Recent advances in the structural biology of modular polyketide synthases and nonribosomal peptide synthetases

Polyketides and nonribosomal peptides are an important class of natural products with useful bioactivities. These compounds are similarly biosynthesized using enzymes with modular structures despite having different physicochemical properties. These enzymes are attractive targets for bioengineering to produce “unnatural” natural products owing to their modular structures. Therefore, their structures have been studied for a long time; however, the main focus was on truncated-single domains. Surprisingly, there is an increasing number of the structures of whole modules reported, most of which have been enabled through the recent advances in cryogenic electron microscopy technology. In this review, we have summarized the recent advances in the structural elucidation of whole modules.     

Recent advances in drug repurposing using machine learning

Drug repurposing aims to find new uses for already existing and approved drugs. We now provide a brief overview of recent developments in drug repurposing using machine learning alongside other computational approaches for comparison. We also highlight several applications for cancer using kinase inhibitors, Alzheimer’s disease as well as COVID-19.     

Deep-lipidotyping by mass spectrometry: recent technical advances and applications

In-depth structural characterization of lipids is an essential component of lipidomics. There has been a rapid expansion of mass spectrometry methods that are capable of resolving lipid isomers at various structural levels over the past decade. These developments finally make deep-lipidotyping possible, which provides new means to study lipid metabolism and discover new lipid biomarkers. In this review, we discuss recent advancements in tandem mass spectrometry (MS/MS) methods for identification of complex lipids beyond the species (known headgroup information) and molecular species (known chain composition) levels. These include identification at the levels of carbon-carbon double bond (C=C) location and sn-position, as well as characterization of acyl chain modifications. We also discuss the integration of isomer-resolving MS/MS methods with different lipid analysis workflows and their applications in lipidomics. The results showcase the distinct capabilities of deep-lipidotyping in untangling the metabolism of individual isomers and sensitive phenotyping by using relative fractional quantitation of the isomers.     

Osilodrostat: A Review of Recent Clinical Studies and Practical Recommendations for its Use in the Treatment of Cushing Disease

ObjectiveCushing disease (CD) is characterized by chronic hypercortisolism caused by an adrenocorticotropic hormone-secreting pituitary adenoma. Surgery remains the first-line treatment option; however, medical therapy is essential if surgery is contraindicated or fails to achieve remission or when recurrence occurs after surgical remission. Osilodrostat (Isturisa), a novel steroidogenic inhibitor, is now approved for the treatment of CD in the United States and Cushing syndrome in Europe. Herein, we review pharmacology and data on the efficacy, safety, and clinical use of osilodrostat and provide guidance on its use in treating patients with CD.MethodsWe reviewed the literature and published clinical trial data of osilodrostat use in patients with Cushing syndrome. Detailed information related to the clinical assessment of osilodrostat use, potential drug-to-drug interactions, drug initiation, dose titration, and the monitoring of drug tolerability were discussed.ResultsClinical trial data demonstrated that osilodrostat, by virtue of inhibiting 11-β hydroxylase, potently and rapidly decreased the 24-hour urinary free cortisol levels and sustained these reductions, with improved glycemia, blood pressure, body weight, and quality of life as well as lessened depression. Osilodrostat may interact with certain drugs, resulting in QT prolongation, which requires careful assessment of concomitant medications and periodic monitoring using electrocardiogram, respectively. The common adverse effects include adrenal insufficiency, hypokalemia, edema, and hyperandrogenic symptoms, which can be minimized using a slower up-titration dosing regimen.ConclusionOsilodrostat is an effective, new treatment option for CD, with positive effects on cardiovascular and quality of life parameters as well as tolerable adverse effects. This article provides a review of the pharmacology of osilodrostat and offers practical recommendations on the use of osilodrostat to treat CD.     

Secondary Adrenal Insufficiency: Recent Updates and New Directions for Diagnosis and Management

Secondary adrenal insufficiency is the most common subtype of adrenal insufficiency; it is caused by certain medications and pituitary destruction (pituitary masses, inflammation, or infiltration) and is rarely associated with certain germline variants. In this review, we discuss the etiology, epidemiology, and clinical presentation of secondary adrenal insufficiency and focus on the diagnostic and management challenges. We also review the management of selected special populations of patients and discuss patient-important outcomes associated with secondary adrenal insufficiency.     

Structure-based analyses of gut microbiome-related proteins by neural networks and molecular dynamics simulations

An imbalance in the gut microbiome is linked to immune disorders, such as autoimmune, allergic, and chronic inflammatory disorders. Elucidation of disease mechanisms is a matter of urgency. It requires precise elucidation of the structure-based mechanisms of protein interactions involved in disease onset. In addition, an understanding of the protein dynamics is vital because these fluctuations affect the function and interaction of disease-associated proteins. Experimental evaluation of not only protein interactions, functions, and structures but also the dynamics are time-consuming; therefore, computational predictions are necessary to elucidate disease mechanisms. Here, we introduce recent studies on structure-based analyses of proteins using computational approaches, particularly artificial intelligence (AI) and molecular dynamics (MD) simulations.     

