A Review of FDA-Approved Anti-HIV-1 Drugs, Anti-Gag Compounds, and Potential Strategies for HIV-1 Eradication.

Acquired immunodeficiency syndrome (AIDS) is an enormous global health threat stemming from human immunodeficiency virus (HIV-1) infection. Up to now, the tremendous advances in combination antiretroviral therapy (cART) have shifted HIV-1 infection from a fatal illness into a manageable chronic disorder. However, the presence of latent reservoirs, the multifaceted nature of HIV-1, drug resistance, severe off-target effects, poor adherence, and high cost restrict the efficacy of current cART targeting the distinct stages of the virus life cycle. Therefore, there is an unmet need for the discovery of new therapeutics that not only bypass the limitations of the current therapy but also protect the body's health at the same time. The main goal for complete HIV-1 eradication is purging latently infected cells from patients' bodies. A potential strategy called "lock-in and apoptosis" targets the budding phase of the life cycle of the virus and leads to susceptibility to apoptosis of HIV-1 infected cells for the elimination of HIV-1 reservoirs and, ultimately, for complete eradication. The current work intends to present the main advantages and disadvantages of United States Food and Drug Administration (FDA)-approved anti-HIV-1 drugs as well as plausible strategies for the design and development of more anti-HIV-1 compounds with better potency, favorable pharmacokinetic profiles, and improved safety issues.

Turkish Plants, Including Quercetin and Oenothein B, Inhibit the HIV-1 Release and Accelerate Cell Apoptosis.

The introduction of combined anti-retroviral therapy (cART) in 1996, along with a continual breakthrough in anti-human immunodeficiency virus-1 (HIV-1) drugs, has improved the life expectancies of HIV-1-infected individuals. However, the incidence of drug-resistant viruses between individuals undergoing cART and treatment-naïve individuals is a common challenge. Therefore, there is a requirement to explore potential drug targets by considering various stages of the viral life cycle. For instance, the late stage, or viral release stage, remains uninvestigated extensively in antiviral drug discovery. In this study, we prepared a natural plant library and selected candidate plant extracts that inhibited HIV-1 release based on our laboratory-established screening system. The plant extracts from Epilobium hirsutum L. and Chamerion angustifolium (L.) Holub, belonging to the family Onagraceae, decreased HIV-1 release and accelerated the apoptosis in HIV-1-infected T cells but not uninfected T cells. A flavonol glycoside quercetin with oenothein B in Onagraceae reduced HIV-1 release in HIV-1-infected T cells. Moreover, extracts from Chamerion angustifolium (L.) Holub and Senna alexandrina Mill. inhibited the infectivity of progeny viruses. Together, these results suggest that C. angustifolium (L.) Holub contains quercetin with oenothein B that synergistically blocks viral replication and kills infected cells via an apoptotic pathway. Consequently, the plant extracts from the plant library of Turkey might be suitable candidates for developing novel anti-retroviral drugs that target the late phase of the HIV-1 life cycle.

Structural Characterization of TRAF6 N-Terminal for Therapeutic Uses and Computational Studies on New Derivatives.

Tumor necrosis factor receptor-associated factors (TRAFs) are a protein family with a wide variety of roles and binding partners. Among them, TRAF6, a ubiquitin ligase, possesses unique receptor binding specificity and shows diverse functions in immune system regulation, cellular signaling, central nervous system, and tumor formation. TRAF6 consists of an N-terminal Really Interesting New Gene (RING) domain, multiple zinc fingers, and a C-terminal TRAF domain. TRAF6 is an important therapeutic target for various disorders and structural studies of this protein are crucial for the development of next-generation therapeutics. Here, we presented a TRAF6 N-terminal structure determined at the Turkish light source "Turkish DeLight" to be 3.2 Å resolution at cryogenic temperature (PDB ID: 8HZ2). This structure offers insight into the domain organization and zinc-binding, which are critical for protein function. Since the RING domain and the zinc fingers are key targets for TRAF6 therapeutics, structural insights are crucial for future research. Separately, we rationally designed numerous new compounds and performed molecular docking studies using this template (PDB ID:8HZ2). According to the results, 10 new compounds formed key interactions with essential residues and zinc ion in the N-terminal region of TRAF6. Molecular dynamic (MD) simulations were performed for 300 ns to evaluate the stability of three docked complexes (compounds 256, 322, and 489). Compounds 256 and 489 was found to possess favorable bindings with TRAF6. These new compounds also showed moderate to good pharmacokinetic profiles, making them potential future drug candidates as TRAF6 inhibitors.

