The war on hTG2: warhead optimization in small molecule human tissue transglutaminase inhibitors.

Human tissue transglutaminase (hTG2) is a multifunctional enzyme with protein cross-linking and G-protein activity, both of which have been implicated in the progression of diseases such as fibrosis and cancer stem cell propagation when dysregulated, prompting the development of small molecule targeted covalent inhibitors (TCIs) possessing a crucial electrophilic 'warhead'. In recent years there have been significant advances in the library of warheads available for the design of TCIs; however, the exploration of warhead functionality in hTG2 inhibitors has remained relatively stagnant. Herein, we describe a structure-activity relationship study entailing rational design and synthesis for systematic variation of the warhead on a previously reported small molecule inhibitor scaffold, and rigorous kinetic evaluation of inhibitory efficiency, selectivity, and pharmacokinetic stability. This study reveals a strong influence on the kinetic parameters k inact and K I with even subtle variation in warhead structure, suggesting that the warhead plays a significant role in not only reactivity, but also binding affinity, which consequently extends to isozyme selectivity. Warhead structure also influences in vivo stability, which we model by measuring intrinsic reactivity with glutathione, as well as stability in hepatocytes and in whole blood, giving insight into degradation pathways and relative therapeutic potential of different functional groups. This work provides fundamental structural and reactivity information highlighting the importance of strategic warhead design for the development of potent hTG2 inhibitors.

A BRD's (BiRD's) eye view of BET and BRPF bromodomains in neurological diseases.

Neurological disorders (NLDs) are among the top leading causes for disability worldwide. Dramatic changes in the epigenetic topography of the brain and nervous system have been found in many NLDs. Histone lysine acetylation has prevailed as one of the well characterised epigenetic modifications in these diseases. Two instrumental components of the acetylation machinery are the evolutionarily conserved Bromodomain and PHD finger containing (BRPF) and Bromo and Extra terminal domain (BET) family of proteins, also referred to as acetylation 'readers'. Several reasons, including their distinct mechanisms of modulation of gene expression and their property of being highly tractable small molecule targets, have increased their translational relevance. Thus, compounds which demonstrated promising results in targeting these proteins have advanced to clinical trials. They have been established as key role players in pathologies of cancer, cardiac diseases, renal diseases and rheumatic diseases. In addition, studies implicating the role of these bromodomains in NLDs are gaining pace. In this review, we highlight the findings of these studies, and reason for the plausible roles of all BET and BRPF members in NLDs. A comprehensive understanding of their multifaceted functions would be radical in the development of therapeutic interventions.

Typhoid in India: An Age-old Problem With an Existing Solution.

Enteric fever continues to impact millions of people who lack adequate access to clean water and sanitation. The typhoid and paratyphoid fever burden in South Asia is broadly acknowledged, but current estimates of incidence, severity, and cost of illness from India are lacking. This supplement addresses this gap in our knowledge, presenting findings from two years of surveillance, conducted at multiple sites between October 2017 and February 2020, in the Surveillance for Enteric Fever in India (SEFI) network. Results provide contemporaneous evidence of high disease burden and cost of illness-the latter borne largely by patients in the absence of universal healthcare coverage in India. Against a backdrop of immediate priorities in the COVID-19 pandemic, these data are a reminder that typhoid, though often forgotten, remains a public health problem in India. Typhoid conjugate vaccines, produced by multiple Indian manufacturers, and recommended for use in high burden settings, ensure that the tools to tackle typhoid are an immediately available solution to this public health problem.

FTO: An Emerging Molecular Player in Neuropsychiatric Diseases.

A myriad of chemical modifications of DNA and histones involved in the epigenetic regulation of neural gene expression have been documented and studied in detail since many years. However, more recently, modifications in RNA and their implications for neural gene functions have been progressively investigated. Of these, the most widely studied is the N6-methyladenosine (m6A) modification. The discovery of the first m6A demethylase, known as the fat, mass and obesity (FTO) associated protein, has further fortified the field of epitranscriptomic regulatory mechanisms, owing to FTO's involvement in several biological processes including brain development and function. Concomitantly, multiple lines of evidence have associated FTO with neuropsychiatric disorders. In this review, we discuss how FTO can exert its effect by acting not only on m6A but also on O, N6-dimethyladenosine (m6Am) in different types of RNA and potentially influence the development of some major neuropsychiatric diseases.

Neurologic complication after a roller coaster ride.