Recent insight into intermediate filament structure

Intermediate filaments (IFs) are key players in multiple cellular processes throughout human tissues. Their biochemical and structural properties are important for understanding filament assembly mechanisms, for interactions between IFs and binding partners, and for developing pharmacological agents that target IFs. IF proteins share a conserved coiled-coil central-rod domain flanked by variable N-terminal ‘head’ and C-terminal ‘tail’ domains. There have been several recent advances in our understanding of IF structure from the study of keratins, glial fibrillary acidic protein, and lamin. These include discoveries of (i) a knob–pocket tetramer assembly mechanism in coil 1B; (ii) a lamin-specific coil 1B insert providing a one-half superhelix turn; (iii) helical, yet flexible, linkers within the rod domain; and (iv) the identification of coil 2B residues required for mature filament assembly. Furthermore, the head and tail domains of some IFs contain low-complexity aromatic-rich kinked segments, and structures of IFs with binding partners show electrostatic surfaces are a major contributor to complex formation. These new data advance the connection between IF structure, pathologic mutations, and clinical diseases in humans.     

Recent advancements in mass spectrometry–based tools to investigate newly synthesized proteins

Tight regulation of protein translation drives the proteome to undergo changes under influence of extracellular or intracellular signals. Despite mass spectrometry–based proteomics being an excellent method to study differences in protein abundance in complex proteomes, analyzing minute or rapid changes in protein synthesis and abundance remains challenging. Therefore, several dedicated techniques to directly detect and quantify newly synthesized proteins have been developed, notably puromycin-based, bio-orthogonal noncanonical amino acid tagging–based, and stable isotope labeling by amino acids in cell culture–based methods, combined with mass spectrometry. These techniques have enabled the investigation of perturbations, stress, or stimuli on protein synthesis. Improvements of these methods are still necessary to overcome various remaining limitations. Recent improvements include enhanced enrichment approaches and combinations with various stable isotope labeling techniques, which allow for more accurate analysis and comparison between conditions on shorter timeframes and in more challenging systems. Here, we aim to review the current state in this field.     

Oral Octreotide: A Review of Recent Clinical Trials and Practical Recommendations for Its Use in the Treatment of Patients With Acromegaly

ObjectiveAcromegaly is characterized by chronic growth hormone (GH) and insulin-like growth factor 1 (IGF-1) hypersecretion, often caused by a GH-secreting pituitary adenoma. Even though surgery remains the first line of treatment, medical therapy is essential if surgery is contraindicated, does not achieve remission, or does not prevent recurrence despite apparent surgical remission. Oral octreotide capsules (OOCs) that combine octreotide with a transient permeability enhancer technology are the first oral somatostatin receptor ligands (SRLs) approved in the United States for acromegaly.MethodsWe review the literature and clinical trial data on OOC therapy in patients with acromegaly and discuss the clinical assessment of OOC use, potential drug–drug interactions, drug initiation, dose titration, and monitoring of drug efficacy and tolerability.ResultsIn 4 pivotal clinical trials involving 238 patients with acromegaly treated with OOC, effective suppression of serum GH and IGF-1 levels, maintenance of disease control, decreased breakthrough symptoms and symptomatic improvement with non-inferiority of OOCs to injectable SRLs in maintaining biochemical response was seen. Additionally, the safety profile of OOC therapy is comparable to that of injectable SRLs. Most patients who completed the clinical trials of OOCs have also expressed preference for oral compared with injectable SRL administration.ConclusionOOCs are an effective treatment option for patients with acromegaly who previously responded to injectable SRLs, with the benefits of avoiding injection-related side effects. This article provides a review of the pharmacology, safety, and efficacy and offers practical recommendations on the use of OOCs to treat injectable SRL-responsive patients with acromegaly.     

Deep generative models for 3D molecular structure

Deep generative models have gained recent popularity for chemical design. Many of these models have historically operated in 2D space; however, more recently explicit 3D molecular generative models have become of interest, which are the topic of this article. Dozens of published models have been developed in the last few years to generate molecules directly in 3D, outputting both the atom types and coordinates, either in one-shot or adding atoms or fragments step-by-step. These 3D generative models can also be guided by structural information such as a binding pocket representation to successfully generate molecules with docking score ranges similar to known actives, but still showing lower computational efficiency and generation throughput than 1D/2D generative models and sometimes producing unrealistic conformations. We advocate for a unified benchmark of metrics to evaluate generation and propose perspectives to be addressed in next implementations.     