Cryogenic X-ray crystallographic studies of biomacromolecules at Turkish Light Source "Turkish DeLight".

X-ray crystallography is a robust and powerful structural biology technique that provides high-resolution atomic structures of biomacromolecules. Scientists use this technique to unravel mechanistic and structural details of biological macromolecules (e.g., proteins, nucleic acids, protein complexes, protein-nucleic acid complexes, or large biological compartments). Since its inception, single-crystal cryocrystallography has never been performed in Türkiye due to the lack of a single-crystal X-ray diffractometer. The X-ray diffraction facility recently established at the University of Health Sciences, Istanbul, Türkiye will enable Turkish and international researchers to easily perform high-resolution structural analysis of biomacromolecules from single crystals. Here, we describe the technical and practical outlook of a state-of-the-art home-source X-ray, using lysozyme as a model protein. The methods and practice described in this article can be applied to any biological sample for structural studies. Therefore, this article will be a valuable practical guide from sample preparation to data analysis.

Evaluation of anti-glioma effects of benzothiazoles as efficient apoptosis inducers and DNA cleaving agents.

Glioma is the fast-growing, aggressive, and prevalent brain cancer with a great level of morbidity and mortality. Current therapy is usually found insufficient for glioma treatment. In the course of our research attempting to identify effective anti-glioma agents, three benzothiazole derivatives (1-3) were examined on U251 glioma cells. Among these derivatives, compound 3 was found to have the strongest cytotoxic effect on glioma cells with an IC50 value of 9.84 ± 0.64 µM in reference to cisplatin (IC50 = 8.41 ± 1.27 µM). Further mechanism of anti-glioma effects of compound 3 was characterized by the determination of its apoptotic effects in glioma cells and DNA cleaving capacity. Compound 3 caused a significant apoptotic death of U251 cell line. Besides, this compound cleaved DNA with FeSO4, H2O2 and ascorbic acid system. Molecular docking results also showed that compound 3 possessed a significant binding potential to DNA via important π-π stacking interaction with DG-16. Some pharmacokinetic determinants of compound 3 complied with standard limits making it as an efficient bioavailable anti-glioma drug candidate for upcoming exploration.

Scaffold Hopping and Structural Modification of NSC 663284: Discovery of Potent (Non)Halogenated Aminobenzoquinones.

The development of new anticancer drugs is still ongoing as a solution to the unsatisfactory results obtained by chemotherapy patients. Our previous studies on natural product-based anticancer agents led us to synthesize a new series of Plastoquinone (PQ) analogs and study their anticancer effects. Four members of PQ analogs (PQ1-4) were designed based on the scaffold hopping strategy; the design was later completed with structural modification. The obtained PQ analogs were synthesized and biologically evaluated against different cancer genotypes according to NCI-60 screening in vitro. According to the NCI results, bromo and iodo-substituted PQ analogs (PQ2 and PQ3) showed remarkable anticancer activities with a wide-spectrum profile. Among the two selected analogs (PQ2 and PQ3), PQ2 showed promising anticancer activity, in particular against leukemia cell lines, at both single- and five-dose NCI screenings. This compound was also detected by MTT assay to reveal significant selectivity between Jurkat cells and PBMC (healthy) compared to imatinib. Further in silico studies indicated that PQ2 was able to occupy the ATP-binding cleft of Abl TK, one of the main targets of leukemia, through key interactions similar to dasatinib and imatinib. PQ2 is also bound to the minor groove of the double helix of DNA. Based on computational pharmacokinetic studies, PQ2 possessed a remarkable drug-like profile, making it a potential anti-leukemia drug candidate for future studies.

Identification of New L-Heptanoylphosphatidyl Inositol Pentakisphosphate Derivatives Targeting the Interaction with HIV-1 Gag by Molecular Modelling Studies.

