A cyclic peptide inhibitor of HIF-1 heterodimerization that inhibits hypoxia signaling in cancer cells.

Hypoxia inducible factor-1 (HIF-1) is a heterodimeric transcription factor that acts as the master regulator of cellular response to reduced oxygen levels, thus playing a key role in the adaptation, survival, and progression of tumors. Here we report cyclo-CLLFVY, identified from a library of 3.2 million cyclic hexapeptides using a genetically encoded high-throughput screening platform, as an inhibitor of the HIF-1α/HIF-1ß protein-protein interaction in vitro and in cells. The identified compound inhibits HIF-1 dimerization and transcription activity by binding to the PAS-B domain of HIF-1α, reducing HIF-1-mediated hypoxia response signaling in a variety of cell lines, without affecting the function of the closely related HIF-2 isoform. The reported cyclic peptide demonstrates the utility of our high-throughput screening platform for the identification of protein-protein interaction inhibitors, and forms the starting point for the development of HIF-1 targeted cancer therapeutics.

Targeting tumour proliferation with a small-molecule inhibitor of AICAR transformylase homodimerization.

Aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is a bifunctional homodimeric enzyme that catalyzes the last two steps of de novo purine biosynthesis. Homodimerization of ATIC, a protein-protein interaction with an interface of over 5000 Å(2), is required for its aminoimidazole carboxamide ribonucleotide (AICAR) transformylase activity, with the active sites forming at the interface of the interacting proteins. Here, we report the development of a small-molecule inhibitor of AICAR transformylase that functions by preventing the homodimerization of ATIC. The compound is derived from a previously reported cyclic hexapeptide inhibitor of AICAR transformylase (with a K(i) of 17 µM), identified by high-throughput screening. The active motif of the cyclic peptide is identified as an arginine-tyrosine dipeptide, a capped analogue of which inhibits AICAR transformylase with a K(i) value of 84 µM. A library of nonnatural analogues of this dipeptide was designed, synthesized, and assayed. The most potent compound inhibits AICAR transformylase with a K(i) value of 685 nM, a 25-fold improvement in activity from the parent cyclic peptide. The potential for this AICAR transformylase inhibitor in cancer therapy was assessed by studying its effect on the proliferation of a model breast cancer cell line. Using a nonradioactive proliferation assay and live cell imaging, a dose-dependent reduction in cell numbers and cell division rates was observed in cells treated with our ATIC dimerization inhibitor.

Modulation of Hypoxia-Induced Chemoresistance to Polymeric Micellar Cisplatin: The Effect of Ligand Modification of Micellar Carrier Versus Inhibition of the Mediators of Drug Resistance.

Hypoxia can induce chemoresistance, which is a significant clinical obstacle in cancer therapy. Here, we assessed development of hypoxia-induced chemoresistance (HICR) against free versus polymeric cisplatin micelles in a triple negative breast cancer cell line, MDA-MB-231. We then explored two strategies for the modulation of HICR against cisplatin micelles: a) the development of actively targeted micelles; and b) combination therapy with modulators of HICR in MDA-MB-231 cells. Actively targeted cisplatin micelles were prepared through surface modification of acetal-poly(ethylene oxide)-poly(α-carboxyl-ε-caprolactone) (acetal-PEO-PCCL) micelles with epidermal growth factor receptor (EGFR)-targeting peptide, GE11 (YHWYGYTPQNVI). Our results showed that hypoxia induced resistance against free and cisplatin micelles in MDA-MB-231 cells. A significant increase in micellar cisplatin uptake was observed in MDA-MB-231 cells that overexpress EGFR, following surface modification of micelles with GE11. This did not lead to increased cytotoxicity of micellar cisplatin, however. On the other hand, the addition of pharmacological inhibitors of key molecules involved in HICR in MDA-MB-231 cells, i.e., inhibitors of hypoxia inducing factor-1 (HIF-1) and signal transducer and activator of transcription 3 (STAT3), substantially enhanced the cytotoxicity of free and cisplatin micelles. The results indicated the potential benefit of combination therapy with HIF-1 and STAT3 inhibitors in overcoming HICR to free or micellar cisplatin.

