Rodrigo J. Carbajo

Polymer Masked−Unmasked Protein Therapy. 1. Bioresponsive Dextrin−Trypsin and −Melanocyte Stimulating Hormone Conjugates Designed for α-Amylase Activation

Polymer-protein conjugation, particularly PEGylation, is well-established as a means of increasing circulation time, reducing antigenicity, and improving the stability of protein therapeutics. However, PEG has limitations including lack of polymer biodegradability, and conjugation can diminish or modify protein activity. The aim of this study was to explore a novel approach for polymer-protein modification called polymer-masking-unmasking-protein therapy (PUMPT), the hypothesis being that conjugation of a biodegradable polymer to a protein would protect it and mask activity in transit, while enabling controlled reinstatement of activity at the target site by triggered degradation of the polymeric component. To test this hypothesis, dextrin (alpha-1,4 polyglucose, a natural polymer degraded by alpha-amylase) was conjugated to trypsin as a model enzyme or to melanocyte stimulating hormone (MSH) as a model receptor-binding ligand. The effect of dextrin molecular weight (7700, and 47200 g/mol) and degree of succinoylation (9-32 mol %) on its ability to mask/unmask trypsin activity was assessed using N-benzoyl-L-arginine-p-nitroanilide (L-BAPNA). Dextrin conjugation reduced enzyme activity by 34-69% depending on the molecular weight and degree of succinoylation of dextrin. However, incubation with alpha-amylase led to reinstatement of activity to a maximum of 92-115%. The highest molecular dextrin (26 mol % succinoylation) gave optimum trypsin masking-unmasking. This intermediate was used to synthesize a dextrin-MSH conjugate (dextrin Mw = 47200 g/mol; MSH content 37 wt %), and its biological activity (+/-alpha-amylase) was assessed by measuring melanin production by murine melanoma (B16F10) cells. Conjugation reduced melanin production to 11%, but addition of alpha-amylase was able to restore activity to 33% of the control value. These were the first studies to confirm the potential of PUMPT for further application to clinically important protein therapeutics. The choice of masking polymer, activation mechanism, and the rate of unmasking can be tailored to therapeutic application.

Polymer conjugates as therapeutics: future trends, challenges and opportunities

Objective: Clinical proof of concept for polymer conjugates has already been achieved over the last 30 years, with a family of polymer–protein conjugates reaching the market and an exponentially growing list of polymer–drug conjugates currently in clinical trials. However, many challenges and opportunities still lie ahead, providing scope to develop this platform technology further. Methods: The delivery of new anticancer agents aimed at novel molecular targets and their combination, the development of both new polymeric materials with defined architectures and the treatment of diseases other than cancer are the most exciting and promising areas. The latest advances and future trends in the polymer conjugate field will be presented in this article, providing an insight into their potential in the clinics and offering a wide range of research approaches within the scientific community. Results/conclusion: Polymer therapeutics is a rapidly emerging field with exponentially growing opportunities to achieve medical treatments with highly enhanced therapeutic value.

Deciphering Biosynthesis of the RNA Polymerase Inhibitor Streptolydigin and Generation of Glycosylated Derivatives

The biosynthetic gene cluster for the dienoyltetramic acid streptolydigin was identified and characterized from the producer organism Streptomyces lydicus NRRL2433. Sequence analysis of an 80.8 kb DNA region revealed the presence of 38 ORFs, 29 of which are probably involved in streptolydigin biosynthesis and would code for all activities required for its biosynthesis. Six insertional inactivation mutants were generated in the sequenced region to prove its involvement in streptolydigin biosynthesis, to define the boundaries of the cluster, to functionally characterize some genes, and to generate novel derivatives. A model for streptolydigin biosynthesis is proposed that includes a probable domain skipping in the streptolydigin PKS and the participation of a free-standing adenylation domain protein. Some bioactive derivatives of streptolydigin with altered glycosylation pattern have been produced by combinatorial biosynthesis showing a certain degree of flexibility of the L-rhodinosyl transferase SlgG for the recognition of 2,3,6-trideoxyhexoses and 2,6-dideoxyhexoses, both in D- and L-configuration.

