Andrew Lemoff

IDH1 and IDH2 mutations in pediatric acute leukemia.

To investigate the frequency of isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) mutations in pediatric acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL), we sequenced these genes in diagnostic samples from 515 patients (227 AMLs and 288 ALLs). Somatic IDH1/IDH2 mutations were rare in ALL (N=1), but were more common in AML, occurring in 3.5% (IDH1 N=3 and IDH2 N=5), with the frequency higher in AMLs with a normal karyotype (9.8%). The identified IDH1 mutations occurred in codon 132 resulting in replacement of arginine with either cysteine (N=3) or histidine (N=1). By contrast, mutations in IDH2 did not affect the homologous residue but instead altered codon 140, resulting in replacement of arginine with either glutamine (N=4) or tryptophan (N=1). Structural modeling of IDH2 suggested that codon 140 mutations disrupt the enzymes ability to bind its substrate isocitrate. Accordingly, recombinant IDH2 R140Q/W were unable to carry out the decarboxylation of isocitrate to α-ketoglutarate (α-KG), but instead gained the neomorphic activity to reduce α-KG to R(–)-2-hydroxyglutarete (2-HG). Analysis of primary leukemic blasts confirmed high levels of 2-HG in AMLs with IDH1/IDH2 mutations. Interestingly, 3/5 AMLs with IDH2 mutations had FLT3-activating mutations, raising the possibility that these mutations cooperate in leukemogenesis.

Synthesis and evaluation of sulfonylnitrophenylthiazoles (SNPTs) as thyroid hormone receptor-coactivator interaction inhibitors.

We previously identified a series of methylsulfonylnitrobenzoates (MSNBs) that block the interaction of the thyroid hormone receptor with its coactivators. MSNBs inhibit coactivator binding through irreversible modification of cysteine 298 of the thyroid hormone receptor (TR). Although MSNBs have better pharmacological features than our first generation inhibitors (β-aminoketones), they contain a potentially unstable ester linkage. Here we report the bioisosteric replacement of the ester linkage with a thiazole moiety, yielding sulfonylnitrophenylthiazoles (SNPTs). An array of SNPTs representing optimal side chains from the MSNB series was constructed using parallel chemistry and evaluated to test their antagonism of the TR-coactivator interaction. Selected active compounds were evaluated in secondary confirmatory assays including regulation of thyroid response element driven transcription in reporter constructs and native genes. In addition the selected SNPTs were shown to be selective for TR relative to other nuclear hormone receptors (NRs).

Functional regulation of cystic fibrosis transmembrane conductance regulator-containing macromolecular complexes: a small-molecule inhibitor approach

CFTR (cystic fibrosis transmembrane conductance regulator) has been shown to form multiple protein macromolecular complexes with its interacting partners at discrete subcellular microdomains to modulate trafficking, transport and signalling in cells. Targeting protein-protein interactions within these macromolecular complexes would affect the expression or function of the CFTR channel. We specifically targeted the PDZ domain-based LPA2 (type 2 lysophosphatidic acid receptor)-NHERF2 (Na+/H+ exchanger regulatory factor-2) interaction within the CFTR-NHERF2-LPA2-containing macromolecular complexes in airway epithelia and tested its regulatory role on CFTR channel function. We identified a cell-permeable small-molecule compound that preferentially inhibits the LPA2-NHERF2 interaction. We show that this compound can disrupt the LPA2-NHERF2 interaction in cells and thus compromises the integrity of macromolecular complexes. Functionally, it elevates cAMP levels in proximity to CFTR and upregulates its channel activity. The results of the present study demonstrate that CFTR Cl- channel function can be finely tuned by modulating PDZ domain-based protein-protein interactions within the CFTR-containing macromolecular complexes. The present study might help to identify novel therapeutic targets to treat diseases associated with dysfunctional CFTR Cl- channels.