Neurologic complications after roller coaster rides are uncommon but potentially catastrophic. Physicians should have a high index of suspicion and prompt appropriate investigation. A 22-year-old healthy African American man presented with a 2-day history of constant occipital headache associated with vertigo, nausea, vomiting, and ambulatory dysfunction. Physical examination showed gait ataxia, slight dysmetria, and vertical nystagmus. Magnetic resonance imaging (MRI) of the brain showed early subacute ischemic infarct in the right cerebellum in the distribution of the right posterior inferior cerebellar artery. Magnetic resonance angiography of the neck showed focal dissection of the right vertebral artery at C1 through C2 level. On subsequent questioning, the patient recollected riding a roller coaster 2 weeks before the onset of symptoms. Anticoagulation with heparin was started, and the patient was bridged to oral warfarin. After a 5-day uneventful hospital course, symptoms improved and patient was discharged on oral anticoagulation. Cervicocephalic arterial dissections after roller coaster rides are rarely described in literature. The acceleration and abrupt changes of direction might lead to indirect trauma that is applied to mobile portions of the cervicocephalic arteries leading to intimal tears. Magnetic resonance angiography combined with axial T1-weighted cervical MRI is preferred because it is a high-sensitive, noninvasive test. The rationale for the use of anticoagulants or antiplatelets in patients with cervicocephalic arterial dissection is to prevent early recurrence and infarction. However, a meta-analysis failed to show significant difference in the rates of disability or death between both groups. Therefore, the decision for medical treatment should be made in a case-by-case basis.

p38γ promotes breast cancer cell motility and metastasis through regulation of RhoC GTPase, cytoskeletal architecture, and a novel leading edge behavior.

Understanding the molecular alterations that confer cancer cells with motile, metastatic properties is needed to improve patient survival. Here, we report that p38γ motogen-activated protein kinase regulates breast cancer cell motility and metastasis, in part, by controlling expression of the metastasis-associated small GTPase RhoC. This p38γ-RhoC regulatory connection was mediated by a novel mechanism of modulating RhoC ubiquitination. This relationship persisted across multiple cell lines and in clinical breast cancer specimens. Using a computational mechanical model based on the finite element method, we showed that p38γ-mediated cytoskeletal changes are sufficient to control cell motility. This model predicted novel dynamics of leading edge actin protrusions, which were experimentally verified and established to be closely related to cell shape and cytoskeletal morphology. Clinical relevance was supported by evidence that elevated expression of p38γ is associated with lower overall survival of patients with breast cancer. Taken together, our results offer a detailed characterization of how p38γ contributes to breast cancer progression. Herein we present a new mechanics-based analysis of cell motility, and report on the discovery of a leading edge behavior in motile cells to accommodate modified cytoskeletal architecture. In summary, these findings not only identify a novel mechanism for regulating RhoC expression but also advance p38γ as a candidate therapeutic target.

From in vitro to in silico and back again: using biological and mathematical synergy to decipher breast cancer cell motility.

The complexity of biological systems is often prohibitive in testing specific hypotheses from first physical principles. To circumvent these limitations we used biological data to inform a mathematical model of breast cancer cell motility. Using this in silico model we were able to accurately assess the influence of actin cytoskeletal architecture on the motility of a genetically modified breast cancer cell line. Furthermore, using the in silico model revealed a biological phenomenon that has not been previously described in live cell movement. Fusing biology and mathematics as presented here represents a new direction for biomedical research in which advances in each field synergistically drive discoveries in the other.

Development of a process to manufacture PEGylated orally bioavailable insulin.

To make insulin orally bioavailable, insulin was modified by covalent attachment (conjugation) of a short-chain methoxy polyethylene glycol (mPEG) derivative to the ε-amino group of a specific amino acid residue (LysB(29)). During the conjugation process, activated PEG can react with any of the free amino groups, the N-terminal of the B chain (PheB(1)), the N-terminal of the A chain (GlyA(1)), and the ε-amino group of amino acid (LysB(29)), resulting in a heterogeneous mixture of conjugated products. The abundance of the desired product (Methoxy-PEG(3)-propionyl--insulin at LysB(29):IN-105) in the conjugation reaction can be controlled by changing the conjugation reaction conditions. Reaction conditions were optimized for maximal yield by varying the proportions of protein to mPEG molecule at various values of pH and different salt and solvent concentrations. The desired conjugated molecule (IN-105) was purified to homogeneity using RP-HPLC. The purified product, IN-105, was crystallized and lyophilized into powder form. The purified product was characterized using multiple analytical methods including ESI-TOF and peptide mapping to verify its chemical structure. In this work, we report the process development of new modified insulin prepared by covalent conjugation of short chain mPEG to the insulin molecule. The attachment of PEG to insulin resulted in a conjugated insulin derivative that was biologically active, orally bioavailable and that showed a dose-dependent glucose lowering effect in Type 2 diabetes patients.

Quantitative determination of oxytocin receptor antagonist atosiban in rat plasma by liquid chromatography-tandem mass spectrometry.

A kinetic study of atosiban was conducted following repeated intravenous administration in Wistar rats. Sample analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) following full validation of an in-house method. Eptifibatide, a cyclic peptide, was used as an internal standard (IS). The analyte and internal standard were extracted using solid phase extraction (SPE) method. Chromatographic separation was carried out using an ACE C18 5 microm 50 mm x 4.6 mm column with gradient elution. Mass spectrometric detection was performed using TSQ Quantum ultra AM. The lower limit of quantification was 0.01 microg/ml when 100 microl rat plasma was used. Plasma concentrations of atosiban were measured at 0 (pre-dose), 2, 15, 30, 45, 60, 120 min at the dosage levels of 0.125 mg/kg (low dose), 0.250 mg/kg (mid dose), and 0.500 mg/kg (high dose), respectively. Atosiban plasma concentration measured at Day 1 showed mean peak atosiban concentration (C(max)) 0.40, 0.57, 1.95 microg/ml for low, mid and high dose treated animals and mean peak concentration on Day 28 was 0.41, 0.88, 1.31microg/ml on Day 28 for low, mid and high dose treated animals.

