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Dihydropteroate synthase (DHPS) is an enzyme of the folate biosynthesis pathway, which catalyzes the formation of 7,8-dihydropteroate (DHPt) from 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (DHPPP) and para-aminobenzoic acid (pABA). DHPS is the long-standing target of the sulfonamide class of antibiotics that compete with pABA. In the wake of sulfa drug resistance, targeting the structurally rigid (and more conserved) pterin site has been proposed as an alternate strategy to inhibit DHPS in wild-type and sulfa drug resistant strains. Following the work on developing pterin-site inhibitors of the adjacent enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), we now present derivatives of 8-mercaptoguanine, a fragment that binds weakly within both enzymes, and quantify sub-μm binding using surface plasmon resonance (SPR) to Escherichia coli DHPS (EcDHPS). Eleven ligand-bound EcDHPS crystal structures delineate the structure-activity relationship observed providing a structural framework for the rational development of novel, substrate-envelope-compliant DHPS inhibitors.

Intermolecular 1H-1H nuclear Overhauser effects (NOE) present a powerful tool to assess contacts between proteins and binding partners, but are difficult to identify for complexes of high molecular weight. We show that intermolecular NOEs can readily be observed following chemical labeling with t-butyl or trimethylsilyl groups. Proteins can be furnished with t-butyl or TMS groups site-specifically using genetically encoded unnatural amino acids or by chemical modification of single cysteine residues. No isotope labeling is required. The approach is demonstrated with the 95 kDa complex between tetrameric E. coli single-stranded DNA binding protein (SSB) and single-stranded DNA.

SummaryIntroduction Growth hormone deficiency is considered the most important factor determining skeletal fragility in hypopituitary patients. Osteoblasts and chondrocytes express growth hormone (GH) receptor. Two GH receptor isoforms (GHRi) have been identified: they differ for the presence/absence of a protein fragment encoded by exon 3 of GHR gene. Consequently, three genotypes were identified: carriers of both the full-length proteins (flfl-GHR), carriers of one full-length protein and one deleted protein (fld3-GHR) and carriers of both deleted proteins (d3d3-GHR). This polymorphism confers a higher sensitivity to endogenous GH and to recombinant human GH (rhGH); its effect on bone metabolism and skeletal fragility is unknown. The aim of this article was to investigate the role of GHRi in predicting skeletal fragility in adult-onset GHD (AO-GHD) patients. Subjects and methods A cross-sectional study was conducted to investigate the association between the d3-GHR isoform and the prevalence of morphometric vertebral fractures (VFs) in AO-GHD. Ninety-three AO-GHD were enrolled. Forty-nine patients carried flfl-GHRi (52·7%), and 44 patients (47·3%) carried at least one allele of the d3-GHR isoform. Thirty-two VFs were documented. Fifty-seven patients underwent rhGH replacement therapy. Results Median age was significantly higher in fractured patients as compared to nonfractured ones; d3-carrier patients showed a lower VF risk as compared to flfl-GHRi (OR: 0·37, 95% IC: 0·24–0·55, P < 0·0001). This finding was also confirmed in AO-GHD undergoing rhGH replacement therapy. Conclusion This study suggests that d3-GHR may protect AO-GHD particularly when treated with rhGH from the risk of VFs.

A reiterative coupling consisting of an ethynyl methyl sulfide synthesis and a iodocyclization sequence is developed and gives access to valuable thieno-fused heterocycles.

As human lifespan increases so does the incidence of age-associated degenerative joint diseases, resulting in significant negative socioeconomic consequences. Osteoarthritis (OA) and intervertebral disc degeneration (IDD) are the most common underlying causes of joint-related chronic disability and debilitating pain in the elderly. Current treatment methods are generally not effective and involve either symptomatic relief with non-steroidal anti-inflammatory drugs and physical therapy or surgery when conservative treatments fail. The limitation in treatment options is due to our incomplete knowledge of the molecular mechanism of degeneration of articular cartilage and disc tissue. Basic understanding of the age-related changes in joint tissue is thus needed to combat the adverse effects of aging on joint health. Aging is caused at least in part by time-dependent accumulation of damaged organelles and macromolecules, leading to cell death and senescence and the eventual loss of multipotent stem cells and tissue regenerative capacity. Studies over the past decades have uncovered a number of important molecular and cellular changes in joint tissues with age. However, the precise causes of damage, cellular targets of damage, and cellular responses to damage remain poorly understood. The objectives of this review are to provide an overview of the current knowledge about the sources of endogenous and exogenous damaging agents and how they contribute to age-dependent degenerative joint disease, and highlight animal models of accelerated aging that could potentially be useful for identifying causes of and therapies for degenerative joint diseases.

Oxidative damage is a well-established driver of aging. Evidence of oxidative stress exists in aged and degenerated discs, but it is unclear how it affects disc metabolism. In this study, we first determined whether oxidative stress negatively impacts disc matrix metabolism using disc organotypic and cell cultures. Mouse disc organotypic culture grown at atmospheric oxygen (20% O(2)) exhibited perturbed disc matrix homeostasis, including reduced proteoglycan synthesis and enhanced expression of matrix metalloproteinases, compared to discs grown at low oxygen levels (5% O(2)). Human disc cells grown at 20% O(2) showed increased levels of mitochondrial-derived superoxide anions and perturbed matrix homeostasis. Treatment of disc cells with the mitochondria-targeted reactive oxygen species (ROS) scavenger XJB-5-131 blunted the adverse effects caused by 20% O(2). Importantly, we demonstrated that treatment of accelerated aging Ercc1(-/Δ) mice, previously established to be a useful in vivo model to study age-related intervertebral disc degeneration (IDD), also resulted in improved disc total glycosaminoglycan content and proteoglycan synthesis. This demonstrates that mitochondrial-derived ROS contributes to age-associated IDD in Ercc1(-/Δ) mice. Collectively, these data provide strong experimental evidence that mitochondrial-derived ROS play a causal role in driving changes linked to aging-related IDD and a potentially important role for radical scavengers in preventing IDD.

A reiterative coupling consisting of an ethynyl methyl sulfide synthesis and a iodocyclization sequence is developed and gives access to valuable thieno-fused heterocycles.

A ready access to a range of different polycyclic scaffolds through minor changes in the reaction conditions and/or substrate substitution patterns is described.

N-Methyl amino acids occur in many natural products. Experimental strategies are presented for a unified approach to the synthesis of N-methyl derivatives through 5-oxazolidinones of the 20 common l-amino acids. The amino acids with reactive side chains that required protecting groups or devoted syntheses for side chain construction for N-methylation to proceed included serine, threonine, tyrosine, cysteine, methionine, tryptophan, asparagine, histidine, and arginine. The studies have provided improved methods for the preparation of N-methyl serine, threonine, and tyrosine. All 20 of the common l-amino acids are now available in suitable forms for solid or solution-phase peptide synthesis.