Structure Based Design of Peptidomimetic Inhibitors of the MLL1-WDR5 Interaction.

MLL1 is a histone-3 lysine-4 (H3K4) methyltransferase, which is misregulated in patients with leukemia and linked with tumorigenicity through upregulation of the target genes HoxA9 and Meis-1. Suppressing expression of these genes by targeting the catalytic activity of MLL1 may be a novel approach in cancer therapy. Methylation of H3K4 by MLL1 requires formation of a core complex consisting of MLL1, WDR5, RbBP5 and ASH2L. The interaction between MLL1 and WDR5 in this complex is essential for its catalytic activity and disruption of the MLL1-WDR5 interaction may provide significant therapeutic benefit to suppress target gene expression and thus tumorigenesis. In this study, the design of peptidomimetic inhibitors that can disrupt the interaction between MLL1 and WDR5 is presented. Starting from a 12mer peptide, the tripeptide -CO-ARA-NH- in MLL1 was identified as the minimal motif for binding to WDR5. Systematic modifications to the Ac-ARA-NH2 tripeptide were performed to elucidate the interaction of WDR5 with its ligands, and a number of peptidomimetic compounds with Ki < 1 nM for WDR5 were developed. These compounds were also demonstrated to effectively inhibit the catalytic activity of the reconstituted MLL1 core complex in vitro. Further modifications to improve cellular permeability of the peptidomimetic inhibitors led to design of MM-101 and MM-102, which have sub-nanomolar binding affinities for WDR5. Crystal structures of MM-101 and MM-102 provide insight for further development of inhibitors. MM-102 inhibits the interaction between MLL1 and WDR5 in vitro, and reduces expression of HoxA9 and Meis-1 genes in MLL1-AF9 transduced bone marrow cells. These findings, together with the selective growth inhibition of leukemia cell lines with MLL1 fusion proteins upon treatment with MM-102, suggest that inhibitors targeting the MLL1-WDR5 interaction have a therapeutic potential in cancer therapy.Ph.D.Medicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/91605/1/hkaratas_1.pd

Targeting Mll1 H3K4 methyltransferase activity to guide cardiac lineage specific reprogramming of fibroblasts

Generation of induced cardiomyocytes (iCMs) directly from fibroblasts offers a great opportunity for cardiac disease modeling and cardiac regeneration. A major challenge of iCM generation is the low conversion rate. To address this issue, we attempted to identify small molecules that could potentiate the reprogramming ability towards cardiac fate by removing inhibitory roadblocks. Using mouse embryonic fibroblasts as the starting cell source, we first screened 47 cardiac development related epigenetic and transcription factors, and identified an unexpected role of H3K4 methyltransferase Mll1 and related factor Men1 in inhibiting iCM reprogramming. We then applied small molecules (MM408 and MI503) of Mll1 pathway inhibitors and observed an improved efficiency in converting embryonic fibroblasts and cardiac fibroblasts into functional cardiomyocyte-like cells. We further observed that these inhibitors directly suppressed the expression of Mll1 target gene Ebf1 involved in adipocyte differentiation. Consequently, Mll1 inhibition significantly decreased the formation of adipocytes during iCM induction. Therefore, Mll1 inhibitors likely increased iCM efficiency by suppressing alternative lineage gene expression. Our studies show that targeting Mll1 dependent H3K4 methyltransferase activity provides specificity in the process of cardiac reprogramming. These findings shed new light on the molecular mechanisms underlying cardiac conversion of fibroblasts and provide novel targets and small molecules to improve iCM reprogramming for clinical applications

TEAD-YAP interaction inhibitors and MDM2 binders from DNA-encoded indole-focused Ugi-peptidomimetics

DNA-encoded combinatorial synthesis provides efficient and dense coverage of chemical space around privileged molecular structures. The indole side chain of tryptophan plays a prominent role in key, or “hot spot”, regions of protein–protein interactions. A DNA-encoded combinatorial peptoid library was designed based on the Ugi four-component reaction by employing tryptophan-mimetic indole side chains to probe the surface of target proteins. Several peptoids were synthesized on a chemically stable hexathymidine adapter oligonucleotide “hexT”, encoded by DNA sequences, and substituted by azide-alkyne cycloaddition to yield a library of 8112 molecules. Selection experiments for the tumor-relevant proteins MDM2 and TEAD4 yielded MDM2 binders and a novel class of TEAD-YAP interaction inhibitors that perturbed the expression of a gene under the control of these Hippo pathway effectors