Generation of potent and selective kinase inhibitors by combinatorial biosynthesis of glycosylated indolocarbazoles.

We report the generation of novel glycosylated indolocarbazoles by combinatorial biosynthesis, and the identification of two novel potent and selective compounds inhibitors of JAK2 and Ikkb kinases.

Contributions of computational chemistry and biophysical techniques to fragment-based drug discovery.

In the last decade, fragment-based drug discovery (FBDD) has evolved from a novel approach in the search of new hits to a valuable alternative to the high-throughput screening (HTS) campaigns of many pharmaceutical companies. The increasing relevance of FBDD in the drug discovery universe has been concomitant with an implementation of the biophysical techniques used for the detection of weak inhibitors, e.g. NMR, X-ray crystallography or surface plasmon resonance (SPR). At the same time, computational approaches have also been progressively incorporated into the FBDD process and nowadays several computational tools are available. These stretch from the filtering of huge chemical databases in order to build fragment-focused libraries comprising compounds with adequate physicochemical properties, to more evolved models based on different in silico methods such as docking, pharmacophore modelling, QSAR and virtual screening. In this paper we will review the parallel evolution and complementarities of biophysical techniques and computational methods, providing some representative examples of drug discovery success stories by using FBDD.

Metabolomic Profile of Umbilical Cord Blood Plasma from Early and Late Intrauterine Growth Restricted (IUGR) Neonates with and without Signs of Brain Vasodilation

Objectives To characterize via NMR spectroscopy the full spectrum of metabolic changes in umbilical vein blood plasma of newborns diagnosed with different clinical forms of intrauterine growth restriction (IUGR). Methods 23 early IUGR cases and matched 23 adequate-for-gestational-age (AGA) controls and 56 late IUGR cases with 56 matched AGAs were included in this study. Early IUGR was defined as a birth weight <10th centile, abnormal umbilical artery (UA) Doppler and delivery <35 weeks. Late IUGR was defined as a birth weight <10th centile with normal UA Doppler and delivery >35 weeks. This group was subdivided in 18 vasodilated (VD) and 38 non-VD late IUGR fetuses. All AGA patients had a birth weight >10th centile. 1H nuclear magnetic resonance (NMR) metabolomics of the blood samples collected from the umbilical vein at delivery was obtained. Multivariate statistical analysis identified several metabolites that allowed the discrimination between the different IUGR subgroups, and their comparative levels were quantified from the NMR data. Results The NMR-based analysis showed increased unsaturated lipids and VLDL levels in both early and late IUGR samples, decreased glucose and increased acetone levels in early IUGR. Non-significant trends for decreased glucose and increased acetone levels were present in late IUGR, which followed a severity gradient when the VD and non-VD subgroups were considered. Regarding amino acids and derivatives, early IUGR showed significantly increased glutamine and creatine levels, whereas the amounts of phenylalanine and tyrosine were decreased in early and late-VD IUGR samples. Valine and leucine were decreased in late IUGR samples. Choline levels were decreased in all clinical subforms of IUGR. Conclusions IUGR is not associated with a unique metabolic profile, but important changes are present in different clinical subsets used in research and clinical practice. These results may help in characterizing comprehensively specific alterations underlying different IUGR subsets.

A Kernel for Open Source Drug Discovery in Tropical Diseases

Background Conventional patent-based drug development incentives work badly for the developing world, where commercial markets are usually small to non-existent. For this reason, the past decade has seen extensive experimentation with alternative R&D institutions ranging from private–public partnerships to development prizes. Despite extensive discussion, however, one of the most promising avenues—open source drug discovery—has remained elusive. We argue that the stumbling block has been the absence of a critical mass of preexisting work that volunteers can improve through a series of granular contributions. Historically, open source software collaborations have almost never succeeded without such “kernels”. Methodology/Principal Findings Here, we use a computational pipeline for: (i) comparative structure modeling of target proteins, (ii) predicting the localization of ligand binding sites on their surfaces, and (iii) assessing the similarity of the predicted ligands to known drugs. Our kernel currently contains 143 and 297 protein targets from ten pathogen genomes that are predicted to bind a known drug or a molecule similar to a known drug, respectively. The kernel provides a source of potential drug targets and drug candidates around which an online open source community can nucleate. Using NMR spectroscopy, we have experimentally tested our predictions for two of these targets, confirming one and invalidating the other. Conclusions/Significance The TDI kernel, which is being offered under the Creative Commons attribution share-alike license for free and unrestricted use, can be accessed on the World Wide Web at http://www.tropicaldisease.org. We hope that the kernel will facilitate collaborative efforts towards the discovery of new drugs against parasites that cause tropical diseases.

