Laura K. Henchey

Contemporary Strategies for the Stabilization of Peptides in the α-Helical Conformation

Herein we review contemporary synthetic and protein design strategies to stabilize the alpha-helical motif in short peptides and miniature proteins. Advances in organometallic catalyst design, specifically for the olefin metathesis reaction, enable the use of hydrocarbon bridges to either crosslink side chains of specific residues or mimic intramolecular hydrogen bonds with carbon-carbon bonds. The resulting hydrocarbon-stapled and hydrogen bond surrogate alpha-helices provide unique synthetic ligands for targeting biomolecules. In the protein design realm, several classes of miniature proteins that display stable helical domains have been engineered and manipulated with powerful in vitro selection technologies to yield libraries of sequences that retain their helical folds. Rational re-design of these scaffolds provide distinctive reagents for the modulation of protein-protein interactions.

Inhibition of Hypoxia Inducible Factor 1–Transcription Coactivator Interaction by a Hydrogen Bond Surrogate α-Helix

Designed ligands that inhibit hypoxia-inducible gene expression could offer new tools for genomic research and, potentially, drug discovery efforts for the treatment of neovascularization in cancers. We report a stabilized alpha-helix designed to target the binding interface between the C-terminal transactivation domain (C-TAD) of hypoxia-inducible factor 1alpha (HIF-1alpha) and cysteine-histidine rich region (CH1) of transcriptional coactivator CBP/p300. The synthetic helix disrupts the structure and function of this complex, resulting in a rapid downregulation of two hypoxia-inducible genes (VEGF and GLUT1) in cell culture.

High Specificity in Protein Recognition by Hydrogen-Bond-Surrogate α-Helices: Selective Inhibition of the p53/MDM2 Complex

Stabilized α-helices and nonpeptidic helix mimetics have emerged as powerful molecular scaffolds for the discovery of protein-protein interaction inhibitors.[1–8] Protein-protein interactions often involve large contact areas, which are often difficult for small molecules to target with high specificity.[9–10] The hypothesis behind the design of stabilized helices and helix mimetics is that these medium-sized molecules may pursue their targets with higher specificity because of a larger number of contacts. We recently introduced a new strategy for the preparation of stabilized α-helices, termed hydrogen bond surrogate (HBS) helices, which involves replacement of one of the main chain hydrogen bonds with a covalent linkage (Figure 1A).[11] The salient feature of the HBS approach is its ability to constrain very short peptides into highly stable α-helical conformation without blocking any molecular recognition surfaces. We have extensively analyzed the conformation adopted by HBS α-helices with 2D NMR, X-ray, and circular dichroism spectroscopies.[12–14] In addition, HBS helices have been shown to target their expected protein partners with high affinity in cell-free and cell culture assays.[15–17]

Direct inhibition of hypoxia-inducible transcription factor complex with designed dimeric epidithiodiketopiperazine.

Selective blockade of hypoxia-inducible gene expression by designed small molecules would prove valuable in suppressing tumor angiogenesis, metastasis and altered energy metabolism. We report the design, synthesis, and biological evaluation of a dimeric epidithiodiketopiperazine (ETP) small molecule transcriptional antagonist targeting the interaction of the p300/CBP coactivator with the transcription factor HIF-1alpha. Our results indicate that disrupting this interaction results in rapid downregulation of hypoxia-inducible genes critical for cancer progression. The observed effects are compound-specific and dose-dependent. Controlling gene expression with designed small molecules targeting the transcription factor-coactivator interface may represent a new approach for arresting tumor growth.

Protein domain mimetics as in vivo modulators of hypoxia-inducible factor signaling

Significance Protein–protein interactions are attractive targets for interfering with processes leading to disease states. Proteins often use folded domains or secondary structures to contact partner proteins. Synthetic molecules that mimic these domains could disrupt protein–protein contacts, thereby inhibiting formation of multiprotein complexes. This article describes protein domain mimetics (PDMs) that modulate interactions between two proteins that control expression of a multitude of genes under hypoxic environments, such as those found inside tumors. The low-oxygen conditions promote angiogenesis—process of formation of new blood vessels—that together with invasion and altered energy metabolism facilitates tumor growth. We find that the PDMs can control expression of target hypoxia-inducible genes in cell culture and reduce tumor burden in mice. Selective blockade of gene expression by designed small molecules is a fundamental challenge at the interface of chemistry, biology, and medicine. Transcription factors have been among the most elusive targets in genetics and drug discovery, but the fields of chemical biology and genetics have evolved to a point where this task can be addressed. Herein we report the design, synthesis, and in vivo efficacy evaluation of a protein domain mimetic targeting the interaction of the p300/CBP coactivator with the transcription factor hypoxia-inducible factor-1α. Our results indicate that disrupting this interaction results in a rapid down-regulation of hypoxia-inducible genes critical for cancer progression. The observed effects were compound-specific and dose-dependent. Gene expression profiling with oligonucleotide microarrays revealed effective inhibition of hypoxia-inducible genes with relatively minimal perturbation of nontargeted signaling pathways. We observed remarkable efficacy of the compound HBS 1 in suppressing tumor growth in the fully established murine xenograft models of renal cell carcinoma of the clear cell type. Our results suggest that rationally designed synthetic mimics of protein subdomains that target the transcription factor–coactivator interfaces represent a unique approach for in vivo modulation of oncogenic signaling and arresting tumor growth.

Abstract 289: Hydrogen bond surrogate (HBS) helices as orthosteric regulator of hypoxia inducible transcription

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Hypoxia-Inducible Factor (HIF-1) is a heterodimeric transcriptional activator which plays a critical role in tumorigenesis and therefore is an important therapeutic target. Transcriptional activation of HIF-1 is known to involve the interaction between cysteine-histidine rich region1 (CH1) of a coactivator protein p300 or Creb Binding Protein (CBP) and C-terminal activation domain (C-TAD) of its α-subunit (HIF-1α C-TAD). Based on the hydrogen bond surrogate (HBS) approach, we have rationally designed stabilized alpha helix that can disrupt the binding interface between C-TAD domain of HIF-1α and CH1 domain of p300/CBP. We have shown by fluorescence polarization that such stabilized alpha helix mimetics can directly bind to the CH1 domain of p300 and also can disrupt the HIF-1α/p300 complex. HBS helix mimetics are also shown to selectively downregulate the hypoxia inducible genes in cell culture. Thus, orthosteric inhibition of the HIF-1 transcriptional complex by rationally designed helix mimetics offers a novel approach to downregulate the expression of key hypoxia inducible genes responsible for tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 289. doi:1538-7445.AM2012-289