Tuesday, July 7, 2015

Aminosterols from the Dogfish Shark: The Discovery of the Allosteric Phosphatase Inhibitor, Trodusquemine

            In 1993, as part of an effort to search for antibiotic host defense agents in the gut of various animals including the dogfish shark (spiny dogfish) Squalus acanthias, the unique antimicrobial aminosterol squalamine (structure shown above) was discovered. Squalamine is essentially the condensation adduct between a C24-sulfated bile salt derivative and the polyamine, spermidine. The steroidal portion of the squalamine structure bears the trans configuration at the A/B ring junction and is additionally hydroxylated at C7 in the a-configuration. It was initially shown that stomach extracts of the dogfish shark exhibited potent antimicrobial activity. Further efforts to purify and identify the bioactive molecule responsible for the observed activity led to the isolation and structure determination of squalamine. The aminosterol was initially identified as a broad-spectrum antimicrobial, as it was found to exhibit potent activity against fungi, protozoa, and both Gram-negative and Gram-positive bacteria. The antimicrobial activity was attributed to squalamine’s ability to modify membrane integrity by increasing permeability. Interestingly, it was later shown that squalamine also possessed antiangiogenic and antitumor properties and the molecule was eventually advanced to Phase II clinical trials for the treatment of patients with advanced nonsmall cell lung cancer.
            Attempts to procure larger amounts of squalamine from Squalus acanthias resulted in the discovery and isolation of a related aminosterol MSI-1436, later dubbed trodusquemine. Trodusquemine, as compared to squalamine, features an invariant steroid skeleton but is conjugated at C3 to spermine (as opposed to spermidine), an elongated tetrabasic polyamine. Rather unexpectedly, trodusquemine was found to induce profound appetite suppression in vivo in mammals (For another example of a natural appetite-suppressant steroid, see here). As a result, it has been speculated that trodusquemine is responsible for the sporadic feeding behavior of the dogfish, which eats only once every two weeks. In vitro screening against a panel of potential cellular targets revealed protein-tyrosine phosphatase 1B (PTP1B) inhibitory activity in cell-free and cell-based assays. PTP1B is a negative regulator of the effects of insulin and leptin signaling through dephosphorylation of the insulin receptor (IR) and IR Substrate 1, thereby inactivating the insulin pathway. Thus, inhibition of PTP1B maintains the insulin and leptin pathways in active, phosphorylated states, which triggers appetite suppression. Indeed, PTP1B expression and activity is increased in obese and insulin-resistant humans and neuronal-specific PTP1B knockout mice have markedly reduced weight.
            The inhibitory activity of trodusquemine at the cellular target PTP1B is significant because it has been notoriously challenging for medicinal chemists to develop effective small molecule inhibitors targeting the active site of this enzyme. One problem is a high degree of active site protein sequence homology, leading to difficulties in achieving selectivity over other off-target phosphatases. Moreover, potent active site tyrosine phosphatase inhibitors were designed to mimic phosphotyrosine (see examples shown above). Consequently, the inhibitor ligands that were developed were highly charged and thus had limited membrane permeability and drug development potential. A known inhibitor of striatal-enriched protein tyrosine phosphatase (or STEP), a potential Alzheimer’s target, is TC-2153, a synthetic molecule that features a rarely encountered heterocyclic ring comprised of five contiguously attached sulfur atoms. Current interest in a structure like TC-2153 reinforces the point that it has been historically difficult to find small molecule phosphatase inhibitors with conventional ‘druglike’ substructures and physicochemical properties. Trodusquemine avoids many of these pitfalls by binding to an inhibitory allosteric site within an intrinsically disordered segment of the C-terminal, noncatalytic region of PTP1B. Many of the previous high-throughput screening campaigns conducted against PTP1B used a recombinant, truncated form of the enzyme lacking this C-terminal segment and, therefore, failed to identify selective allosteric inhibitors such as trodusquemine. As a selective, non-competitive inhibitor of PT1B, trodusquemine has tremendous potential as an anti-obesity and anti-diabetic therapeutic agent. The molecule has also been shown to be able to cross the blood brain barrier (BBB), and thus may be centrally active, opening a new range of potential indications. Trodusquemine has been well tolerated in dose escalation and dose ranging clinical studies completed to date in over 65 patients.
            The antimicrobial natural product squalamine has been synthesized by processes starting from b-stigmasterol, as well as chenodeoxycholic acid (CDCA, see Scheme above). One interesting and critical transformation that has been utilized to generate gram-quantities of squalamine is a regio- and stereoselective reductive amination with a spermidine equivalent, wherein the distal primary amine is masked as a non-nucleophilic nitrile. In this conversion, the primary amine reacts preferentially over the internal secondary amine to generate an intermediary imine species, upon elimination of water with assistance from trimethyl orthoformate. Sodium borohydride is then used to reduce the imine in a stereoselective fashion from the bottom face of the molecule to give, predominantly, the desired b-orientation at the C3 position (d.r. 6:1). The nitrile is then easily converted to the requisite amine (spermidine) by catalytic hydrogenation, with both operations performed in the presence of the apparently unreactive C24-sulfate. The hydrogenation must be conducted under acidic conditions (TFA) to avoid cyclization of the internal secondary amine onto the pendant nitrile to give a cyclic amidine impurity. In this example, the semisynthetic squalamine was purified by HPLC and isolated as the trifluoroacetate salt with a final purity of 97%. One presumes that a similar process is currently used to manufacture the related clinical candidate, trodusquemine.