Biosynthesis of the RNA polymerase inhibitor streptolydigin in Streptomyces lydicus: tailoring modification of 3-methyl-aspartate.

The asparaginyl-tRNA synthetase-like SlgZ and methyltransferase SlgM enzymes are involved in the biosynthesis of the tetramic acid streptolydigin in Streptomyces lydicus. Inactivation of slgZ led to a novel streptolydigin derivative. Overexpression of slgZ, slgM, or both in S. lydicus led to a considerable increase in streptolydigin production.

Amino Acid Precursor Supply in the Biosynthesis of the RNA Polymerase Inhibitor Streptolydigin by Streptomyces lydicus

Biosynthesis of the hybrid polyketide-nonribosomal peptide antibiotic streptolydigin, 3-methylaspartate, is utilized as precursor of the tetramic acid moiety. The three genes from the Streptomyces lydicus streptolydigin gene cluster slgE1-slgE2-slgE3 are involved in 3-methylaspartate supply. SlgE3, a ferredoxin-dependent glutamate synthase, is responsible for the biosynthesis of glutamate from glutamine and 2-oxoglutarate. In addition to slgE3, housekeeping NADPH- and ferredoxin-dependent glutamate synthase genes have been identified in S. lydicus. The expression of slgE3 is increased up to 9-fold at the onset of streptolydigin biosynthesis and later decreases to ∼2-fold over the basal level. In contrast, the expression of housekeeping glutamate synthases decreases when streptolydigin begins to be synthesized. SlgE1 and SlgE2 are the two subunits of a glutamate mutase that would convert glutamate into 3-methylaspartate. Deletion of slgE1-slgE2 led to the production of two compounds containing a lateral side chain derived from glutamate instead of 3-methylaspartate. Expression of this glutamate mutase also reaches a peak increase of up to 5.5-fold coinciding with the onset of antibiotic production. Overexpression of either slgE3 or slgE1-slgE2 in S. lydicus led to an increase in the yield of streptolydigin.

Synthesis and 1H-, 15N-, 31P-, 183W-Multinuclear Magnetic Resonance Study of the Cyclotriphosphazenes [N3P3(dobp)2(OC5H4N-4)2] and [N3P3(dobp)(OC5H4N-4)4] and Their W(CO)5 Complexes (dobp = 2,2′-dioxybiphenyl)

The reactions of [N3P3(dobp)2Cl2] and [N3P3(dobp)Cl4] with a mixture of HOC5H4N-4 and K2CO3 in acetone give the cyclotriphosphazenes [N3P3(dobp)2(OC5H4N-4)2] and [N3P3(dobp)(OC5H4N-4)4], respectively. These compounds react with [W(MeOH)(CO)5] in methanol to give mixtures of the polymetallic complexes [N3P3(dobp)2(OC5H4N-4)2{W(CO)5}x] (x = 1, 2) and [N3P3(dobp)(OC5H4N-4)4{W(CO)5}x] (x = 1–4), which are unstable in solution, slowly undergoing loss of the pentacarbonyl moiety. A complete characterization by multinuclear 1H, 15N, 31P, 183W magnetic resonance has revealed that the complexation of the N atom of one 4-oxypyridine ligand by the W(CO)5 fragment has a measurable effect on other parts of the phosphazene molecule very far away from the coordination site. The changes observed in δ183W have been used to identify the components in mixtures of compounds incorporating different numbers of tungsten atoms in the molecule. The characterization of less sensitive nuclei has been accomplished by means of indirect detection methods.