Targeting Bacillus anthracis toxicity with a genetically selected inhibitor of the PA/CMG2 protein-protein interaction.

The protein-protein interaction between the human CMG2 receptor and the Bacillus anthracis protective antigen (PA) is essential for the transport of anthrax lethal and edema toxins into human cells. We used a genetically encoded high throughput screening platform to screen a SICLOPPS library of 3.2 million cyclic hexapeptides for inhibitors of this protein-protein interaction. Unusually, the top 3 hits all contained stop codons in the randomized region of the library, resulting in linear rather than cyclic peptides. These peptides disrupted the targeted interaction in vitro; two act by binding to CMG2 while one binds PA. The efficacy of the most potent CMG2-binding inhibitor was improved through the incorporation of non-natural phenylalanine analogues. Cell based assays demonstrated that the optimized inhibitor protects macrophages from the toxicity of lethal factor.

AMPK Activation via Modulation of De Novo Purine Biosynthesis with an Inhibitor of ATIC Homodimerization.

5-Aminoimidazole-4-carboxamide ribonucleotide (known as ZMP) is a metabolite produced in de novo purine biosynthesis and histidine biosynthesis, but only utilized in the cell by a homodimeric bifunctional enzyme (called ATIC) that catalyzes the last two steps of de novo purine biosynthesis. ZMP is known to act as an allosteric activator of the cellular energy sensor adenosine monophosphate-activated protein kinase (AMPK), when exogenously administered as the corresponding cell-permeable ribonucleoside. Here, we demonstrate that endogenous ZMP, produced by the aforementioned metabolic pathways, is also capable of activating AMPK. Using an inhibitor of ATIC homodimerization to block the ninth step of de novo purine biosynthesis, we demonstrate that the subsequent increase in endogenous ZMP activates AMPK and its downstream signaling pathways. We go on to illustrate the viability of using this approach to AMPK activation as a therapeutic strategy with an in vivo mouse model for metabolic disorders.

Binding of acetylcholine and tetramethylammonium to flexible cyclophane receptors: improving on binding ability by optimizing host's geometry.

The structure of a cyclophanic tetraester (1), previously employed for investigations on the cation-pi interaction, has been optimized to better accommodate acetylcholine (ACh) and tetramethylammonium (TMA) guests. Following indications from molecular modeling calculations, a flexible cyclophane receptor of significantly improved binding properties has been obtained by removing the four carbonyl groups of the parent host. 2,11,20,29-Tetraoxa[3.3.3.3]paracyclophane (2) was prepared by an improved procedure, which was conveniently devised to avoid the formation of contiguous cyclooligomers that caused serious separation issues. Association of 2 with TMA picrate was measured in CDCl(3) at T = 296 K by (1)H NMR titrations and compared to binding data obtained for a set of reference hosts, including the parent tetraester 1, the corresponding cyclophanic tetraamine, the open-chain counterpart of 2, and its cyclooligomers from pentamer to octamer. Binding enhancements ranging from 15-fold (with respect to the tetraester and the tetraamine) to over 80-fold (with respect to the open-chain tetraether) were achieved by geometry optimization of the host. Binding of 2 to ACh and TMA was investigated for a variety of counterions. A constant binding free energy increment of nearly 8 kJ mol(-1) with respect to 1 was observed, independent from the anion and irrespective of the different structure of the cationic guests. Results showed that the electrostatic inhibiting contribution of the counterion to the cation's binding is a characteristic constant of each anion. The value of -Delta G degrees = 44.9 kJ mol(-1) extrapolated for TMA in the absence of a counterion indicates that 28-34 kJ mol(-1) of binding free energy are lost in ion pairing.