The HIV-1 Gag protein binds to the host cell membrane and assembles into immature particles. Then, in the course of immature virion budding, activated protease cleaves Gag into its main components: MA, CA, NC, and p6 proteins. The highly basic residues of MA predominantly interact with the acidic head of phosphatidyl-inositol-4,5-bisphosphate (PI(4,5)P2) inserted into the membrane. Our research group developed L-Heptanoylphosphatidyl Inositol Pentakisphosphate (L-HIPPO) and previously confirmed that this compound bound to the MA more strongly than PI(4,5)P2 and inositol hexakisphosphate (IP6) did. Therefore, herein we rationally designed eight new L-HIPPO derivatives based on the fact that the most changeable parts of L-HIPPO were two acyl chains. After that, we employed molecular docking for eight compounds via Maestro software using high-resolution crystal structures of MA in complex with IP6 (PDB IDs: 7E1I, 7E1J, and 7E1K), which were recently elucidated by our research group. The most promising docking scores were obtained with benzene-inserted compounds. Thus, we generated a library containing 213 new aromatic group-inserted L-HIPPO derivatives and performed the same molecular docking procedure. According to the results, we determined the nine new L-HIPPO derivatives most effectively binding to the MA with the most favorable scoring functions and pharmacokinetic properties for further exploration.

In Vitro Cytotoxicity Evaluation of Plastoquinone Analogues against Colorectal and Breast Cancers along with In Silico Insights.

Colorectal cancer (CRC) and breast cancer are leading causes of death globally, due to significant challenges in detection and management. The late-stage diagnosis and treatment failures require the discovery of potential anticancer agents to achieve a satisfactory therapeutic effect. We have previously reported a series of plastoquinone analogues to understand their cytotoxic profile. Among these derivatives, three of them (AQ-11, AQ-12, and AQ-15) were selected by the National Cancer Institute (NCI) to evaluate their in vitro antiproliferative activity against a panel of 60 human tumor cell lines. AQ-12 exhibited significant antiproliferative activity against HCT-116 CRC and MCF-7 breast cancer cells at a single dose and further five doses. MTT assay was also performed for AQ-12 at different concentrations against these two cells, implying that AQ-12 exerted notable cytotoxicity toward HCT-116 (IC50 = 5.11 ± 2.14 µM) and MCF-7 (IC50 = 6.06 ± 3.09 µM) cells in comparison with cisplatin (IC50 = 23.68 ± 6.81 µM and 19.67 ± 5.94 µM, respectively). This compound also augmented apoptosis in HCT-116 (62.30%) and MCF-7 (64.60%) cells comparable to cisplatin (67.30% and 78.80%, respectively). Molecular docking studies showed that AQ-12 bound to DNA, forming hydrogen bonding through the quinone scaffold. In silico pharmacokinetic determinants indicated that AQ-12 demonstrated drug-likeness with a remarkable pharmacokinetic profile for future mechanistic anti-CRC and anti-breast cancer activity studies.

Design, semi-synthesis and examination of new gypsogenin derivatives against leukemia via Abl tyrosine kinase inhibition and apoptosis induction.

Chronic myelogenous leukemia (CML) is characterized by Philadelphia translocation arising from Bcr-Abl fusion gene, which encodes abnormal oncoprotein showing tyrosine kinase (TK) function. Certain mutations in kinase domain, off-target effects and resistance problems of current TK inhibitors require the discovery of novel Abl TK inhibitors. For this purpose, herein, we synthesized new gypsogenin derivatives (6a-l) and evaluated their anticancer effects towards CML cells along with healthy cell line and different leukemic cells. Among these compounds, compound 6l was found as the most active anti-leukemic agent against K562 CML cells compared to imatinib exerting less cytotoxicity towards PBMCs (healthy). This compound also revealed significant anti-leukemic effects against Jurkat cell line. Besides, compound 6l enhanced apoptosis in CML cells with 52.4 % when compared with imatinib (61.8 %) and inhibited Abl TK significantly with an IC50 value of 13.04 ± 2.48 µM in a large panel of kinases accentuating Abl TK-mediated apoptosis of compound 6l in CML cells. Molecular docking outcomes showed that compound 6l formed mainly crucial interactions in the ATP-binding cleft of Abl TK similar to that of imatinib. Ultimately, in silico pharmacokinetic evaluation of compound 6l indicated that this compound was endowed with anti-leukemic drug candidate features.

A New 1,2-Naphthoquinone Derivative with Anti-lung Cancer Activity.

