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ABSTRACT Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an emerging class of small molecules that disrupt viral maturation by inducing the aberrant multimerization of IN. Here, we present cocrystal structures of HIV-1 IN with two potent ALLINIs, namely, BI-D and the drug candidate Pirmitegravir. The structures reveal atomistic details of the ALLINI-induced interface between the HIV-1 IN catalytic core and carboxyl-terminal domains (CCD and CTD). Projecting from their principal binding pocket on the IN CCD dimer, the compounds act as molecular glue by engaging a triad of invariant HIV-1 IN CTD residues, namely, Tyr226, Trp235, and Lys266, to nucleate the CTD-CCD interaction. The drug-induced interface involves the CTD SH3-like fold and extends to the beginning of the IN carboxyl-terminal tail region. We show that mutations of HIV-1 IN CTD residues that participate in the interface with the CCD greatly reduce the IN-aggregation properties of Pirmitegravir. Our results explain the mechanism of the ALLINI-induced condensation of HIV-1 IN and provide a reliable template for the rational development of this series of antiretrovirals through the optimization of their key contacts with the viral target. IMPORTANCE Despite the remarkable success of combination antiretroviral therapy, HIV-1 remains among the major causes of human suffering and loss of life in poor and developing nations. To prevail in this drawn-out battle with the pandemic, it is essential to continue developing advanced antiviral agents to fight drug resistant HIV-1 variants. Allosteric integrase inhibitors (ALLINIs) are an emerging class of HIV-1 antagonists that are orthogonal to the current antiretroviral drugs. These small molecules act as highly specific molecular glue, which triggers the aggregation of HIV-1 integrase. In this work, we present high-resolution crystal structures that reveal the crucial interactions made by two potent ALLINIs, namely, BI-D and Pirmitegravir, with HIV-1 integrase. Our results explain the mechanism of drug action and will inform the development of this promising class of small molecules for future use in antiretroviral regimens.

Abstract All organisms are constantly exposed to various stresses, necessitating adaptive strategies for survival. In bacteria, the main stress-coping mechanism is the stringent response triggered by the accumulation of “alarmone” (p)ppGpp to arrest proliferation and reprogram transcriptome. While mammalian genomes encode MESH1—the homolog of the (p)ppGpp hydrolase SpoT, current knowledge about its function remains limited. We found MESH1 expression tended to be higher in tumors and associated with poor patient outcomes. Consistently, MESH1 knockdown robustly inhibited proliferation, depleted dNTPs, reduced tumor sphere formation, and retarded xenograft growth. These antitumor phenotypes associated with MESH1 knockdown were accompanied by a significantly altered transcriptome, including the repressed expression of TAZ, a HIPPO coactivator, and proliferative gene. Importantly, TAZ restoration mitigated many anti-growth phenotypes of MESH1 knockdown, including proliferation arrest, reduced sphere formation, tumor growth inhibition, dNTP depletion, and transcriptional changes. Furthermore, TAZ repression was associated with the histone hypo-acetylation at TAZ regulatory loci due to the induction of epigenetic repressors HDAC5 and AHRR. Together, MESH1 knockdown in human cells altered the genome-wide transcriptional patterns and arrested proliferation that mimicked the bacterial stringent response through the epigenetic repression of TAZ expression.

SAMHD1 is a cellular dNTPase proposed to inhibit HIV-1 reverse transcription in non-cycling immune cells by limiting dNTP substrate supply; its anti-viral but not dNTPase function is downregulated by phosphorylation of T592. Here, Martinat et al. describe an additional SUMOylation at residue K595, which promotes the dNTPase-independent restriction activity.

Periodontitis is caused by dysbiotic subgingival bacterial communities that may lead to increased bacterial invasion into gingival tissues. Although shifts in community structures associated with transition from health to periodontitis have been well characterized, the nature of bacteria present within the gingival tissue of periodontal lesions is not known. To characterize microbiota within tissues of periodontal lesions and compare them with plaque microbiota, gingival tissues and subgingival plaques were obtained from 7 patients with chronic periodontitis. A sequencing analysis of the 16S rRNA gene revealed that species richness and diversity were not significantly different between the 2 groups. However, intersubject variability of intratissue communities was smaller than that of plaque communities. In addition, when compared with the plaque communities, intratissue communities were characterized by decreased abundance of Firmicutes and increased abundance of Fusobacteria and Chloroflexi. In particular, Fusobacterium nucleatum and Porphyromonas gingivalis were highly enriched within the tissue, composing 15% to 40% of the total bacteria. Furthermore, biofilms, as visualized by alcian blue staining and atomic force microscopy, were observed within the tissue where the degradation of connective tissue fibers was prominent. In conclusion, very complex bacterial communities exist in the form of biofilms within the gingival tissue of periodontal lesions, which potentially serve as a reservoir for persistent infection. This novel finding may prompt new research on therapeutic strategies to treat periodontitis. [ABSTRACT FROM AUTHOR]

