Current Topics in Medicinal Chemistry - Volume 12, Issue 14, 2012

Microbial metabolites are remarkable versatile as potent and selective drug lead candidates, and as in situ molecular probes, capable of interrogating key signalling, transport and developmental pathways. Microbial biodiscovery as a drug discovery paradigm has served science and society extremely well, and with appropriate modernisation and reinvestment is well placed to continue to do so into the future. Advances across many disciplines have revealed an untapped silent microbial secondary metabolism, which promises access to unprecedented bioactive chemical space. This renewed capacity can be further enhanced by recognition of the critical importance of widening the search parameters from narrow single bioassay/indication directed programs, to target both active and (seemingly) inactive metabolites, as well as new and known compounds, and a diversity of non-enzymatic chemical transformation products (all too often dismissed as artefacts). Many of the technical and commercial challenges that confronted microbial biodiscovery late last century have been resolved. The need is great and the time is right to re-plumb microbial biodiscovery back into the drug discovery pipeline.

Histone deacetylase (HDAC) enzymes have emerged as promising targets for the treatment of a wide range of human diseases, including cancers, inflammatory and metabolic disorders, immunological, cardiovascular, and infectious diseases. At present, such applications are limited by the lack of selective inhibitors available for each of the eighteen HDAC enzymes, with most currently available HDAC inhibitors having broad-spectrum activity against multiple HDAC enzymes. Such broad-spectrum activity maybe useful in treating some diseases like cancers, but can be detrimental due to cytotoxic side effects that accompany prolonged treatment of chronic diseased states. Here we summarize progress towards the design and discovery of HDAC inhibitors that are selective for some of the eleven zinc-containing classical HDAC enzymes, and identify opportunities to use such isozyme-selective inhibitors as chemical probes for interrogating the biological roles of individual HDAC enzymes in diseases.

Most drug discovery programs today originate by selection of ‘hit’ molecules resulting from assays against large compound screening libraries. The chemical space in which these hits reside has implications for its biological activity in vivo and likelihood of progression to a drug candidate. We have created a database of commercially available screening compounds and natural products in order to analyse the drug- and lead-likeness of commercial screening compounds and compare them with i) orally administered drugs, ii) non-orally administered drugs, and iii) compounds with significant biological activity but unspecified or not yet determined route of administration from the public databases DrugBank and ChEMBL. The data set contained 15.5 million entries from 102 vendors, which resulted in just over 8 million unique chemical structures. We review these data for current drug/lead-likeness, then utilise substructure-based filters for promiscuity and unwanted groups, and finally compare chemical properties for structures within the different sub-sets. While the majority of the commercial compounds satisfy various drug-likeness rules, they show a larger molecular weight and higher hydrophobicity compared to orally available drugs, with generally higher aromaticity and lower solubility. This ‘right shift’ of chemical properties has also been found in the majority of the compounds with significant biological activity in ChEMBL, reflecting a common trend in current drug discovery, towards larger, more hydrophobic compounds and fewer drug-like compounds. In particular, successful drugs were found to possess much lower median logD values than those found for compound collections. In addition, commercial compounds show a quite narrow distribution in molecular weight, with a median absolute deviation of only 78 Da around a median of 387 Da. For high-throughput screening a highly stringent combination of several lead-likeness and substructure filters against unwanted groups could be applied, resulting in 2 million lead-like structures. For fragment based screening approaches the rule of three (Ro3) would select around 400,000 structures.

Understanding the relationship between structure and function underpins both biochemistry and chemical biology, and has enabled the discovery of numerous agricultural and therapeutic agents. Small cysteine-rich proteins, which form a unique set of protein frameworks and folds, are found in all living organisms and often play crucial roles as hormones, growth factors, ion channel modulators and enzyme inhibitors in various biological pathways. Here we review secreted human cysteine-rich mini-proteins, classify them into broad families and briefly describe their structure and function. To systematically investigate this protein sub-class we designed a step-wise high throughput algorithm that is able to isolate the mature and active forms of human secreted cysteine-rich proteins (up to 200 amino acids in length) and extract their cysteine scaffolds. We limited our search to frameworks that contain an even number of cysteine residues (< 20), all of which are engaged in intra-molecular disulfide bonds. We found 53 different cysteine-rich frameworks spread over 378 secreted cysteine-rich mini-proteins. Restricting our search to those that contain >5% cysteine residues led to the identification of 22 cysteine-rich frameworks representing 21 protein families. Analysis of their molecular targets showed that these mini-proteins are frequently ligands for G protein- and enzyme-coupled receptors, transporters, extracellular enzyme inhibitors, and antimicrobial peptides. It is clear that these human secreted mini-proteins possess a wide diversity of frameworks and folds, some of which are conserved across the phylogenetic spectrum. Further study of these proteins will undoubtedly lead to insights into unresolved questions of basic biology, and the development of system-specific human therapeutics.

