is currently being developed to prevent HIV infections, benefiting from infrequent dosing and high efficacy. The crystal structure can affect the bioavailability and efficacy of cabotegravir. However, the stability determination of crystal structures of GSK744 have remained a challenge. Here, we introduced an ab initio protocol to determine the stability of the crystal structures of pharmaceutical molecules, which were obtained from crystal structure prediction process starting from the molecular diagram. Using GSK744 as a case study, the ab initio predicted that Gibbs free energy provides reliable further refinement of the predicted crystal structures and presents its capability for becoming a crystal stability determination approach in the future. The proposed work can assist in the comprehensive screening of pharmaceutical design and can provide structural predictions and stability evaluation for pharmaceutical crystals.

Anti-HIV Integrase Inhibitors as New Candidates for the Treatment of COVID-19: A Narrative Literature Review

Background: The initial reports of a contagious novel Severe Acute Respiratory Syndrome – Coronavirus-2 (SARS-CoV-2) were proclaimed by Wuhan, Hubei province, China. This pathogen quickly became a health concern due to the World Health Organization's (WHO) alarm of its pandemic essence. Hence, there is an urgent need for efficacious and curative therapy against COVID-19. Objective: Theoretically, repurposing anti-viral drugs, specifically HIV treatments, could help the urgent need for treating COVID-19 due to the structural similarities of their critical enzyme substrates. Integrase inhibitors are a category of anti-HIV drugs that inhibit integrase strand transfer. In this review, we investigate the binding affinity and stability of raltegravir, dolutegravir, bictegravir, and elvitegravir in interactions with crucial enzymes of coronavirus. Methods: A literature search was conducted using scientific databases such as Web of Science, Medline (PubMed), Scopus, Google Scholar, and Embase from commencement to September 2020. The most relevant articles regarding the potential effects of integrase inhibitors against COVID-19 were gathered. Ultimately, ten original articles related to the searched terms were selected for this narrative review. Results: Apparently, in addition to the recent drugs prescribed to cure SARS-CoV-2, integrase inhibitors are promising drugs for repurposing in COVID-19 treatment. Several studies on raltegravir, dolutegravir, bictegravir and elvitegravir were conducted using virtual screening to guess either they are effective or not. Encouraging results were mostly reported for raltegravir and dolutegravir. Nevertheless, bictegravir and elvitegravir need more investigations. Conclusion: Further experimental and clinical studies of antiviral drugs are necessary to introduce appropriate treatment options for COVID-19.

Targeting the Integrated Stress Response Kinase GCN2 to Modulate Retroviral Integration

Multiple viral targets are now available in the clinic to fight HIV infection. Even if this targeted therapy is highly effective at suppressing viral replication, caregivers are facing growing therapeutic failures in patients due to resistance, with or without treatment-adherence glitches. Accordingly, it is important to better understand how HIV and other retroviruses replicate in order to propose alternative antiviral strategies. Recent studies have shown that multiple cellular factors are implicated during the integration step and, more specifically, that integrase can be regulated through post-translational modifications. We have shown that integrase is phosphorylated by GCN2, a cellular protein kinase of the integrated stress response, leading to a restriction of HIV replication. In addition, we found that this mechanism is conserved among other retroviruses. Accordingly, we developed an in vitro interaction assay, based on the AlphaLISA technology, to monitor the integrase-GCN2 interaction. From an initial library of 133 FDA-approved molecules, we identified nine compounds that either inhibited or stimulated the interaction between GCN2 and HIV integrase. In vitro characterization of these nine hits validated this pilot screen and demonstrated that the GCN2-integrase interaction could be a viable solution for targeting integrase out of its active site.

Prevalence of resistance mutations associated with integrase inhibitors in therapy-naive HIV-positive patients in Hungary

Widespread introduction of HIV integrase inhibitors into clinical care may result in appearance of drug resistance mutations affecting treatment outcome. The aim of our study was to monitor the resistance patterns of integrase inhibitors beside protease and reverse transcriptase inhibitors in newly diagnosed therapy-naive HIV-positive patients in Hungary between 2017 and 2019.Genotype-based resistance testing of HIV integrase, protease and reverse transcriptase was performed by amplification and Sanger population sequencing from plasma samples. Drug resistance mutations were identified by the algorithm of Stanford HIV Drug Resistance Database.Potentially transmitted, non-polymorphic integrase major mutation was detected in 1 out of 249 samples, while accessory mutations were observed in further 31 patients (12.4%). The overall prevalence of transmitted drug resistance (TDR) mutations related to protease and reverse transcriptase inhibitors was 5.8% (10/173) between the end of 2017 and 2019. Nucleoside reverse transcriptase inhibitor associated resistance mutations were the most frequent indicators of TDR (6/173; 3.5%), followed by resistance mutations associated with protease (3/173; 1.7%) and non-nucleoside reverse transcriptase inhibitors (2/173, 1.2%).The first detection of integrase major mutation and the changing patterns of other resistance mutations in Hungarian untreated HIV-positive population indicate the necessity of continuous molecular surveillance of Hungarian HIV epidemic.

