Robert K. Campbell

Genomic-scale prioritization of drug targets: the TDR Targets database

The increasing availability of genomic data for pathogens that cause tropical diseases has created new opportunities for drug discovery and development. However, if the potential of such data is to be fully exploited, the data must be effectively integrated and be easy to interrogate. Here, we discuss the development of the TDR Targets database (http://tdrtargets.org), which encompasses extensive genetic, biochemical and pharmacological data related to tropical disease pathogens, as well as computationally predicted druggability for potential targets and compound desirability information. By allowing the integration and weighting of this information, this database aims to facilitate the identification and prioritization of candidate drug targets for pathogens.

Target repurposing for neglected diseases

Infectious diseases are an enormous burden to global health and ,since drug discovery is costly, those infectious diseases that affect the developing world are often not pursued by commercial drug-discovery efforts. Therefore, pragmatic means by which new therapeutics can be discovered are needed. One such approach is target repurposing, where pathogen targets are matched with homologous human targets that have been pursued for drug discovery for other indications. In many cases, the medicinal chemistry, structural biology and biochemistry knowledge around these human targets can be directly repurposed to launch and accelerate new drug-discovery efforts against the pathogen targets. This article describes the overarching strategy of target repurposing as a tool for initiating and prosecuting neglected disease drug-discovery programs, highlighting this approach with three case studies.

Threading of a glycosylated protein loop through a protein hole: implications for combination of human chorionic gonadotropin subunits.

Chorionic gonadotropin (hCG) is a heterodimeric placental glycoprotein hormone essential for human reproduction. Twenty hCG β‐subunit residues, termed the seatbelt, are wrapped around α‐subunit loop 2 (α2) and their positions “latched” by a disulfide formed by cysteines at the end of the seatbelt (Cys 110) and in the β‐subunit core (Cys 26). This unique arrangement explains the stability of the heterodimer but raises questions as to how the two subunits combine. The seatbelt is latched in the free β‐subunit. If the seatbelt remained latched during the process of subunit combination, formation of the heterodimer would require α2 and its attached oligosaccharide to be threaded through a small β‐subunit hole. The subunits are known to combine during oxidizing conditions in vitro, and studies described here tested the idea that this requires transient disruption of the latch disulfide, possibly as a consequence of the thioredoxin activity reported in hCG. We observed that alkylating agents did not modify either cysteine in the latch disulfide (Cys 26 or Cys 110) during heterodimer formation in several oxidizing conditions and had minimal influence on these cysteines during combination in the presence of mild reductants (1–3 mM β‐mercaptoethanol). Reducing agents appeared to accelerate subunit combination by disrupting a disulfide (Cys 93–Cys 100) that forms a loop within the seatbelt, thereby increasing the size of the β‐subunit hole. We propose a mechanism for hCG assembly in vitro that depends on movements of α2 and the seatbelt and suggest that the process of glycoprotein hormone subunit combination may be useful for studying the movements of loops during protein folding.

Pharmacological Validation of Trypanosoma brucei Phosphodiesterases B1 and B2 as Druggable Targets for African Sleeping Sickness

Neglected tropical disease drug discovery requires application of pragmatic and efficient methods for development of new therapeutic agents. In this report, we describe our target repurposing efforts for the essential phosphodiesterase (PDE) enzymes TbrPDEB1 and TbrPDEB2 of Trypanosoma brucei , the causative agent for human African trypanosomiasis (HAT). We describe protein expression and purification, assay development, and benchmark screening of a collection of 20 established human PDE inhibitors. We disclose that the human PDE4 inhibitor piclamilast, and some of its analogues, show modest inhibition of TbrPDEB1 and B2 and quickly kill the bloodstream form of the subspecies T. brucei brucei . We also report the development of a homology model of TbrPDEB1 that is useful for understanding the compound-enzyme interactions and for comparing the parasitic and human enzymes. Our profiling and early medicinal chemistry results strongly suggest that human PDE4 chemotypes represent a better starting point for optimization of TbrPDEB inhibitors than those that target any other human PDEs.

Myelin Tetraspan Family Proteins but No Non-Tetraspan Family Proteins Are Present in the Ascidian (Ciona intestinalis) Genome

Several of the proteins used to form and maintain myelin sheaths in the central nervous system (CNS) and the peripheral nervous system (PNS) are shared among different vertebrate classes. These proteins include one-to-several alternatively spliced myelin basic protein (MBP) isoforms in all sheaths, proteolipid protein (PLP) and DM20 (except in amphibians) in tetrapod CNS sheaths, and one or two protein zero (P0) isoforms in fish CNS and in all vertebrate PNS sheaths. Several other proteins, including 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNP), myelin and lymphocyte protein (MAL), plasmolipin, and peripheral myelin protein 22 (PMP22; prominent in PNS myelin), are localized to myelin and myelin-associated membranes, though class distributions are less well studied. Databases with known and identified sequences of these proteins from cartilaginous and teleost fishes, amphibians, reptiles, birds, and mammals were prepared and used to search for potential homologs in the basal vertebrate, Ciona intestinalis. Homologs of lipophilin proteins, MAL/plasmolipin, and PMP22 were identified in the Ciona genome. In contrast, no MBP, P0, or CNP homologs were found. These studies provide a framework for understanding how myelin proteins were recruited during evolution and how structural adaptations enabled them to play key roles in myelination.

