The discovery and clinical development of heat shock protein 90 (Hsp90) inhibitors continue to progress. A number of Hsp90 inhibitors are in clinical trials, and preclinical discoveries of new chemotypes that bind to distinct regions in the protein as well as isoform selective compounds are active areas of research. This review will highlight progress in the field since 2010.

Sigma receptors (σ-1 and σ-2) are non-opioid proteins implicated in the pathophysiology of various neurological disorders and cancer. The σ-1 subtype is a chaperon protein widely distributed in the CNS and peripheral tissues. These receptors are involved in the modulation of K(+)- and Ca(2+)-dependent signaling cascades at the endoplasmic reticulum and modulation of neurotransmitter release. σ-1 receptors are emerging targets for the treatment of neurophychiatric diseases (schizophrenia and depression) and cocaine addiction. σ-2 receptors are lipid raft proteins. They are highly expressed on many tumor cells and hence considered potential targets for anticancer drugs. σ receptors bind to a diverse class of pharmacological compounds like cocaine, methamphetamine, benzomorphans like (±)-pentazocine, (±)-SKF-10,047 and endogenous neurosteroids and sphingolipids. In this review we focus on the early development of σ receptor-specific ligands and radiolabeling agents.

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σ Receptors represent a potential drug target for numerous therapeutic indications including cancer, depression, psychostimulant abuse, and stroke. Most published radioligand binding studies for σ receptors utilize a low throughput method employing a “cell harvester.” Higher throughput methods are required to facilitate efficient screening of large numbers of novel compounds. In this study, a series of reference compounds was analyzed with a new medium-throughput 96-well filtration method and the results were compared to those obtained using the conventional cell harvester-based method. The 96-well assay utilized rat liver membranes for the determination of both known σ receptor subtypes (σ1 and σ2) because this tissue contains high densities of both subtypes and fulfills criteria required for reliable use with the 96-well format. The new method gave comparable Ki values for reference ligands analyzed in parallel with samples prepared in rat brain membranes and processed on the traditional cell harvester. For σ1 receptors, equivalent affinity values were observed for both methods/tissues. For σ2 receptors, approximately 2-fold higher affinities were observed for most compounds in liver, as compared to brain membranes, but excellent correlation with brain-derived values was maintained. To further demonstrate the utility of the new method it was used to screen a novel series of 2(3H)-benzothiazolone compounds, resulting in the identification of several analogues with nanomolar affinity and greater than 50-fold specificity for σ1 versus σ2 receptors.

Filariasis is a tropical disease caused by the parasitic nematodes Wuchereria bancrofti and Brugia malayi. Known inhibitors of dihydrofolate reductase (DHFR) have been previously shown to kill Brugia malayi nematodes and to inhibit Brugia malayi DHFR (BmDHFR) at nanomolar concentrations. These data suggest that BmDHFR is a potential target for the treatment of filariasis. Here, protocols for cloning, expression and purification of Wuchereria bancrofti DHFR (WbDHFR) were developed. The Uniprot entry J9F199-1 predicts a 172 amino acid protein for WbDHFR but alignment of this sequence to the previously described BmDHFR shows that this WbDHFR sequence lacks a crucial, conserved 13 amino acid loop. The presence of the loop in WbDHFR is supported by a noncanonical splicing event and the loop sequence was therefore included in the gene design. Subsequently, the KM for dihydrofolate (3.7 ± 2 μM), kcat (7.4 ± 0.6 s-1), and pH dependence of activity were determined. IC50 values of methotrexate, trimethoprim, pyrimethamine, raltitrexed, aminopterin, (-)-epicatechin gallate, (-)-epicatechin, and vitexin were measured for WbDHFR and BmDHFR. Methotrexate and structurally related aminopterin were found to be effective inhibitors of WbDHFR, with an KI of 1.2 ± 0.2 nM and 2.1 ± 0.5 nM, respectively, suggesting that repurposing of known antifolate compound may be an effective strategy to treating filariasis. Most compounds showed similar inhibition profiles toward both enzymes, suggesting that the two enzymes have important similarities in their active site environments and can be targeted with the same compound, once a successful inhibitor is identified.