RETRACTED: A low-cost in-house HIV integrase strand transfer inhibitor drug resistance test for resource-limited settings [version 1; peer review: awaiting peer review]

At the request of the authors, the article titled 'A low-cost in-house HIV integrase strand transfer inhibitor drug resistance test for resource-limited settings' ([version 1; peer review: awaiting peer review]. F1000Research 2021, 10:260, https://doi.org/10.12688/f1000research.28404.1) has been retracted from F1000Research. Since publication, it has come to the attention of the authors that the primers described in Table 1 were incorrect. As this article contains information which should not be publicly available the content of the article has been removed. The authors apologise for this honest error, and intend to republish the article with the correct primer information. Unfortunately, Dr Graeme B. Jacobs has passed away since publication of version 1 of this article.

Six-Step Gram Scale Synthesis of the HIV Integrase Inhibitor Dolutegravir Sodium

A short and practical synthesis for preparing the active pharmaceutical ingredient dolutegravir sodium was investigated. The convergent strategy developed herein starts from 3-(R)-amino-1- butanol and builds up the BC ring system in 76% isolated yield over four steps. Ring A was constructed by a one-pot 1,4-addition to diethyl-(2E/Z)-2-(ethoxymethylidene)-3-oxobutandioate and subsequent MgBr2·OEt2-mediated regioselective cyclization. Amide formation with 2,4- difluorobenzylamine was either performed from the carboxylic acid or through aminolysis of the corresponding ester precursor. Final salt formation afforded dolutegravir sodium in 48–51% isolated yield (HPLC-purity: 99.7–99.9%) over six linear steps.<br>

Six-Step Gram Scale Synthesis of the HIV Integrase Inhibitor Dolutegravir Sodium

A short and practical synthesis for preparing the active pharmaceutical ingredient dolutegravir sodium was investigated. The convergent strategy developed herein starts from 3-(R)-amino-1- butanol and builds up the BC ring system in 76% isolated yield over four steps. Ring A was constructed by a one-pot 1,4-addition to diethyl-(2E/Z)-2-(ethoxymethylidene)-3-oxobutandioate and subsequent MgBr2·OEt2-mediated regioselective cyclization. Amide formation with 2,4- difluorobenzylamine was either performed from the carboxylic acid or through aminolysis of the corresponding ester precursor. Final salt formation afforded dolutegravir sodium in 48–51% isolated yield (HPLC-purity: 99.7–99.9%) over six linear steps.<br>

A low-cost in-house HIV integrase strand transfer inhibitor drug resistance test for resource-limited settings

Background: HIV-1 drug resistance testing (DRT) is vital for monitoring of individual patient treatment outcomes and for public health surveillance. Access to HIV-1 DRT is limited in resource-poor settings, including Kenya due to its costly nature. Recent inclusion of integrase strand transfer inhibitors (INSTIs) in first-line treatment for all people living with HIV-1 (PLHIV-1) underscores the need for an INSTI DRT. This study aims to validate a cost-effective in-house DRT method to detect HIV-1 Integrase resistance-associated mutations (RAMs) using HIV positive plasma derived samples Methods: Thirty-six plasma derived samples were used to assess the performance characteristics including accuracy, precision, reproducibility and amplification sensitivity of an in-house method in comparison with a reference assay. Cost estimation per test followed an incremental ingredient costing approach. Clinical application of the in-house test was evaluated on a plasma sample from a patient failing an INSTI-based regimen. Results: Comparison of the in-house and reference assay gave mean nucleotide and amino acid sequence identity of 99.49 %, CI [99.21- 99.77] and 99%, CI [98.58, 99.42] respectively. Complete concordance was observed by both assays in detection of the T97TA INSTI RAM. Precision and reproducibility assessment revealed mean nucleotide sequence identities of 100% and 99.14% respectively. The amplification sensitivity was 100% for samples with VL> 1000 copies/mL (n=8) and 50% for samples with VL<1000 copies/mL. Two major (G118R and E188K) and two accessory INSTI mutations (G149A and E157Q) were detected from the clinical sample of a patient failing an INSTI-based therapy. Cost analysis estimated the cost per test at $50.31. Conclusion: The developed HIV-Integrase assay met the validation acceptance criteria and demonstrates the ability to detect clinically relevant INSTI-resistance-associated mutations, highlighting its potential use as an alternative to commercial INSTI tests in resource-limited settings.