Eric M. Bennett

Morpholine Derivatives Greatly Enhance the Selectivity of Mammalian Target of Rapamycin (mTOR) Inhibitors

Dramatic improvements in mTOR-targeting selectivity were achieved by replacing morpholine in pyrazolopyrimidine inhibitors with bridged morpholines. Analogues with subnanomolar mTOR IC(50) values and up to 26000-fold selectivity versus PI3Kalpha were prepared. Chiral morpholines gave inhibitors whose enantiomers had different selectivity and potency profiles. Molecular modeling suggests that a single amino acid difference between PI3K and mTOR (Phe961Leu) accounts for the profound selectivity seen by creating a deeper pocket in mTOR that can accommodate bridged morpholines.

Nicotinamide adenine dinucleotide based therapeutics.

Nicotinamide adenine dinucleotide (NAD), generally considered a key component involved in redox reactions, has been found to participate in an increasingly diverse range of cellular processes, including signal transduction, DNA repair, and post-translational protein modifications. In recent years, medicinal chemists have become interested in the therapeutic potential of molecules affecting interactions of NAD with NAD-dependent enzymes. Also, enzymes involved in de novo biosynthesis, salvage pathways, and down-stream utilization of NAD have been extensively investigated and implicated in a wide variety of diseases. These studies have bolstered NAD-based therapeutics as a new avenue for the discovery and development of novel treatments for medical conditions ranging from cancer to aging. Industrial and academic groups have produced structurally diverse molecules which target NAD metabolic pathways, with some candidates advancing into clinical trials. However, further intensive structural, biological, and medical studies are needed to facilitate the design and evaluation of new generations of NAD-based therapeutics. At this time, the field of NAD-therapeutics is most likely at a stage similar to that of the early successful development of protein kinase inhibitors, where analogs of ATP (a more widely utilized metabolite than NAD) began to show selectivity against target enzymes. This review focuses on key representative opportunities for research in this area, which extends beyond the scope of this article.

Quantitative Three Dimensional Structure Linear Interaction Energy Model of 5'-O-[N-(Salicyl)sulfamoyl]adenosine and the Aryl Acid Adenylating Enzyme MbtA

MbtA (a salicyl AMP ligase) is a key target for the design of new antitubercular agents. On the basis of structure-activity relationship (SAR) data generated in our laboratory, a structure-based model is developed to predict the binding affinities of aryl acid-AMP bisubstrate inhibitors of MbtA. The approach described takes advantage of the linear interaction energy (LIE) technique to derive linear equations relating ligand structure to function. With only two parameters derived from molecular dynamics simulations, good correlation (R(2) = 0.70) was achieved for a set of 31 inhibitors with binding affinities spanning 6 orders of magnitude. The results were applied to understand the effect of steric and heteroatom substitutions on bisubstrate ligand binding and to predict second generation inhibitors of MbtA. The resulting model was further validated by chemical synthesis of a novel inhibitor with a predicted LIE binding affinity of 1.6 nM and a subsequently determined experimental K(i)(app) of 0.7 nM.

Selective inhibition of nicotinamide adenine dinucleotide kinases by dinucleoside disulfide mimics of nicotinamide adenine dinucleotide analogues.

Diadenosine disulfide (5) was reported to inhibit NAD kinase from Listeria monocytogenes and the crystal structure of the enzyme-inhibitor complex has been solved. We have synthesized tiazofurin adenosine disulfide (4) and the disulfide 5, and found that these compounds were moderate inhibitors of human NAD kinase (IC(50)=110 microM and IC(50)=87 microM, respectively) and Mycobacterium tuberculosis NAD kinase (IC(50)=80 microM and IC(50)=45 microM, respectively). We also found that NAD mimics with a short disulfide (-S-S-) moiety were able to bind in the folded (compact) conformation but not in the common extended conformation, which requires the presence of a longer pyrophosphate (-O-P-O-P-O-) linkage. Since majority of NAD-dependent enzymes bind NAD in the extended conformation, selective inhibition of NAD kinases by disulfide analogues has been observed. Introduction of bromine at the C8 of the adenine ring restricted the adenosine moiety of diadenosine disulfides to the syn conformation making it even more compact. The 8-bromoadenosine adenosine disulfide (14) and its di(8-bromoadenosine) analogue (15) were found to be the most potent inhibitors of human (IC(50)=6 microM) and mycobacterium NAD kinase (IC(50)=14-19 microM reported so far. None of the disulfide analogues showed inhibition of lactate-, and inosine monophosphate-dehydrogenase (IMPDH), enzymes that bind NAD in the extended conformation.

Abstract B145: Highly selective pyrazolopyrimidine mTOR inhibitors with profound in vivo antitumor activity

The mammalian target of rapamycin (mTOR) is an important target for cancer chemotherapy due to the deregulation of its signaling pathway in a wide spectrum of human tumors. mTOR exists in at least two functional protein complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). While rapamycin and its analogs (rapalogs) inhibit only mTORC1, ATP‐competitive inhibitors of mTOR target both mTORC1 and mTORC2 and therefore have the potential for improved anticancer efficacy. We have previously reported on the pyrazolopyrimidine series of ATP‐competitive mTOR inhibitors (J. Med. Chem. 2009, 52, 5013 – 5016). We now report that derivatization of the morpholine ring in these inhibitors leads to greatly enhanced mTOR selectivity versus PI3K. Selective mTOR inhibitors have the potential to achieve a higher therapeutic index in the clinic due to better tolerability. Testing these inhibitors in tumor cells and evaluating the most potent analogs in human tumor xenograft models in the nude mouse led to the identification of WYE‐125132, a candidate for clinical development. WYE‐125132 is also selective versus a wide range of other kinases including the closely‐related PIKKs. Detailed structure activity relationships in the pyrazolopyrimidine series will be presented. In conclusion, modification of the morpholine ring of a series of pyrazolopyrimidines leads to analogs with enhanced mTOR selectivity over PI3K and potent efficacy in human tumor xenograft models in the nude mouse. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B145.