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When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Here, we describe the use of peptide backbone N-methylation as a new strategy to transform membrane-lytic peptides (MLPs) into cytocompatible intracellular delivery vehicles. The ability of lytic peptides to engage with cell membranes has been exploited for drug delivery to carry impermeable cargo into cells, but their inherent toxicity results in narrow therapeutic windows that limit their clinical translation. For most linear MLPs, a prerequisite for membrane activity is their folding at cell surfaces. Modification of their backbone with N-methyl amides inhibits folding, which directly correlates to a reduction in lytic potential but only minimally affects cell entry. We synthesized a library of N-methylated peptides derived from MLPs and conducted structure-activity studies that demonstrated the broad utility of this approach across different secondary structures, including both β-sheet and helix-forming peptides. Our strategy is highlighted by the delivery of a notoriously difficult class of protein-protein interaction inhibitors that displayed on-target activity within cells.

Zika virus has spread rapidly in the Americas and has caused devastation of human populations affected in these regions. The virus causes teratogenic effects involving the nervous system, and in adults and children can cause a neuropathy similar to Guillain-Barre syndrome, an anterior myelitis, or, rarely, an encephalitis. While major efforts have been undertaken to control mosquito populations that spread the virus and to develop a vaccine, drug development that directly targets the virus in an infected individual to prevent or treat the neurological manifestations is necessary. Rational and targeted drug development is possible since the viral life cycle and the structure of the key viral proteins are now well understood. While several groups have identified therapeutic candidates, their approaches differ in the types of screening processes and viral assays used. Animal studies are available for only a few compounds. Here we provide an exhaustive review and compare each of the classes of drugs discovered, the methods used for drug discovery, and their potential use in humans for the prevention or treatment of neurological complications of Zika virus infection.

A 1,2-dioxolane (FINO2) was identified as a lead compound from a screen of organic peroxides. FINO2 does not induce apoptosis, but instead initiates ferroptosis, an iron-dependent, oxidative cell death pathway. Few compounds are known to induce primarily ferroptosis. In contrast to the perceived instability of peroxides, FINO2 was found to be thermally stable to at least 150 °C. FINO2 was more potent in cancer cells than nonmalignant cells of the same type. One of the enantiomers was found to be more responsible for the observed activity.

Despite being predicted to be stereoelectronically favorable by the Baldwin rules, efficient formation of a C-C bond through a 5-endo-dig radical cyclization remained unknown for more than 40 years. This work reports a remarkable increase in the efficiency of this process upon beta-Ts substitution, which led to the development of an expedient approach to densely functionalized cyclic 1,3-dienes. Good qualitative agreement between the increased efficiency and stereoselectivity for the 5-endo-dig cyclization of Ts-substituted vinyl radicals and the results of density functional theory analysis further confirms the utility of computational methods in the design of new radical processes. Although reactions of Br atoms generated through photochemical Ts-Br bond homolysis lead to the formation of cyclic dibromide side products, the yields of target bromosulfones in the photochemically induced reactions can be increased by recycling the dibromide byproduct into the target bromosulfones through a sequence of addition/elimination reactions at the exocyclic double bond. Discovery of a relatively efficient radical 5-endo-dig closure, accompanied by a C-C bond formation, provides further support to stereoelectronic considerations at the heart of the Baldwin rules and fills one of the last remaining gaps in the arsenal of radical cyclizations.

Mutations in p53 and elevated expression of Bcl-2 proteins are associated with therapy resistance in many human tumors. A lead compound capable of bypassing these resistance pathways, RA1, was identified from several synthetic peroxides tested at the National Cancer Institute through the In Vitro Cell Line Screening Project. The 1,2-dioxolane scaffold of RA1 differs from that of the peroxide containing anti-malarial agent artemisinin. The lowest concentration of RA1 to cause 50% growth inhibition (GI50) was observed in an ovarian cancer line, IGROV-1 (GI50 435 nM), and the highest in a lung cancer line, NCI-H322M (GI50 42 μM). Eighteen out of the fifty-nine cell lines tested showed a GI50 below 2.5 μM. In cell lines with altered p53 status, RA1 exhibited a potent effect (GI50 for NCI-H522, HOP-92, and HL-60: 1.2 μM, 1.4 μM, and 2.2 μM, respectively). RA1 showed a high selectivity index, inducing >90% cell death at 40 μM in a transformed cancerous cell line (BJ-ELR: LC50 30 μM); while only ∼20% cell death was observed in non-transformed cells of the same origin (BJ-hTERT: LC50 55 μM). Using a B-lymphoblastic cell leukemia line, RS4:11 (LC50 ∼7 μM; wild type p53), as a model system, we investigated the mechanism of action of RA1. No increase in p53 protein expression was observed upon treatment with RA1. Unlike conventional chemotherapy drugs, overexpression of the Bcl-2 protein to prevent mitochondrial outer membrane permeabilization (MOMP) had no effect on RA1's ability to induce cell death. The LC50 of GFP control and Bcl-2 overexpressing cells with RA1 is 7.7 and 7.4 μM, respectively, compared to the control (etoposide: LC50 0.6 μM and >4 μM). No change in cell cycle distribution was observed. No activation of the executioner caspases was seen by Western Blot; however, pretreatment with a caspase-2 inhibitor (z-VDVAD-FMK) prevented cell death. Electron microscopy images showed no morphological features characteristic of apoptosis, necrosis, or autophagy. Ferroptosis, an iron and reactive oxygen species (ROS) dependent form of cell death also lacks these morphological characteristics. While no organic peroxides are known to induce ferroptosis, the use of an iron chelator and Ferrostatin (a lipophillic anti-oxidant known to inhibit ferroptosis) inhibited cell death induced by RA1. Similar to some ferroptosis inducing compounds, a water soluble anti-oxidant had no effect on cell death; however, use of a ROS detection kit indicated oxidative stress is occurring. RA1 demonstrates potential therapeutic activity for the treatment of cancer. It is capable of overcoming two common chemo-resistance pathways: p53 mutation and inhibition of MOMP by modulation of the Bcl-2 family of proteins. RA1 targets cancer cells by a unique mechanism distinct from other peroxide compounds, as well as conventional chemotherapies. Current experimental data suggests RA1 is inducing a form of ferroptotic cell death. Citation Format: Rachel P. Abrams, K. A. Woerpel, William L. Carroll. Development of a synthetic organic peroxide, RA1, that overcomes p53 and Bcl-2 resistance mechanisms. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4545. doi:10.1158/1538-7445.AM2015-4545