Mary Liang

Structure-Activity Study of Bioisosteric Trifluoromethyl and Pentafluorosulfanyl Indole Inhibitors of the AAA ATPase p97.

Exploratory SAR studies of a new phenyl indole chemotype for p97 inhibition revealed C-5 indole substituent effects in the ADPGlo assay that did not fully correlate with either electronic or steric factors. A focused series of methoxy-, trifluoromethoxy-, methyl-, trifluoromethyl-, pentafluorosulfanyl-, and nitro-analogues was found to exhibit IC50s from low nanomolar to double-digit micromolar. Surprisingly, we found that the trifluoromethoxy-analogue was biochemically a better match of the trifluoromethyl-substituted lead structure than a pentafluorosulfanyl-analogue. Moreover, in spite of their almost equivalent strongly electron-depleting effect on the indole core, pentafluorosulfanyl- and nitro-derivatives were found to exhibit a 430-fold difference in p97 inhibitory activities. Conversely, the electronically divergent C-5 methyl- and nitro-analogues both showed low nanomolar activities.

Evaluation of a Novel Calcium Channel Agonist for Therapeutic Potential in Lambert–Eaton Myasthenic Syndrome

We developed a novel calcium (Ca2+) channel agonist that is selective for N- and P/Q-type Ca2+ channels, which are the Ca2+ channels that regulate transmitter release at most synapses. We have shown that this new molecule (GV-58) slows the deactivation of channels, resulting in a large increase in presynaptic Ca2+ entry during activity. GV-58 was developed as a modification of (R)-roscovitine, which was previously shown to be a Ca2+ channel agonist, in addition to its known cyclin-dependent kinase activity. In comparison with the parent molecule, (R)-roscovitine, GV-58 has a ∼20-fold less potent cyclin-dependent kinase antagonist effect, a ∼3- to 4-fold more potent Ca2+ channel agonist effect, and ∼4-fold higher efficacy as a Ca2+ channel agonist. We have further evaluated GV-58 in a passive transfer mouse model of Lambert–Eaton myasthenic syndrome and have shown that weakened Lambert–Eaton myasthenic syndrome-model neuromuscular synapses are significantly strengthened following exposure to GV-58. This new Ca2+ channel agonist has potential as a lead compound in the development of new therapeutic approaches to a variety of disorders that result in neuromuscular weakness.

Allosteric Indole Amide Inhibitors of p97: Identification of a Novel Probe of the Ubiquitin Pathway

A high-throughput screen to discover inhibitors of p97 ATPase activity identified an indole amide that bound to an allosteric site of the protein. Medicinal chemistry optimization led to improvements in potency and solubility. Indole amide 3 represents a novel uncompetitive inhibitor with excellent physical and pharmaceutical properties that can be used as a starting point for drug discovery efforts.

Synthesis and Biological Evaluation of a Selective N- and P/Q-Type Calcium Channel Agonist

The acute effect of the potent cyclin-dependent kinase (cdk) inhibitor (R)-roscovitine on Ca(2+) channels inspired the development of structural analogues as a potential treatment for motor nerve terminal dysfunction. On the basis of a versatile chlorinated purine scaffold, we have synthesized ca. 20 derivatives and characterized their N-type Ca(2+) channel agonist action. Agents that showed strong agonist effects were also characterized in a kinase panel for their off-target effects. Among several novel compounds with diminished cdk activity, we identified a new lead structure with a 4-fold improved N-type Ca(2+) channel agonist effect and a 22-fold decreased cdk2 activity as compared to (R)-roscovitine. This compound was selective for agonist activity on N- and P/Q-type over L-type calcium channels.

Optimization of pyrazole-containing 1,2,4-triazolo-[3,4-b]thiadiazines, a new class of STAT3 pathway inhibitors.

Structure-activity relationship studies of a 1,2,4-triazolo-[3,4-b]thiadiazine scaffold, identified in an HTS campaign for selective STAT3 pathway inhibitors, determined that a pyrazole group and specific aryl substitution on the thiadiazine were necessary for activity. Improvements in potency and metabolic stability were accomplished by the introduction of an α-methyl group on the thiadiazine. Optimized compounds exhibited anti-proliferative activity, reduction of phosphorylated STAT3 levels and effects on STAT3 target genes. These compounds represent a starting point for further drug discovery efforts targeting the STAT3 pathway.

New calcium channel agonists as potential therapeutics in Lambert–Eaton myasthenic syndrome and other neuromuscular diseases

Lambert–Eaton myasthenic syndrome (LEMS) causes neuromuscular weakness as a result of an autoimmune attack on the calcium channels that normally regulate chemical transmitter release at the neuromuscular junction. Currently there are limited treatment options for patients with this and other forms of neuromuscular weakness. A novel, first‐in‐class calcium channel agonist that is selective for the types of voltage‐gated calcium channels that regulate transmitter release at neuromuscular synapses has recently been developed. This compound (GV‐58) slows deactivation (closing) of the channel, resulting in a large increase in total calcium entry during motor nerve action potential activity. This new calcium channel agonist is currently being evaluated for the treatment of neuromuscular weakness. Potential applications include development as single therapeutics, or for combination treatments.