Dual Detection Approach to a More Accurate Measure of Relative Purity in High-Throughput Characterization of Compound Collections

The accurate determination of compound purity is crucial for characterizing library purity, monitoring the stability of storage compounds, and obtaining meaningful high-throughput screening results. However, current high-throughput techniques for the determination of compound purity are inadequate. We evaluated on-line chromatography detectors, including UV(TWC), UV(214), and ELSD detectors, in a series of studies of 233 compound mixtures prepared with known compositions. Results indicate that both UV(TWC) and UV(214) overestimate the minor component in a mixture whereas ELSD underestimates the minor component. An average of UV(TWC) and ELSD purities gives a more accurate measure of the relative purity for a wide range of compounds in various purity ranges. This technique was applied to 959 compounds from our compound collection to more accurately determine their relative purity.

Optimization of propafenone analogues as antimalarial leads.

Propafenone, a class Ic antiarrythmic drug, inhibits growth of cultured Plasmodium falciparum. While the drugs potency is significant, further development of propafenone as an antimalarial would require divorcing the antimalarial and cardiac activities as well as improving the pharmacokinetic profile of the drug. A small array of propafenone analogues was designed and synthesized to address the cardiac ion channel and PK liabilities. Testing of this array revealed potent inhibitors of the 3D7 (drug sensitive) and K1 (drug resistant) strains of P. falciparum that possessed significantly reduced ion channel effects and improved metabolic stability. Propafenone analogues are unusual among antimalarial leads in that they are more potent against the multidrug resistant K1 strain of P. falciparum compared to the 3D7 strain.

Optimization of a Novel Series of Ataxia-Telangiectasia Mutated Kinase Inhibitors as Potential Radiosensitizing Agents

We previously reported a novel inhibitor of the ataxia-telangiectasia mutated (ATM) kinase, which is a target for novel radiosensitizing drugs. While our initial lead, compound 4, was relatively potent and nontoxic, it exhibited poor stability to oxidative metabolism and relatively poor selectivity against other kinases. The current study focused on balancing potency and selectivity with metabolic stability through structural modification to the metabolized site on the quinazoline core. We performed extensive structure-activity and structure-property relationship studies on this quinazoline ATM kinase inhibitor in order to identify structural variants with enhanced selectivity and metabolic stability. We show that, while the C-7-methoxy group is essential for potency, replacing the C-6-methoxy group considerably improves metabolic stability without affecting potency. Promising analogues 20, 27g, and 27n were selected based on in vitro pharmacology and evaluated in murine pharmacokinetic and tolerability studies. Compound 27g possessed significantly improve pharmacokinetics relative to that of 4. Compound 27g was also significantly more selective against other kinases than 4. Therefore, 27g is a good candidate for further development as a potential radiosensitizer.

Lead Optimization of Antimalarial Propafenone Analogues

Previously reported studies identified analogues of propafenone that had potent antimalarial activity, reduced cardiac ion channel activity, and properties that suggested the potential for clinical development for malaria. Careful examination of the bioavailability, pharmacokinetics, toxicology, and efficacy of this series of compounds using rodent models revealed orally bioavailable compounds that are nontoxic and suppress parasitemia in vivo. Although these compounds possess potential for further preclinical development, they also carry some significant challenges.

Optimization of purine-nitrile TbcatB inhibitors for use in vivo and evaluation of efficacy in murine models

There are currently only four clinical drugs available for treating human African trypanosomiasis (HAT), three of which were developed over 60 years ago. Despite years of effort, there has been relatively little progress towards identifying orally available chemotypes active against the parasite in vivo. Here, we report the lead optimization of a purine-nitrile scaffold that inhibits the essential TbcatB protease and its evaluation in murine models. A lead inhibitor that had potent activity against the trypanosomal protease TbcatB in vitro and cultured parasites ex vivo was optimized by rationally driven medicinal chemistry to an inhibitor that is orally available, penetrates the CNS, has a promising pharmacokinetic profile, and is non-toxic at 200mg/kg in a repeat dosage study. Efficacy models using oral administration of this lead inhibitor showed a significantly increased survival time in Trypanosoma brucei brucei infected mice but little effect on Trypanosoma brucei rhodesiense infected mice.