Identification of a new class of proteasome inhibitors based on a naphthyl-azotricyclic-urea-phenyl scaffold.

Proteasomes play an important role in protein degradation and regulation of many cellular pathways by maintaining protein balance. Inhibitors of proteasomes disrupt this balance affecting proteins that are key in malignancies and as such have found applications in the treatment of multiple myeloma and mantle cell lymphoma. However, resistance mechanisms have been reported for these proteasome inhibitors including mutations at the ß5 site which necessitates the constant development of new inhibitors. In this work, we report the identification of a new class of proteasome inhibitors, polycyclic molecules bearing a naphthyl-azotricyclic-urea-phenyl scaffold, from screening of the ZINC library of natural products. The most potent of these compounds showed evidence of dose dependency through proteasome assays with IC50 values in the low micromolar range, and kinetic analysis revealed competitive binding at the ß5c site with an estimated inhibition constant, K i, of 1.15 µM. Inhibition was also shown for the ß5i site of the immunoproteasome at levels similar to those of the constitutive proteasome. Structure-activity relationship studies identified the naphthyl substituent to be crucial for activity and this was attributed to enhanced hydrophobic interactions within ß5c. Further to this, halogen substitution within the naphthyl ring enhanced the activity and allowed for π-π interactions with Y169 in ß5c and Y130 and F124 in ß5i. The combined data highlight the importance of hydrophobic and halogen interactions in ß5 binding and assist in the design of next generation inhibitors of proteasomes.

Argyrin B, a non-competitive inhibitor of the human immunoproteasome exhibiting preference for ß1i.

Inhibitors of the proteasome have found broad therapeutic applications; however, they show severe toxicity due to the abundance of proteasomes in healthy cells. In contrast, inhibitors of the immunoproteasome, which is upregulated during disease states, are less toxic and have increased therapeutic potential including against autoimmune disorders. In this project, we report argyrin B, a natural product cyclic peptide to be a reversible, non-competitive inhibitor of the immunoproteasome. Argyrin B showed selective inhibition of the ß5i and ß1i sites of the immunoproteasome over the ß5c and ß1c sites of the constitutive proteasome with nearly 20-fold selective inhibition of ß1i over the homologous ß1c. Molecular modelling attributes the ß1i over ß1c selectivity to the small hydrophobic S1 pocket of ß1i and ß5i over ß5c to site-specific amino acid variations that enable additional bonding interactions and stabilization of the binding conformation. These findings facilitate the design of immunoproteasome selective and reversible inhibitors that may have a greater therapeutic potential and lower toxicity.

Evaluation of geometrical effects of microneedles on skin penetration by CT scan and finite element analysis.

Computerized tomography scan (CT scan) imaging and finite element analysis were employed to investigate how the geometric composition of microneedles affects their mechanical strength and penetration characteristics. Simulations of microneedle arrays, comprising triangular, square and hexagonal microneedle base, revealed a linear dependence of the mechanical strength to the number of vertices in the polygon base. A laser-enabled, micromoulding technique was then used to fabricate 3×3 microneedle arrays, each individual microneedle having triangular, square or hexagonal base geometries. Their penetration characteristics into ex-vivo porcine skin, were investigated for the first time by CT scan imaging. This revealed greater penetration depths for the triangular and square-based microneedles, demonstrating CT scan as a powerful and reliable technique for studying microneedle skin penetration.

Structural characterisation and transdermal delivery studies on sugar microneedles: experimental and finite element modelling analyses.

Dissolving microneedles are especially attractive for transdermal drug delivery as they are associated with improved patient compliance and safety. Furthermore, microneedles made of sugars offer the added benefit of biomolecule stabilisation making them ideal candidates for delivering biological agents such as proteins, peptides and nucleic acids. In this study, we performed experimental and finite element analyses to study the mechanical properties of sugar microneedles and evaluate the effect of sugar composition on microneedle ability to penetrate and deliver drug to the skin. Results showed that microneedles made of carboxymethylcellulose/maltose are superior to those made of carboxymethylcellulose/trehalose and carboxymethylcellulose/sucrose in terms of mechanical strength and the ability to deliver drug. Buckling was predicted to be the main mode of microneedle failure and the order of buckling was positively correlated to the Young's modulus values of the sugar constituents of each microneedle.