Holstein-Friesian steer beef production is renowned globally as a secondary product of the milk industry. Grass feeding is a common practice in raising Holstein steers because of its low cost. Furthermore, grass feeding is an alternative way to produce beef with a balanced n-6 to n-3 fatty acids (FAs) ratio. However, the performance and meat quality of Holstein-Friesian cattle is more likely to depend on a high-quality diet. The aim of this study was to observe whether feeding two mixed diets; a corn-based total mixed ration (TMR) with winter ryegrass (Lolium perenne) or flaxseed oil-supplemented pellets with reed canary grass haylage (n-3 mix) provided benefits on carcass weight, meat quality and FA composition compared with cattle fed with reed canary grass (Phalaris arundinacea) haylage alone. In all, 15 21-month-old Holstein-Friesian steers were randomly assigned to three group pens, were allowed free access to water and were fed different experimental diets for 150 days. Blood samples were taken a week before slaughter. Carcass weight and meat quality were evaluated after slaughter. Plasma lipid levels and aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT), creatine kinase (CK) and alkaline phosphatase (ALP) activities were determined. Diet did not affect plasma triglyceride levels and GGT activity. Plasma cholesterol levels, including low-density and high-density lipoproteins, were higher in both mixed-diet groups than in the haylae group. The highest activities of plasma AST, CK and ALP were observed in the haylage group, followed by n-3 mix and TMR groups, respectively. Carcass weight was lower in the haylage group than in the other groups and no differences were found between the TMR and n-3 mix groups. Although the n-3 mix-fed and haylage-fed beef provided lower n-6 to n-3 FAs ratio than TMR-fed beef, the roasted beef obtained from the TMR group was more acceptable with better overall meat physicochemical properties and sensory scores. According to daily cost, carcass weight and n-6 to n-3 FAs ratio, the finishing diet containing flaxseed oil-supplemented pellets and reed canary grass haylage at the as-fed ratio of 40 : 60 could be beneficial for the production of n-3-enriched beef. [ABSTRACT FROM PUBLISHER]

Abstract Poly ADP-ribose polymerase (PARP) inhibitors are promising targeted therapy for epithelial ovarian cancer (EOC) with BRCA mutations or defective homologous recombination (HR) repair. However, reversion of BRCA mutation and restoration of HR repair in EOC lead to PARP inhibitor resistance and reduced clinical efficacy of PARP inhibitors. We have previously shown that triapine, a small molecule inhibitor of ribonucleotide reductase (RNR), impaired HR repair and sensitized HR repair-proficient EOC to PARP inhibitors. In this study, we performed in silico screening of small molecule libraries to identify novel compounds that bind to the triapine-binding pocket on the R2 subunit of RNR and inhibit RNR in EOC cells. Following experimental validation of selected top-ranking in silico hits for inhibition of dNTP and DNA synthesis, we identified, DB4, a putative RNR pocket-binding inhibitor markedly abrogated HR repair and sensitized BRCA-wild-type EOC cells to the PARP inhibitor olaparib. Furthermore, we demonstrated that the combination of DB4 and olaparib deterred the progression of BRCA-wild type EOC xenografts and significantly prolonged the survival time of tumor-bearing mice. Herein we report the discovery of a putative small molecule inhibitor of RNR and HR repair for combination with PARP inhibitors to treat PARP inhibitor-resistant and HR repair-proficient EOC.

SAMHD1 catalyses the dephosphorylation of deoxynucleotide triphosphates (dNTPs) and has antiretroviral activity. Here, the authors present the crystal structures of SAMHD1-oligonucleotide complexes, which reveal that the allosteric binding sites of SAMHD1 are plastic and can fit oligonucleotides in place of the two allosteric activators GTP and dNTP, and they also show that SAMHD1 recognises GpsN phosphorothioation modifications in nucleic acids, which is of interest in drug design.

Despite some well-characterized functions in cancer, the impact of most long non-coding RNAs remains unknown. Here, the authors discover the lncRNA lincNMR which is upregulated in cancer and drives cell proliferation by interacting with YBX1 and controlling nucleotide metabolism.

Ribonucleoside monophosphates are incorporated by DNA polymerases into double-stranded DNA. Here, the authors use ribose-seq and Ribose-Map techniques to reveal that signatures and patterns of ribonucleotide incorporation in yeast mitochondrial and nuclear DNA show preference for cytosine and guanosine preceded by deoxyadenosine.

The effects of complex Korean medicine treatment including acupotomy, on irritable bowel syndrome (IBS) are reported in this case study. A 54-year-old woman with diarrheal symptoms that alternatively improved and worsened for many years and worsening abdominal pain visited the emergency room and was diagnosed with IBS; she was admitted for approximately 2 weeks at the Acupuncture and Moxibustion Department of National Medical Center (NMC). Numerical rating scale (NRS), irritable bowel syndrome?quality of life (IBS-QOL), and gastrointestinal symptom rating scale (GSRS) scores were evaluated on the day of hospitalization, midway through the hospitalization period, and on the day of discharge. Abdominal pain was measured daily using NRS, and the score decreased from 8 at admission to 0 at discharge. The IBS-QOL percentile score improved from 42 points to 100 points. The total GSRS score also improved, from 30 points to 2 points. These results suggest that complex Korean medicine treatment with acupotomy, is useful for treating internal diseases, such as IBS, as well as musculoskeletal disorders.