TEAD-YAP Interaction Inhibitors and MDM2 Binders from DNA-Encoded Indole-Focused Ugi Peptidomimetics

DNA-encoded combinatorial synthesis provides efficient and dense coverage of chemical space around privileged molecular structures. The indole side chain of tryptophan plays a prominent role in key, or “hot spot”, regions of protein–protein interactions. A DNA-encoded combinatorial peptoid library was designed based on the Ugi four-component reaction by employing tryptophan-mimetic indole side chains to probe the surface of target proteins. Several peptoids were synthesized on a chemically stable hexathymidine adapter oligonucleotide “hexT”, encoded by DNA sequences, and substituted by azide-alkyne cycloaddition to yield a library of 8112 molecules. Selection experiments for the tumor-relevant proteins MDM2 and TEAD4 yielded MDM2 binders and a novel class of TEAD-YAP interaction inhibitors that perturbed the expression of a gene under the control of these Hippo pathway effectors

In Vivo Molecular Bioluminescence Imaging: New Tools and Applications

in vivo bioluminescence imaging (BLi) is an optical molecular imaging technique used to visualize molecular and cellular processes in health and diseases and to follow the fate of cells with high sensitivity using luciferase-based gene reporters. The high sensitivity of this technique arises from efficient photon production, followed by the reaction between luciferase enzymes and luciferin substrates. Novel discoveries and developments of luciferase reporters, substrates, and gene-editing techniques, and emerging fields of applications, promise a new era of deeper and more sensitive molecular imaging

Real-Time Imaging and Quantification of Peptide Uptake in Vitro and in Vivo

Peptides constitute an important class of drugs for the treatment of multiple metabolic, ontological, and neurodegenerative diseases, and several hundred novel therapeutic peptides are currently in the preclinical and clinical stages of development. However, many leads fail to advance clinically because of poor cellular membrane and tissue permeability. Therefore, assessment of the ability of a peptide to cross cellular membranes is critical when developing novel peptide-based therapeutics. Current methods to assess peptide cellular permeability are limited by multiple factors, such as the need to introduce rather large modifications (e.g., fluorescent dyes) that require complex chemical reactions as well as an inability to provide kinetic information on the internalization of a compound or distinguish between internalized and membrane-bound compounds. In addition, many of these methods are based on end point assays and require multiple sample manipulation steps. Herein, we report a novel "Split Luciferin Peptide" (SLP) assay that enables the real-time noninvasive imaging and quantification of peptide uptake both in vitro and in vivo using a very sensitive bioluminescence readout. This method is based on a straightforward, stable chemical modification of the peptide of interest with a D-cysteine tag that preserves the overall peptidic character of the original molecule. This method can be easily adapted for screening peptide libraries and can thus become an important tool for preclinical peptide drug development

The effect of octreotide, an analog of somatostatin, on bleomycin-induced interstitial pulmonary fibrosis in rats

In this study, octreotide (OCT), a synthetic sonnatostatin analog, was tested for its beneficial effects in the prevention of interstitial pulmonary fibrosis (IPF) induced by bleomycin (BLM) in rats by histological examination and by evaluating tissue OH-proline levels. Thirty male Wistar rats were divided randomly into three groups: group I: intratracheal (i.t.) BLM (7.5 mg/kg, single dose) + saline solution [0.9% NaCl, subcutaneously (s.c.), once-daily for 7 days]; group II: i.t. BLM (7.5 mg/kg, single dose) + OCT acetate (82.5 mu g/kg, s.c., once-daily for 7 days); and the control group. At the end of the 7 days, lung tissues were excised and examined by histopathological methods. Levels of tissue hydroxyproline (OH-proline) were determined. BLM administration resulted in prominent histopathologic findings, such as diffuse alveolar damage and interstitial pulmonary fibrosis, as well as a significant increase in OH-proline level, as compared to controls. OCT application explicitly attenuated the histopathologic changes to a significant extent. OCT decreased paranchymal fibrosis and structural deformities in BLM-induced lung fibrosis. These results suggest that OCT administration to rats with BLM-induced IPF has a protective effect. Further studies are necessary to reveal the molecular mechanism(s) of OCT-induced protective effect