Saturday, July 4, 2015

Dragmacidolide A, A New Oxysterol Metabolite from the Dragmacidon Genus of Marine Sponges

            Deep-water sponges of the Dragmacidon genus typically yield structurally complex bis-indole alkaloid compounds such as dragmacidins D, E and F. Some Dragmacidon alkaloids have been shown to be potent inhibitors of the serine/threonine phosphatases PP1 and PP2A. In addition, the dragmacidins exhibit antiviral, antibacterial and antifungal bioactivities, as well as cytotoxicity towards various cancer cell lines. Very recently, a Dragmacidon australe specimen, collected by SCUBA off the coast of the Whitsunday Islands in Queensland, Australia, was investigated to gain a better understanding of the chemistry of this poorly investigated genus. Surprisingly, a new steroidal secondary metabolite, dragmacidolide A, was isolated and characterized using 1D/2D NMR and MS data. The isolation of a steroid from this sponge genus is unusual given that previous reports of Dragmacidon metabolites were comprised exclusively of indole and b-carboline alkaloids (with the unique exception of the nucleoside, dragmacidoside). At this time, it cannot be ruled out that a microbial symbiont could be responsible for the biosynthetic production of the oxysterol, within the microenvironment of the sponge.

            In order to obtain a small sample of dragmacidolide A, the methanol/dichloromethane extract of a freeze-dried and ground specimen of sponge was subjected to RP-HPLC purification and analysis of the purified fraction suggested the presence of a steroid. Unfortunately, the relative configuration of C20 and C22 could not be assigned on the basis of the 2D NMR data. Both H21, as well as H20, showed ROESY cross-peaks to protons on the angular methylene projecting from the C/D ring junction (H18), indicating that free-rotation about the C17-C20 bond was operative. The C20 configuration depicted in the structure shown above is based solely on analogy to the stereogenic disposition of the side chains of other archetypal cholestane-type steroids (e.g. lanosterol or OSW-1). The limited availability from natural sources and highly oxidized nature of the dragmacidolide A skeletal framework makes it an ideal target for chemical synthesis. New synthetic technologies that provide access to large quantities of molecules like dragmacidolide A are likely to lead to the discovery of new biological targets and properties of pharmacotherapeutic significance.