Recent progress in dissecting ubiquitin signals with chemical biology tools

Protein ubiquitination regulates almost all eukaryotic cellular processes, and is of very high complexity due to the diversity of ubiquitin (Ub) modifications including mono-, multiply mono-, homotypic poly-, and even heterotypic poly-ubiquitination. To accurately elucidate the role of each specific Ub signal in different cells with spatiotemporal resolutions, a variety of chemical biology tools have been developed. In this review, we summarize some recently developed chemical biology tools for ubiquitination studies and their applications in molecular and cellular biology.     

The intriguing molecular dynamics of Cer[EOS] in rigid skin barrier lipid layers requires improvement of the model

Omega-O-acyl ceramides such as 32-linoleoyloxydotriacontanoyl sphingosine (Cer[EOS]) are essential components of the lipid skin barrier, which protects our body from excessive water loss and the penetration of unwanted substances. These ceramides drive the lipid assembly to epidermal-specific long periodicity phase (LPP), structurally much different than conventional lipid bilayers. Here, we synthesized Cer[EOS] with selectively deuterated segments of the ultralong N-acyl chain or deuterated or ¹³C-labeled linoleic acid and studied their molecular behavior in a skin lipid model. Solid-state ²H NMR data revealed surprising molecular dynamics for the ultralong N-acyl chain of Cer[EOS] with increased isotropic motion toward the isotropic ester-bound linoleate. The sphingosine moiety of Cer[EOS] is also highly mobile at skin temperature, in stark contrast to the other LPP components, N-lignoceroyl sphingosine acyl, lignoceric acid, and cholesterol, which are predominantly rigid. The dynamics of the linoleic chain is quantitatively described by distributions of correlation times and using dynamic detector analysis. These NMR results along with neutron diffraction data suggest an LPP structure with alternating fluid (sphingosine chain-rich), rigid (acyl chain-rich), isotropic (linoleate-rich), rigid (acyl-chain rich), and fluid layers (sphingosine chain-rich). Such an arrangement of the skin barrier lipids with rigid layers separated with two different dynamic “fillings” i) agrees well with ultrastructural data, ii) satisfies the need for simultaneous rigidity (to ensure low permeability) and fluidity (to ensure elasticity, accommodate enzymes, or antimicrobial peptides), and iii) offers a straightforward way to remodel the lamellar body lipids into the final lipid barrier.     

In silico reconstitution of DNA replication. Lessons from single-molecule imaging and cryo-tomography applied to single-particle cryo-EM

DNA replication has been reconstituted in vitro with yeast proteins, and the minimal system requires the coordinated assembly of 16 distinct replication factors, consisting of 42 polypeptides. To understand the molecular interplay between these factors at the single residue level, new structural biology tools are being developed. Inspired by advances in single-molecule fluorescence imaging and cryo-tomography, novel single-particle cryo-EM experiments have been used to characterise the structural mechanism for the loading of the replicative helicase. Here, we discuss how in silico reconstitution of single-particle cryo-EM data can help describe dynamic systems that are difficult to approach with conventional three-dimensional classification tools.     

Relationship between hepatic and mitochondrial ceramides: a novel in vivo method to track ceramide synthesis

Ceramides (CERs) are key intermediate sphingolipids implicated in contributing to mitochondrial dysfunction and the development of multiple metabolic conditions. Despite the growing evidence of CER role in disease risk, kinetic methods to measure CER turnover are lacking, particularly using in vivo models. The utility of orally administered ¹³C₃, ¹⁵N l-serine, dissolved in drinking water, was tested to quantify CER 18:1/16:0 synthesis in 10-week-old male and female C57Bl/6 mice. To generate isotopic labeling curves, animals consumed either a control diet or high-fat diet (HFD; n = 24/diet) for 2 weeks and varied in the duration of the consumption of serine-labeled water (0, 1, 2, 4, 7, or 12 days; n = 4 animals/day/diet). Unlabeled and labeled hepatic and mitochondrial CERs were quantified using liquid chromatography tandem MS. Total hepatic CER content did not differ between the two diet groups, whereas total mitochondrial CERs increased with HFD feeding (60%, P < 0.001). Within hepatic and mitochondrial pools, HFD induced greater saturated CER concentrations (P < 0.05) and significantly elevated absolute turnover of 16:0 mitochondrial CER (mitochondria: 59%, P < 0.001 vs. liver: 15%, P = 0.256). The data suggest cellular redistribution of CERs because of the HFD. These data demonstrate that a 2-week HFD alters the turnover and content of mitochondrial CERs. Given the growing data on CERs contributing to hepatic mitochondrial dysfunction and the progression of multiple metabolic diseases, this method may now be used to investigate how CER turnover is altered in these conditions.     