MLL1 and MLL1 fusion proteins play distinct roles in regulating leukemic transcription program

MLL1 plays a critical role in mixed lineage leukemia (MLL) and is a validated therapeutic target. However, its role in global gene regulation and its functional interaction with MLL1 fusion proteins in MLL remain unclear. Here we show that despite shared DNA binding and cofactor interacting domains at N-terminus, MLL1 and MLL-AF9 are recruited to distinct chromatin regions and have divergent functions in regulating the leukemic transcription program. We demonstrate that MLL1, probably through C-terminal interaction with WDR5, is recruited to regulatory enhancers that are enriched for binding sites of ETS family transcription factors while MLL-AF9 binds to chromatin regions that have no enrichment for H3K4me1. Transcriptome-wide changes induced by different small molecule inhibitors also highlight the distinct functions of MLL1 and MLL-AF9. Taken together, our studies provide novel insights on how MLL1 and MLL-fusion proteins contribute to leukemic gene expression, which have implications in developing effective therapies in future

Structure-Based Design of High-Affinity Macrocyclic Peptidomimetics to Block the Menin-Mixed Lineage Leukemia 1 (MLL1) Protein–Protein Interaction

Menin is an essential oncogenic cofactor for mixed lineage leukemia 1 (MLL1)-mediated leukemogenesis through its direct interaction with MLL1. Targeting the menin–MLL1 protein–protein interaction represents a promising strategy to block MLL1-mediated leukemogenesis. Employing a structure-based approach and starting from a linear MLL1 octapeptide, we have designed a class of potent macrocyclic peptidomimetic inhibitors of the menin–MLL1 interaction. The most potent macrocyclic peptidomimetic (MCP-1), <b>34</b>, binds to menin with a <i>K</i>_i value of 4.7 nM and is >600 times more potent than the corresponding acyclic peptide. Compound <b>34</b> is also less peptide-like and has a lower molecular weight than the initial MLL1 peptide. Therefore, compound <b>34</b> serves as a promising lead structure for the design of potent and cell-permeable inhibitors of the menin–MLL1 interaction

High-Affinity, Small-Molecule Peptidomimetic Inhibitors of MLL1/WDR5 Protein–Protein Interaction

Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase, and targeting the MLL1 enzymatic activity has been proposed as a novel therapeutic strategy for the treatment of acute leukemia harboring MLL1 fusion proteins. The MLL1/WDR5 protein–protein interaction is essential for MLL1 enzymatic activity. In the present study, we designed a large number of peptidomimetics to target the MLL1/WDR5 interaction based upon −CO-ARA-NH–, the minimum binding motif derived from MLL1. Our study led to the design of high-affinity peptidomimetics, which bind to WDR5 with <i>K</i>_i < 1 nM and function as potent antagonists of MLL1 activity in a fully reconstituted <i>in vitro</i> H3K4 methyltransferase assay. Determination of co-crystal structures of two potent peptidomimetics in complex with WDR5 establishes their structural basis for high-affinity binding to WDR5. Evaluation of one such peptidomimetic, MM-102, in bone marrow cells transduced with <i>MLL1-AF9</i> fusion construct shows that the compound effectively decreases the expression of <i>HoxA9</i> and <i>Meis-1</i>, two critical MLL1 target genes in MLL1 fusion protein mediated leukemogenesis. MM-102 also specifically inhibits cell growth and induces apoptosis in leukemia cells harboring MLL1 fusion proteins. Our study provides the first proof-of-concept for the design of small-molecule inhibitors of the WDR5/MLL1 protein–protein interaction as a novel therapeutic approach for acute leukemia harboring MLL1 fusion proteins