The hydroxylase EgIN2 contributes to triple negative breast cancers. Here, using an enzyme-substrate trapping strategy, the authors identify ASDL as a bona fide substrate of EgIN2 promoting aggressive properties of TNBC via the activation of cMYC signaling.

Abstract Background Terminally differentiated/nondividing macrophages, a key target cell type of HIV-1, harbor extremely low dNTP concentrations established by a host dNTP triphosphohydrolase, SAM domain and HD domain containing protein 1 (SAMHD1). We tested whether the induction of dNTP pool imbalance can affect HIV-1 replication in macrophages. For this test, we induced a large dNTP pool imbalance by treating human primary monocyte derived macrophages with either one or three of the four deoxynucleosides (dNs), which are phosphorylated to dNTPs in cells, to establish two different dNTP imbalance conditions in macrophages. Results The transduction efficiency and 2-LTR circle copy number of HIV-1 GFP vector were greatly diminished in human primary macrophages treated with the biased dN treatments, compared to the untreated macrophages. We also observed the induced dNTP bias blocked the production of infectious dual tropic HIV-1 89.6 in macrophages. Moreover, biochemical DNA synthesis by HIV-1 reverse transcriptase was significantly inhibited by the induced dNTP pool imbalance. Third, the induced dNTP bias increased the viral mutant rate by approximately 20–30% per a single cycle infection. Finally, unlike HIV-1, the single dN treatment did not significantly affect the transduction of SIVmac239-based GFP vector encoding Vpx in macrophages. This is likely due to Vpx, which can elevate all four dNTP levels even with the single dN treatment. Conclusion Collectively, these data suggest that the elevated dNTP pool imbalance can induce kinetic block and mutation synthesis of HIV-1 in macrophages.

Metabolic dysfunctions enabling increased nucleotide biosynthesis are necessary for supporting malignant proliferation. Our investigations indicate that upregulation of fatty acid synthase (FASN) and de novo lipogenesis, commonly observed in many cancers, are associated with nucleotide metabolic dysfunction in lymphoma. The results from our experiments showed that ribonucleotide and deoxyribonucleotide pool depletion, suppression of global RNA/DNA synthesis, and cell cycle inhibition occurred in the presence of FASN inhibition. Subsequently, we observed that FASN inhibition caused metabolic blockade in the rate-limiting step of the oxidative branch of the pentose phosphate pathway (oxPPP) catalyzed by phosphogluconate dehydrogenase (PGDH). Furthermore, we determined that FASN inhibitor treatment resulted in NADPH accumulation and inhibition of PGDH enzyme activity. NADPH is a cofactor utilized by FASN, also a known allosteric inhibitor of PGDH. Through cell-free enzyme assays consisting of FASN and PGDH, we delineated that the PGDH-catalyzed ribulose-5-phosphate synthesis is enhanced in the presence of FASN and is suppressed by increasing concentrations of NADPH. Additionally, we observed that FASN and PGDH were colocalized in the cytosol. The results from these experiments led us to conclude that NADP–NADPH turnover and the reciprocal stimulation of FASN and PGDH catalysis are involved in promoting oxPPP and nucleotide biosynthesis in lymphoma. Finally, a transcriptomic analysis of non-Hodgkin’s lymphoma (n = 624) revealed the increased expression of genes associated with metabolic functions interlinked with oxPPP, while the expression of genes participating in oxPPP remained unaltered. Together we conclude that FASN–PGDH enzymatic interactions are involved in enabling oxPPP and nucleotide metabolic dysfunction in lymphoma tumors.

Allosteric integrase inhibitors (ALLINIs) are a class of experimental anti-HIV agents that target the noncatalytic sites of the viral integrase (IN) and interfere with the IN-viral RNA interaction during viral maturation. Here, we report a highly potent and safe pyrrolopyridine-based ALLINI, STP0404, displaying picomolar IC50 in human PBMCs with a >24,000 therapeutic index against HIV-1. X-ray structural and biochemical analyses revealed that STP0404 binds to the host LEDGF/p75 protein binding pocket of the IN dimer, which induces aberrant IN oligomerization and blocks the IN-RNA interaction. Consequently, STP0404 inhibits proper localization of HIV-1 RNA genomes in viral particles during viral maturation. Y99H and A128T mutations at the LEDGF/p75 binding pocket render resistance to STP0404. Extensive in vivo pharmacological and toxicity investigations demonstrate that STP0404 harbors outstanding therapeutic and safety properties. Overall, STP0404 is a potent and first-in-class ALLINI that targets LEDGF/p75 binding site and has advanced to a human trial.