1,2-Naphthoquinone (2-NQ) is a nucleophile acceptor that non-selectively makes covalent bonds with cysteine residues in various cellular proteins, and is also found in diesel exhaust, an air pollutant. This molecule has rarely been considered as a pharmacophore of bioactive compounds, in contrast to 1,4-naphthoquinone. We herein designed and synthesized a compound named N-(7,8-dioxo-7,8-dihydronaphthalen-1-yl)-2-methoxybenzamide (MBNQ), in which 2-NQ was hybridized with the nuclear factor-κB (NF-κB) inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) as a nucleophile acceptor. Although 50 µM MBNQ did not inhibit NF-κB signaling, 10 µM MBNQ induced cell death in the lung cancer cell line A549, which was insensitive to 2-NQ (10 µM). In contrast, MBNQ was less toxic in normal lung cells than 2-NQ. A mechanistic study showed that MBNQ mainly induced apoptosis, presumably via the activation of p38 mitogen-activated protein kinase (MAPK). Collectively, the present results demonstrate that the introduction of an appropriate substituent into 2-NQ constitutes a new biologically active entity, which will lead to the development of 2-NQ-based drugs.

Comprehensive Research on Past and Future Therapeutic Strategies Devoted to Treatment of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rapidly debilitating fatal neurodegenerative disorder, causing muscle atrophy and weakness, which leads to paralysis and eventual death. ALS has a multifaceted nature affected by many pathological mechanisms, including oxidative stress (also via protein aggregation), mitochondrial dysfunction, glutamate-induced excitotoxicity, apoptosis, neuroinflammation, axonal degeneration, skeletal muscle deterioration and viruses. This complexity is a major obstacle in defeating ALS. At present, riluzole and edaravone are the only drugs that have passed clinical trials for the treatment of ALS, notwithstanding that they showed modest benefits in a limited population of ALS. A dextromethorphan hydrobromide and quinidine sulfate combination was also approved to treat pseudobulbar affect (PBA) in the course of ALS. Globally, there is a struggle to prevent or alleviate the symptoms of this neurodegenerative disease, including implementation of antisense oligonucleotides (ASOs), induced pluripotent stem cells (iPSCs), CRISPR-9/Cas technique, non-invasive brain stimulation (NIBS) or ALS-on-a-chip technology. Additionally, researchers have synthesized and screened new compounds to be effective in ALS beyond the drug repurposing strategy. Despite all these efforts, ALS treatment is largely limited to palliative care, and there is a strong need for new therapeutics to be developed. This review focuses on and discusses which therapeutic strategies have been followed so far and what can be done in the future for the treatment of ALS.

In Vitro and In Silico Study of Analogs of Plant Product Plastoquinone to Be Effective in Colorectal Cancer Treatment.

Plants have paved the way for the attainment of molecules with a wide-range of biological activities. However, plant products occasionally show low biological activities and/or poor pharmacokinetic properties. In that case, development of their derivatives as drugs from the plant world has been actively performed. As plant products, plastoquinones (PQs) have been of high importance in anticancer drug design and discovery; we have previously evaluated and reported the potential cytotoxic effects of a series of PQ analogs. Among these analogs, PQ2, PQ3 and PQ10 were selected for National Cancer Institute (NCI) for in vitro screening of anticancer activity against a wide range of cancer cell lines. The apparent superior anticancer potency of PQ2 on the HCT-116 colorectal cancer cell line than that of PQ3 and PQ10 compared to other tested cell lines has encouraged us to perform further mechanistic studies to enlighten the mode of anti-colorectal cancer action of PQ2. For this purpose, its apoptotic effects on the HCT-116 cell line, DNA binding capacity and several crucial pharmacokinetic properties were investigated. Initially, MTT assay was conducted for PQ2 at different concentrations against HCT-116 cells. Results indicated that PQ2 exhibited significant cytotoxicity in HCT-116 cells with an IC50 value of 4.97 ± 1.93 µM compared to cisplatin (IC50 = 26.65 ± 7.85 µM). Moreover, apoptotic effects of PQ2 on HCT-116 cells were investigated by the annexin V/ethidium homodimer III staining method and PQ2 significantly induced apoptosis in HCT-116 cells compared to cisplatin. Based on the potent DNA cleavage capacity of PQ2, molecular docking studies were conducted in the minor groove of the double helix of DNA and PQ2 presented a key hydrogen bonding through its methoxy moiety. Overall, both in vitro and in silico studies indicated that effective, orally bioavailable drug-like PQ2 attracted attention for colorectal cancer treatment. The most important point to emerge from this study is that appropriate derivatization of a plant product leads to unique biologically active compounds.