Imidazolium compounds are active against all stages of Trypanosoma cruzi

Imidazolium salts are best known for their applications in organic synthesis as room-temperature ionic liquids, or as precursors of stable carbenes, but they also show important biological properties such as anti-oxidative effects, induction of mitochondrial membrane permeabilisation and inhibition of the infection cycle of Plasmodium falciparum. For these reasons, and since chemotherapy for Chagas disease is inefficient, the aim of this study was to test the use of imidazolium compounds against the kinetoplastid haemoflagellate aetiological agent for this disease, namely Trypanosoma cruzi. The results show that five of the tested compounds are more effective than the reference drug benznidazole against the epimastigote and trypomastigote forms of T. cruzi. Moreover, intracellular amastigotes were also affected by the compounds, which showed lower toxicity in host cells. Transmission electron microscopy analysis demonstrated that the tested agents induced alterations of the kinetoplast and particularly of the mitochondria, leading to extraordinary swelling of the organelle. These results further demonstrate that the test agents with the best profile are those bearing symmetrical bulky substituents at N(1) and N(3), displaying promising activity against all forms of T. cruzi, interesting selectivity indexes and exceptional activity at low doses. Accordingly, these agents represent promising candidates for the treatment of Chagas disease.

Complete reversal of Lambert–Eaton myasthenic syndrome synaptic impairment by the combined use of a K+ channel blocker and a Ca2+ channel agonist

Lambert–Eaton myasthenic syndrome (LEMS) is characterized by an autoimmune‐mediated attack on presynaptic P/Q‐type Ca2+ channels at the neuromuscular junction (NMJ). The current common symptomatic treatment option is 3,4‐diaminopyridine (3,4‐DAP), a potassium channel blocker that widens the presynaptic action potential, causing an increase in the amount of neurotransmitter release. This approach, however, does not completely reverse symptoms and can have dose‐limiting side‐effects. Thus, there is a need for additional treatment options. We show that GV‐58, a Ca2+ channel agonist developed from the cyclin‐dependent kinase inhibitor (R)‐roscovitine, does not significantly inhibit cell division at physiological levels of ATP. We further show that GV‐58 has a greater agonist effect when more Ca2+ channels are open, and combining GV‐58 and 3,4‐DAP elicits a supra‐additive effect that completely restores the magnitude of neurotransmitter release in LEMS model NMJs. These results suggest that a combination of GV‐58 and 3,4‐DAP is promising as a possible alternative treatment approach to LEMS and other neuromuscular diseases.

Synthesis and structure–activity relationships of small molecule inhibitors of the simian virus 40 T antigen oncoprotein, an anti-polyomaviral target.

Polyomavirus infections are common and relatively benign in the general human population but can become pathogenic in immunosuppressed patients. Because most treatments for polyomavirusassociated diseases nonspecifically target DNA replication, existing treatments for polyomavirus infection possess undesirable side effects. However, all polyomaviruses express Large Tumor Antigen (T Ag), which is unique to this virus family and may serve as a therapeutic target. Previous screening of pyrimidinone–peptoid hybrid compounds identified MAL2-11B and a MAL2-11B tetrazole derivative as inhibitors of viral replication and T Ag ATPase activity (IC50 of ~20-50 μM. To improve upon this scaffold and to develop a structure–activity relationship for this new class of antiviral agents, several iterative series of MAL2-11B derivatives were synthesized. The replacement of a flexible methylene chain linker with a benzyl group or, alternatively, the addition of an ortho-methyl substituent on the biphenyl side chain in MAL2-11B yielded an IC50 of 50 μM, which retained antiviral activity. After combining both structural motifs, a new lead compound was identified that inhibited T Ag ATPase activity with an IC50 of 50 μM. We suggest that the knowledge gained from the structure–activity relationship and a further refinement cycle of the MAL2-11B scaffold will provide a specific, novel therapeutic treatment option for polyomavirus infections and their associated diseases.

New Cav2 calcium channel gating modifiers with agonist activity and therapeutic potential to treat neuromuscular disease

ABSTRACT Voltage‐gated calcium channels (VGCCs) are critical regulators of many cellular functions, including the activity‐dependent release of chemical neurotransmitter from nerve terminals. At nerve terminals, the Cav2 family of VGCCs are closely positioned with neurotransmitter‐containing synaptic vesicles. The relationship between calcium ions and transmitter release is such that even subtle changes in calcium flux through VGCCs have a strong influence on the magnitude of transmitter released. Therefore, modulators of the calcium influx at nerve terminals have the potential to strongly affect transmitter release at synapses. We have previously developed novel Cav2‐selective VGCC gating modifiers (notably GV‐58) that slow the deactivation of VGCC current, increasing total calcium ion flux. Here, we describe ten new gating modifiers based on the GV‐58 structure that extend our understanding of the structure‐activity relationship for this class of molecules and extend the range of modulation of channel activities. In particular, we show that one of these new compounds (MF‐06) was more efficacious than GV‐58, another (KK‐75) acts more quickly on VGCCs than GV‐58, and a third (KK‐20) has a mix of increased speed and efficacy. A subset of these new VGCC agonist gating modifiers can increase transmitter release during action potentials at neuromuscular synapses, and as such, show potential as therapeutics for diseases with a presynaptic deficit that results in neuromuscular weakness. Further, several of these new compounds can be useful tool compounds for the study of VGCC gating and function. Graphical abstract Figure. No Caption available. HighlightsGV‐58 and novel analogs are first‐in‐class Cav2 voltage‐gated calcium channel gating modifiers with therapeutic potential.These analogs contribute to our understanding of the structure‐activity relationship of the GV‐58 scaffold.Cav2 gating modifiers that act quickly, and are potent and efficacious, have therapeutic potential.GV‐58 and novel analogs are also tools for the study of calcium channel gating and calcium‐dependent pathways in cells.