Computational inhibition studies of the human proteasome by argyrin-based analogues with subunit specificity.

A computational procedure was developed to study the subunit-specific interactions of the proteasome inhibitors argyrin A and F, with the aim of indentifying the determinants of subunit selectivity. Three-dimensional models of humanized proteasome active sites ß1 , ß2 and ß5 were developed and subsequently used in molecular docking simulations with the argyrin analogues. The subunit selectivity exhibited by each analogue could be explained based on the site-specific interactions and a probability-based specificity parameter derived in this study. A rational approach that involved maximizing site-specific interactions was followed to guide the design of new argyrin analogues as specific inhibitors of the caspase-like (ß1 site) activity.

Receptor-attached amphiphilic terpolymer for selective drug recognition in aqueous solutions.

In the present work, a combination of binding studies and molecular docking were employed to demonstrate drug encapsulation and host-guest chemistry in self-assembled micelles consisting of amphiphilic terpolymers. The terpolymer is composed of poly(3-sulfopropyl methacrylate), as the hydrophilic component, poly(n-dodecyl acrylate), as the hydrophobic component and poly(barbiturate receptor), as the component for drug recognition. The combined approach was tested on four model compounds from the family of barbiturates, phenobarbital, mephobarbital, secobarbital, and thiopental, chosen based on their differential hydrogen bonding capabilities. Drug encapsulation and hydrogen-bonding based recognition within the micellar core of the receptor-terpolymer was demonstrated by micellar electrokinetic chromatography. The resulting trends in the binding affinity of the barbiturates to the receptor-terpolymer, were correlated to the trends obtained from computational docking simulations. This receptor-modified polymeric micelle is intended to serve as a model for the design of novel, versatile, and highly selective molecular scaffolds that will provide suitable environment for host-guest chemistry and act as simplified mimics to more complex biological systems.

Analysis of binding parameters of HIV-1 integrase inhibitors: correlates of drug inhibition and resistance.

This study undertook an exploratory data analysis of the binding parameters of HIV-1 integrase inhibitors. The study group involved inhibitors in preclinical development from the diketo acid, pyrroloquinoline and naphthyridine carboxamide families and the most advanced inhibitors Raltegravir and Elvitegravir. Distinct differences were observed in the energetics of binding between the studied classes of inhibitors that also correlated with drug resistant patterns. Quantitative-property-activity-relationships correlated experimental IC(50) values to the binding energy and the logarithm of the partition coefficient between n-octanol and water (clogP). The approach followed here serves as an improved basis for the development of 'second generation' integrase inhibitors.

Implications of HIV-1 M group polymorphisms on integrase inhibitor efficacy and resistance: genetic and structural in silico analyses.

The extensive polymorphisms among HIV-1 subtypes have been implicated in drug resistance development. Integrase inhibitors represent the latest addition to the treatment of HIV-1, and their efficacy and resistance patterns among M group strains are currently under investigation. This study analyzed the intersubtype variation within 108 integrase sequences from seven subtypes. The residues associated with catalytic activity and primary resistance to raltegravir were highly conserved among all strains. Variations were observed in residues associated with secondary resistance. Molecular modeling studies indicated a two-way binding mode of raltegravir that explains the resistance pathways and the implication of nonconservative mutations in integrase-raltegravir interactions.

Second-generation total synthesis of azaspiracids-1, -2, and -3.

The naturally occurring but scarce marine neurotoxins azaspiracids-1, -2, and -3 have been synthesized from five key building blocks by a convergent strategy that involved dithiane and Stille coupling reactions. The ABCD fragments were constructed through a cascade reaction involving deprotection/self-assembly of the precursors, while the FGHI fragment was forged by a neodymium triflate-induced cyclization. The final ring closure (ring G) was achieved, after the union of all fragments, through an iodoetherification reaction followed by reductive removal of the facilitating iodine residue. These improved, second-generation routes confirm the absolute structures and render all three azaspiracids readily available for biological studies.