A New Series of Indeno[1,2-c]pyrazoles as EGFR TK Inhibitors for NSCLC Therapy.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death throughout the world. Due to the shortcomings of traditional chemotherapy, targeted therapies have come into prominence for the management of NSCLC. In particular, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapy has emerged as a first-line therapy for NSCLC patients with EGFR-activating mutations. In this context, new indenopyrazoles, which were prepared by an efficient microwave-assisted method, were subjected to in silico and in vitro assays to evaluate their potency as EGFR TK-targeted anti-NSCLC agents. Compound 4 was the most promising antitumor agent towards A549 human lung adenocarcinoma cells, with an IC50 value of 6.13 µM compared to erlotinib (IC50 = 19.67 µM). Based on its low cytotoxicity to peripheral blood mononuclear cells (PBMCs), it can be concluded that compound 4 exerts selective antitumor action. This compound also inhibited EGFR TK with an IC50 value of 17.58 µM compared to erlotinib (IC50 = 0.04 µM) and induced apoptosis (56.30%). Taking into account in silico and in vitro data, compound 4 stands out as a potential EGFR TKI for the treatment of NSCLC.

Studies on 1,4-Quinone Derivatives Exhibiting Anti-Leukemic Activity along with Anti-Colorectal and Anti-Breast Cancer Effects.

Colorectal cancer (CRC), breast cancer, and chronic myeloid leukemia (CML) are life-threatening malignancies worldwide. Although potent therapeutic and screening strategies have been developed so far, these cancer types are still major public health problems. Therefore, the exploration of more potent and selective new agents is urgently required for the treatment of these cancers. Quinones represent one of the most important structures in anticancer drug discovery. We have previously identified a series of quinone-based compounds (ABQ-1-17) as anti-CML agents. In the current work, ABQ-3 was taken to the National Cancer Institute (NCI) for screening to determine its in vitro antiproliferative effects against a large panel of human tumor cell lines at five doses. ABQ-3 revealed significant growth inhibition against HCT-116 CRC and MCF-7 breast cancer cells with 2.00 µM and 2.35 µM GI50 values, respectively. The MTT test also showed that ABQ-3 possessed anticancer effects towards HCT-116 and MCF-7 cells with IC50 values of 5.22 ± 2.41 µM and 7.46 ± 2.76 µM, respectively. Further experiments indicated that ABQ-3 induced apoptosis in both cell lines, and molecular docking studies explicitly suggested that ABQ-3 exhibited DNA binding in a similar fashion to previously reported compounds. Based on in silico pharmacokinetic prediction, ABQ-3 might display drug-like features enabling this compound to become a lead molecule for future studies.

Structural insight into host plasma membrane association and assembly of HIV-1 matrix protein.

Oligomerization of Pr55Gag is a critical step of the late stage of the HIV life cycle. It has been known that the binding of IP6, an abundant endogenous cyclitol molecule at the MA domain, has been linked to the oligomerization of Pr55Gag. However, the exact binding site of IP6 on MA remains unknown and the structural details of this interaction are missing. Here, we present three high-resolution crystal structures of the MA domain in complex with IP6 molecules to reveal its binding mode. Additionally, extensive Differential Scanning Fluorimetry analysis combined with cryo- and ambient-temperature X-ray crystallography and GNM-based transfer entropy calculations identify the key residues that participate in IP6 binding. Our data provide novel insights about the multilayered HIV-1 virion assembly process that involves the interplay of IP6 with PIP2, a phosphoinositide essential for the binding of Pr55Gag to membrane. IP6 and PIP2 have neighboring alternate binding sites within the same highly basic region (residues 18-33). This indicates that IP6 and PIP2 bindings are not mutually exclusive and may play a key role in coordinating virion particles' membrane localization. Based on our three different IP6-MA complex crystal structures, we propose a new model that involves IP6 coordination of the oligomerization of outer MA and inner CA domain's 2D layers during assembly and budding.

Effects of Glycyrrhetic Acid on Human Chronic Myelogenous Leukemia Cells.