Main-chain conformational tendencies of amino acids.

A Ramachandran plot is a visual representation of the main‐chain conformational tendencies of an amino acid. Despite forty years of research, the shape of Ramachandran plots is still a matter of debate. The issue in making a Ramachandran plot based on experimental data is deciding whether sparse data represent genuine conformations. We present here a simple solution to settle the ambiguities of the sparse data, and explain how we verified the accuracies of our plots using an independent dataset. To obtain our results, we then measured the pair‐wise distances of main‐chain conformational tendencies among amino acids, and showed that the conformational relationships of amino acids are well preserved in a two‐dimensional map, leading to the conclusion that the conformational diversity space of amino acids is largely two dimensional. We further noticed that amino acids in early and late evolutionary stages are located in different zones in the two‐dimensional map. In addition to these conclusions, we here present an amino acid substitution table derived from experimental data. Proteins 2005.

Influence of Subunit Interactions on Lutropin Specificity IMPLICATIONS FOR STUDIES OF GLYCOPROTEIN HORMONE FUNCTION

Bovine lutropin (bLH) and human chorionic gonadotropin (hCG) are heterodimeric glycoprotein hormones required for reproduction. Both bind rat LH receptors (rLHRs), but hCG binds human LH receptors (hLHRs) 1000-10,000 fold better than bLH. We tested the premise that this difference in affinity could be used to identify lutropin receptor contacts. Heterodimers containing hCG/bLH α- or β-subunit chimeras that bound hLHR like hCG (or bLH) were expected to have hCG (or bLH) residues at the receptor contact sites. Analogs containing one subunit derived from hCG bound hLHR much more like hCG than bLH, indicating that each bLH subunit contains all the residues sufficient for high affinity hLHR binding. Indeed, the presence of bovine α-subunit residues increased the activities of some hCG analogs. The low hLHR activity of bLH was due primarily to an interaction between its α-subunit and β-subunit residue Leu95. Leu95 does not appear to contact the hLHR since it did not influence the hLHR activity of heterodimers containing human α-subunit. These observations show that interactions within and between the subunits can significantly influence the activities of lutropins, thereby confounding efforts to identify ligand residues that contact these receptors.

Synthesis and evaluation of human phosphodiesterases (PDE) 5 inhibitor analogs as trypanosomal PDE inhibitors. Part 1. Sildenafil analogs

Parasitic diseases, such as African sleeping sickness, have a significant impact on the health and well-being in the poorest regions of the world. Pragmatic drug discovery efforts are needed to find new therapeutic agents. In this Letter we describe target repurposing efforts focused on trypanosomal phosphodiesterases. We outline the synthesis and biological evaluation of analogs of sildenafil (1), a human PDE5 inhibitor, for activities against trypanosomal PDEB1 (TbrPDEB1). We find that, while low potency analogs can be prepared, this chemical class is a sub-optimal starting point for further development of TbrPDE inhibitors.

Repurposing human PDE4 inhibitors for neglected tropical diseases: design, synthesis and evaluation of cilomilast analogues as Trypanosoma brucei PDEB1 inhibitors.

A medicinal chemistry exploration of the human phosphodiesterase 4 (hPDE4) inhibitor cilomilast (1) was undertaken in order to identify inhibitors of phosphodiesterase B1 of Trypanosoma brucei (TbrPDEB1). T. brucei is the parasite which causes African sleeping sickness, a neglected tropical disease that affects thousands each year, and TbrPDEB1 has been shown to be an essential target of therapeutic relevance. Noting that 1 is a weak inhibitor of TbrPDEB1, we report the design and synthesis of analogs of this compound, culminating in 12b, a sub-micromolar inhibitor of TbrPDEB1 that shows modest inhibition of T. brucei proliferation.

Inferring protein function from genomic sequence: Giardia lamblia expresses a phosphatidylinositol kinase-related kinase similar to yeast and mammalian TOR

Functional assays of genes have historically led to insights about the activities of a protein or protein cascade. However, the rapid expansion of genomic and proteomic information for a variety of diverse taxa is an alternative and powerful means of predicting function by comparing the enzymes and metabolic pathways used by different organisms. As part of the Giardia lamblia genome sequencing project, we routinely survey the complement of predicted proteins and compare those found in this putatively early diverging eukaryote with those of prokaryotes and more recently evolved eukaryotic lineages. Such comparisons reveal the minimal composition of conserved metabolic pathways, suggest which proteins may have been acquired by lateral transfer, and, by their absence, hint at functions lost in the transition from a free-living to a parasitic lifestyle. Here, we describe the use of bioinformatic approaches to investigate the complement and conservation of proteins in Giardia involved in the regulation of translation. We compare an FK506 binding protein homologue and phosphatidylinositol kinase-related kinase present in Giardia to those found in other eukaryotes for which complete genomic sequence data are available. Our investigation of the Giardia genome suggests that PIK-related kinases are of ancient origin and are highly conserved.