Probing deamidation in therapeutic immunoglobulin gamma (IgG1) by 'bottom-up' mass spectrometry with electron transfer dissociation.

Aspartic acid formed by nonenzymatic deamidation of asparagine often isomerizes to isoaspartic acid through a succinimide intermediate. Accumulation of isoaspartic acid initiates aggregation and degradation in proteins. Deamidation at the antigen-binding region reduces the efficacy and also upregulates immunogenicity of monoclonal antibodies. We report an improved 'bottom-up' tandem mass spectrometric method to detect and decipher the position of isoaspartate formation in therapeutic immunoglobulin gamma in a single chromatographic run. Differentiation between aspartate and isoaspartate residues through collision-induced tandem mass spectrometry is formidable due to their identical mass. Signature backbone cleavage ions, c(n) + 57 and z(l-n) - 57, produced upon radical-mediated fragmentation, were used to delineate the site of isomerization. It is more conclusive than monitoring the relative peak intensity and the decrease in hydrophobicity of the isoaspartate-containing peptide in a chromatographic elution. Collectively, this methodology provides a useful tool to monitor deamidation and isomerization in biopharmaceuticals during their production, downstream processing and storage.

Oral insulin - a review of current status.

Oral insulin is one of the most exciting areas of development in the treatment of diabetes because of its potential benefit in patient convenience, rapid insulinization of liver, adequate insulin delivery avoiding peripheral hyperinsulinaemia while potentially avoiding adverse effects of weight gain and hypoglycaemia. Growing evidence that earlier initiation of intensive insulin therapy produces sustained tight glycaemic control resulting in substantial delay in complications makes an effective oral insulin product even more vital for the management of patients with diabetes. Despite knowledge of this unmet medical need, oral delivery of insulin has been unsuccessful because of several barriers. For several decades, researchers have tried to develop oral insulin using various technologies without much clinical or commercial success. This review summarizes the development status of oral insulins which are publicly reported to be undergoing clinical studies. Currently, two oral insulin products are in an advanced stage of clinical development and first data from long-term therapy are expected to be available in the second half of 2010.

A tandem mass spectrometric approach to the identification of O-glycosylated glargine glycoforms in active pharmaceutical ingredient expressed in Pichia pastoris.

Glycoforms of glargine expressed in Pichia pastoris were isolated by high-performance liquid chromatography and analyzed by a series of chemical and mass spectrometric methods for the identification of various glycoforms, glycosylation position, nature and structure of glycans. Reduction and alkylation, peptide mapping techniques were used to decipher the amino acid site at which glycosylation had taken place. Chemical methods were coupled with mass spectrometry techniques such as electrospray ionization and matrix-assisted laser desorption/ionization for identification of the glycosylation site.

Nimotuzumab, a promising therapeutic monoclonal for treatment of tumors of epithelial origin.

Nimotuzumab is a humanized therapeutic monoclonal antibody against epidermal growth factor receptor (EGFR). Clinical trials are ongoing globally to evaluate nimotuzumab in different indications. Nimotuzumab has been granted approval for use in squamous cell carcinoma of head and neck (SCCHN), glioma and nasopharyngeal cancer in different countries. This review focuses on the unique functional characteristics of nimotuzumab. Also, it discusses the safety and efficacy data obtained from the Phase IIb clinical trial conducted in India in SCCHN. Post marketing surveillance data from Cuba for the use of nimotuzumab in pediatric and adult glioma is also discussed. Overall, nimotuzumab has immense therapeutic potential in cancers of epithelial origin.

European Medicines Agency workshop on biosimilar monoclonal antibodies: July 2, 2009, London, UK.

The European Medicines Agency (EMEA) workshop on biosimilar monoclonal antibodies (mAbs), held July 2, 2009 at the EMEA headquarters in London, was a harbinger with potentially far-reaching implications for all groups interested in antibody therapeutics development. These groups include not only regulators and the innovator and generic biopharmaceutical industries, but also physicians, patients and payers. The objective of the workshop was to discuss and assess the feasibility of the development and authorization of mAbs using EMEA's biosimilar regulatory pathways. The workshop sequentially focused on questions relevant to three areas: (1) chemistry, manufacturing and controls (CMC), (2) non-clinical issues and (3) clinical issues, including outcome measures. Proceedings of the workshop are presented in Part 1 of this report, and discussed within the context of the legal, regulatory and business environments of the European Union, Asia and the United States in Parts 2, 3 and 4, respectively.