The Evaluation of Low- and High-Dose Steroid Treatments in Subacute Thyroiditis: A Retrospective Observational Study

ObjectiveThe optimal steroid regimen in the treatment of subacute thyroiditis (SAT) is controversial. This study aims to compare low- and high-dose steroid regimens in the treatment of SAT.MethodsA single-center, retrospective observational cohort study with up to 1 year of follow-up was conducted. A total of 44 patients in the 16-mg methylprednisolone (MPS) group and 47 patients in the 48-mg MPS group were enrolled. Clinical and laboratory findings from the time of diagnosis to 1-year of the follow-up were assessed. Treatment response, recurrence, and hypothyroidism (HPT) rates were evaluated.ResultsClinical symptoms, sedimentation rates, C-reactive protein, and thyroid hormone levels of the patients were similar in the 2 groups. Recovery was achieved in all patients at the end of the treatments; however, treatment duration needed to be extended for 6 (13.6%) and 1 (2.1%) of the patients in the 16-mg and 48-mg MPS groups, respectively. The 48-mg MPS group had a higher SAT recurrence rate than the 16-mg MPS group (P = .04). Logistic regression analysis suggested that a lower thyroid-stimulating hormone level at the end of the treatment was a predictor of recurrence (β = –0.544, P = .014, 95% CI: 0.376-0.895). While the transient HPT rate was 10 (21.3%) and 10 (22.7%) in the 48-mg and 16-mg MPS groups, respectively, a permanent HPT developed in 5 (10.6%) of patients in the 48-mg MPS and 3 (6.8%) in the 16-mg MPS group. The permanent and transient HPT rates were determined to be similar in the low- and high-dose groups (P > .05).ConclusionLow-dose steroid therapy may be sufficient to achieve a complete recovery and better outcomes in SAT.     

The Effect of Gender-Affirming Hormone Therapy on the Risk of Subclinical Atherosclerosis in the Transgender Population: A Systematic Review

ObjectiveThe impact of gender-affirming hormone therapy (GAHT) on cardiovascular (CV) health is still not entirely established. A systematic review was conducted to summarize the evidence on the risk of subclinical atherosclerosis in transgender people receiving GAHT.MethodsA systematic review was performed following Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines, and data were searched in PubMed, LILACS, EMBASE, and Scopus databases for cohort, case-control, and cross-sectional studies or randomized clinical trials, including transgender people receiving GAHT. Transgender men and women before and during/after GAHT for at least 2 months, compared with cisgender men and women or hormonally untreated transgender persons. Studies reporting changes in variables related to endothelial function, arterial stiffness, autonomic function, and blood markers of inflammation/coagulation associated with CV risk were included.ResultsFrom 159 potentially eligible studies initially identified, 12 were included in the systematic review (8 cross-sectional and 4 cohort studies). Studies of trans men receiving GAHT reported increased carotid thickness, brachial-ankle pulse wave velocity, and decreased vasodilation. Studies of trans women receiving GAHT reported decreased interleukin 6, plasminogen activator inhibitor-1, and tissue plasminogen activator levels and brachial-ankle pulse wave velocity, with variations in flow-mediated dilation and arterial stiffness depending on the type of treatment and route of administration.ConclusionsThe results suggest that GAHT is associated with an increased risk of subclinical atherosclerosis in transgender men but may have either neutral or beneficial effects in transgender women. The evidence produced is not entirely conclusive, suggesting that additional studies are warranted in the context of primary prevention of CV disease in the transgender population receiving GAHT.Systematic Review RegistrationPROSPERO, identifier CRD42022323757.     

Geranylgeranoic acid,a bioactive and endogenous fatty acid in mammals: a review

Geranylgeranoic acid (GGA) was first reported in 1983 as one of the mevalonic acid metabolites, but its biological significance was not studied for a long time. Our research on the antitumor effects of retinoids led us to GGA, one of the acyclic retinoids that induce cell death in human hepatoma-derived cell lines. We were able to demonstrate the presence of endogenous GGA in various tissues of male rats, including the liver, testis, and cerebrum, by LC-MS/MS. Furthermore, the biosynthesis of GGA from mevalonic acid in mammals including humans was confirmed by isotopomer spectral analysis using ¹³C-labeled mevalonolactone and cultured hepatoma cells, and the involvement of hepatic monoamine oxidase B in the biosynthesis of GGA was also demonstrated. The biological activity of GGA was analyzed from the retinoid (differentiation induction) and nonretinoid (cell death induction) aspects, and in particular, the nonretinoid mechanism by which GGA induces cell death in hepatoma cells was found to involve pyroptosis via ER stress responses initiated by TLR4 signaling. In addition to these effects of GGA, we also describe the in vivo effects of GGA on reproduction. In this review, based mainly on our published papers, we have shown that hepatic monoamine oxidase B is involved in the biosynthesis of GGA and that GGA induces cell death in human hepatoma-derived cell lines by noncanonical pyroptosis, one of the mechanisms of sterile inflammatory cell death.