OBJECTIVES: Chronic myelogenous leukemia (CML) is a type of blood cancer that is initially treated with imatinib (first Abl kinase inhibitor). However, some patients with CML develop imatinib resistance. Several new generation drugs have been developed, but do not overcome this problem. Glycyrrhetic acid (GA) is a plant-derived pentacyclic triterpenoid that exhibits multiple pharmacological properties for the treatment of cancers. The current study aimed to investigate the effects of GA on the K562 cell line (Bcr-Abl positive leukemia). MATERIALS AND METHODS: The MTT cell proliferation assay was employed to evaluate the cytotoxic effect of GA compared with imatinib (positive control) against leukemia and normal blood cells. For detection of cell death, an apoptotic/necrotic/healthy assay was performed against the K562 cell line. To investigate the kinase inhibitory activity of GA, the Abl1 kinase profiling assay and a molecular docking study were performed. RESULTS: GA showed Abl kinase inhibitory activity with an IC50 value of 29.2 µM and induced apoptosis in the K562 cell line after 6 h of treatment. CONCLUSION: The current findings indicate that this class of plant extract could be a potential candidate for treatment of CML.

Aminoglycoside ribosome interactions reveal novel conformational states at ambient temperature.

The bacterial 30S ribosomal subunit is a primary antibiotic target. Despite decades of discovery, the mechanisms by which antibiotic binding induces ribosomal dysfunction are not fully understood. Ambient temperature crystallographic techniques allow more biologically relevant investigation of how local antibiotic binding site interactions trigger global subunit rearrangements that perturb protein synthesis. Here, the structural effects of 2-deoxystreptamine (paromomycin and sisomicin), a novel sisomicin derivative, N1-methyl sulfonyl sisomicin (N1MS) and the non-deoxystreptamine (streptomycin) aminoglycosides on the ribosome at ambient and cryogenic temperatures were examined. Comparative studies led to three main observations. First, individual aminoglycoside-ribosome interactions in the decoding center were similar for cryogenic versus ambient temperature structures. Second, analysis of a highly conserved GGAA tetraloop of h45 revealed aminoglycoside-specific conformational changes, which are affected by temperature only for N1MS. We report the h44-h45 interface in varying states, i.e. engaged, disengaged and in equilibrium. Third, we observe aminoglycoside-induced effects on 30S domain closure, including a novel intermediary closure state, which is also sensitive to temperature. Analysis of three ambient and five cryogenic crystallography datasets reveal a correlation between h44-h45 engagement and domain closure. These observations illustrate the role of ambient temperature crystallography in identifying dynamic mechanisms of ribosomal dysfunction induced by local drug-binding site interactions. Together, these data identify tertiary ribosomal structural changes induced by aminoglycoside binding that provides functional insight and targets for drug design.

Se-SAD serial femtosecond crystallography datasets from selenobiotinyl-streptavidin.

We provide a detailed description of selenobiotinyl-streptavidin (Se-B SA) co-crystal datasets recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS) for selenium single-wavelength anomalous diffraction (Se-SAD) structure determination. Se-B SA was chosen as the model system for its high affinity between biotin and streptavidin where the sulfur atom in the biotin molecule (C10H16N2O3S) is substituted with selenium. The dataset was collected at three different transmissions (100, 50, and 10%) using a serial sample chamber setup which allows for two sample chambers, a front chamber and a back chamber, to operate simultaneously. Diffraction patterns from Se-B SA were recorded to a resolution of 1.9 Å. The dataset is publicly available through the Coherent X-ray Imaging Data Bank (CXIDB) and also on LCLS compute nodes as a resource for research and algorithm development.

Selenium single-wavelength anomalous diffraction de novo phasing using an X-ray-free electron laser.

Structural information about biological macromolecules near the atomic scale provides important insight into the functions of these molecules. To date, X-ray crystallography has been the predominant method used for macromolecular structure determination. However, challenges exist when solving structures with X-rays, including the phase problem and radiation damage. X-ray-free electron lasers (X-ray FELs) have enabled collection of diffraction information before the onset of radiation damage, yet the majority of structures solved at X-ray FELs have been phased using external information via molecular replacement. De novo phasing at X-ray FELs has proven challenging due in part to per-pulse variations in intensity and wavelength. Here we report the solution of a selenobiotinyl-streptavidin structure using phases obtained by the anomalous diffraction of selenium measured at a single wavelength (Se-SAD) at the Linac Coherent Light Source. Our results demonstrate Se-SAD, routinely employed at synchrotrons for novel structure determination, is now possible at X-ray FELs.