Cyclic peptides typically have much higher stability and improved biopharmaceutical properties over their linear counterparts. Our work focuses on the discovery of naturally occurring disulfide-rich cyclic peptides and their applications in drug design. These peptides provide a design basis for re-engineering natural acyclic peptides to improve their biopharmaceutical properties by chemically linking their termini. Here we describe examples of the discovery of the cyclotide family of peptides, their chemical re-engineering to introduce desired pharmaceutical activities, studies of their biopharmaceutical properties and applications of cyclization technologies to naturally occurring toxins, including conotoxins and scorpion toxins. In the case of the conotoxin Vc1.1, we produced an orally active peptide with potential for the treatment of neuropathic pain by cyclising the native peptide. In the case of the scorpion toxin chlorotoxin, a cyclised derivative had improved biopharmaceutical properties as a tumour imaging agent over the naturally occurring linear chlorotoxin. Ongoing chemical and structural studies of these classes of disulfide-rich peptides promise to increase their value for use in dissecting biological processes in plants and mammals while also providing leads to new classes of biopharmaceuticals.

Cone snails have evolved many 1000s of small, structurally stable venom peptides (conopeptides) for prey capture and defense. Whilst <0.1% have been pharmacologically characterised, those with known function typically target membrane proteins of therapeutic importance, including ion channels, transporters and GPCRs. Several conopeptides reduce pain in animals models, with one in clinical development (χ-conopeptide analogue Xen2174) and one marketed (ω- conotoxin MVIIA or Prialt) for the treatment of severe pain. In addition to their therapeutic potential, conopeptides have been valuable probes for studying the role of a number of key membrane proteins in normal and disease physiology.

Chemical information can be used to inform biology through being employed to develop bioinformatic tools. One area where bioinformatic tools are valuable is the study of linear motif-mediated protein interactions. Linear motifs are short sequences found mostly in disordered regions of proteins that function in cellular signaling and regulation, by binding to protein interaction domains or by being the target of post-translational modifications. Linear motifs pose difficulty not only to experimental study, but also computational methods; they are difficult to identify due to their small size; and their binding specificity is affected by several factors acting in concert. We discuss the different ways linear motifs can be represented computationally, and how computational approaches can integrate the different specificity-determining factors. We illustrate these issues on our own work focusing on the use of three-dimensional structural information in predicting protein phosphorylation sites, and the integration of diverse types of data in predicting nuclear localization. Computational approaches will play an increasing role in the future, allowing new relationships and system-wide understanding to be unearthed from the large datasets becoming available through high-throughput studies.

Many biologically active compounds are unsuitable for development as drugs due to their poor bioavailability. For hydrophilic compounds, modifications to increase lipophilicity can increase passive diffusion or increase uptake into the lymphatic system. Alternatively, improved bioavailability of hydrophilic drug candidates may be achieved by formulation with absorption promoters such as surfactants, penetration enhancers, or ion pairing agents. This approach to enhancing bioavailability also has the potential to widen the range of compound categories that can be used as chemical probes to study biological systems in cells and in vivo where membrane permeability would otherwise be a significant limitation. Lipidic amino acids, which combine the structural properties of lipids with those of α-amino acids, represent a relatively unexplored class of agents that can improve drug adsorption. This review discusses the potential of absorption promoters possessing lipoamino acids for improving drug bioavailability.

Cervical cancer is the second leading cause of cancer in women worldwide. Human papillomavirus (HPV) is responsible for all cases of cervical cancer. Commercial prophylactic HPV vaccines are now available, but unfortunately these vaccines have no therapeutic effect against established HPV infections. In order to accelerate the control of cervical cancer and treat established HPV infections, it is necessary to develop therapeutic vaccines to eradicate HPV by generating cell-mediated immunity against HPV infected cells. Two HPV-encoded early proteins, the E6 and E7 oncoproteins, are the preferred targets because they are consistently expressed in virtually all cervical cancer cells and are necessary for the induction and maintenance of HPV-associated disease. A variety of vaccine strategies have been employed targeting immune responses to these proteins. Peptide-based vaccines are a promising strategy for the development of therapeutic HPV vaccines because of their safety, stability, and ease of production. This review summarizes the prospects of peptidebased vaccines for the treatment of established HPV infections. We address the challenges that scientists currently face for developing peptide-based vaccines and explore feasible strategies for improving the potency of the induced immune response with the aim of treating established HPV infections.