Computational protein design is a reverse procedure of protein folding and structure prediction, where constructing structures from evolutionarily related proteins has been demonstrated to be the most reliable method for protein 3-dimensional structure prediction. Following this spirit, we developed a novel method to design new protein sequences based on evolutionarily related protein families. For a given target structure, a set of proteins having similar fold are identified from the PDB library by structural alignments. A structural profile is then constructed from the protein templates and used to guide the conformational search of amino acid sequence space, where physicochemical packing is accommodated by single-sequence based solvation, torsion angle, and secondary structure predictions. The method was tested on a computational folding experiment based on a large set of 87 protein structures covering different fold classes, which showed that the evolution-based design significantly enhances the foldability and biological functionality of the designed sequences compared to the traditional physics-based force field methods. Without using homologous proteins, the designed sequences can be folded with an average root-mean-square-deviation of 2.1 Å to the target. As a case study, the method is extended to redesign all 243 structurally resolved proteins in the pathogenic bacteria Mycobacterium tuberculosis, which is the second leading cause of death from infectious disease. On a smaller scale, five sequences were randomly selected from the design pool and subjected to experimental validation. The results showed that all the designed proteins are soluble with distinct secondary structure and three have well ordered tertiary structure, as demonstrated by circular dichroism and NMR spectroscopy. Together, these results demonstrate a new avenue in computational protein design that uses knowledge of evolutionary conservation from protein structural families to engineer new protein molecules of improved fold stability and biological functionality.

ASH1L histone methyltransferase plays a crucial role in the pathogenesis of different diseases, including acute leukemia. While ASH1L represents an attractive drug target, developing ASH1L inhibitors is challenging, as the catalytic SET domain adapts an inactive conformation with autoinhibitory loop blocking the access to the active site. Here, by applying fragment-based screening followed by medicinal chemistry and a structure-based design, we developed first-in-class small molecule inhibitors of the ASH1L SET domain. The crystal structures of ASH1L-inhibitor complexes reveal compound binding to the autoinhibitory loop region in the SET domain. When tested in MLL leukemia models, our lead compound, AS-99, blocks cell proliferation, induces apoptosis and differentiation, downregulates MLL fusion target genes, and reduces the leukemia burden in vivo. This work validates the ASH1L SET domain as a druggable target and provides a chemical probe to further study the biological functions of ASH1L as well as to develop therapeutic agents.

Destabilizing mutations in small heat shock proteins (sHsps) are linked to multiple diseases; however, sHsps are conformationally dynamic, lack enzymatic function and have no endogenous chemical ligands. These factors render sHsps as classically "undruggable" targets and make it particularly challenging to identify molecules that might bind and stabilize them. To explore potential solutions, we designed a multi-pronged screening workflow involving a combination of computational and biophysical ligand-discovery platforms. Using the core domain of the sHsp family member Hsp27/HSPB1 (Hsp27c) as a target, we applied mixed solvent molecular dynamics (MixMD) to predict three possible binding sites, which we confirmed using NMR-based solvent mapping. Using this knowledge, we then used NMR spectroscopy to carry out a fragment-based drug discovery (FBDD) screen, ultimately identifying two fragments that bind to one of these sites. A medicinal chemistry effort improved the affinity of one fragment by ~50-fold (16 µM), while maintaining good ligand efficiency (~0.32 kcal/mol/non-hydrogen atom). Finally, we found that binding to this site partially restored the stability of disease-associated Hsp27 variants, in a redox-dependent manner. Together, these experiments suggest a new and unexpected binding site on Hsp27, which might be exploited to build chemical probes.

Nucleophosmin (NPM1) is a ubiquitously expressed nucleolar protein with a wide range of biological functions. In 30% of acute myeloid leukemia (AML), the terminal exon of NPM1 is often found mutated, resulting in the addition of a nuclear export signal and a shift of the protein to the cytoplasm (NPM1c). AMLs carrying this mutation have aberrant expression of the HOXA/B genes, whose overexpression leads to leukemogenic transformation. Here, for the first time, we comprehensively prove that NPM1c binds to a subset of active gene promoters in NPM1c AMLs, including well-known leukemia-driving genes-HOXA/B cluster genes and MEIS1. NPM1c sustains the active transcription of key target genes by orchestrating a transcription hub and maintains the active chromatin landscape by inhibiting the activity of histone deacetylases. Together, these findings reveal the neomorphic function of NPM1c as a transcriptional amplifier for leukemic gene expression and open up new paradigms for therapeutic intervention.

Aberrant activation of Wnt/β-catenin pathway is a key driver of colorectal cancer (CRC) growth and of great therapeutic importance. In this study, we performed comprehensive CRISPR screens to interrogate the regulatory network of Wnt/β-catenin signaling in CRC cells. We found marked discrepancies between the artificial TOP reporter activity and β-catenin-mediated endogenous transcription and redundant roles of T cell factor/lymphoid enhancer factor transcription factors in transducing β-catenin signaling. Compiled functional genomic screens and network analysis revealed unique epigenetic regulators of β-catenin transcriptional output, including the histone lysine methyltransferase 2A oncoprotein (KMT2A/Mll1). Using an integrative epigenomic and transcriptional profiling approach, we show that KMT2A loss diminishes the binding of β-catenin to consensus DNA motifs and the transcription of β-catenin targets in CRC. These results suggest that KMT2A may be a promising target for CRCs and highlight the broader potential for exploiting epigenetic modulation as a therapeutic strategy for β-catenin-driven malignancies.

CDC25 phosphatases are key cell cycle regulators and represent very attractive but challenging targets for anticancer drug discovery. Here, we explored whether fragment-based screening represents a valid approach to identify inhibitors of CDC25B. This resulted in identification of 2-fluoro-4-hydroxybenzonitrile, which directly binds to the catalytic domain of CDC25B. Interestingly, NMR data and the crystal structure demonstrate that this compound binds to the pocket distant from the active site and adjacent to the protein-protein interaction interface with CDK2/Cyclin A substrate. Furthermore, we developed a more potent analogue that disrupts CDC25B interaction with CDK2/Cyclin A and inhibits dephosphorylation of CDK2. Based on these studies, we provide a proof of concept that targeting CDC25 phosphatases by inhibiting their protein-protein interactions with CDK2/Cyclin A substrate represents a novel, viable opportunity to target this important class of enzymes.

Mixed lineage leukemia (MLL) fusion proteins cause oncogenic transformation of hematopoietic cells by constitutive recruitment of elongation factors to HOX promoters, resulting in overexpression of target genes. The structural basis of transactivation by MLL fusion partners remains undetermined. We show that the ANC1 homology domain (AHD) of AF9, one of the most common MLL translocation partners, is intrinsically disordered and recruits multiple transcription factors through coupled folding and binding. We determined the structure of the AF9 AHD in complex with the elongation factor AF4 and show that aliphatic residues, which are conserved in each of the AF9 binding partners, form an integral part of the hydrophobic core of the complex. Nuclear magnetic resonance relaxation measurements show that AF9 retains significant dynamic behavior which may facilitate exchange between disordered partners. We propose that AF9 functions as a signaling hub that regulates transcription through dynamic recruitment of cofactors in normal hematopoiesis and in acute leukemia.

Transcription factors have traditionally been viewed with skepticism as viable drug targets, but they offer the potential for completely novel mechanisms of action that could more effectively address the stem cell like properties, such as self-renewal and chemo-resistance, that lead to the failure of traditional chemotherapy approaches. Core binding factor is a heterodimeric transcription factor comprised of one of 3 RUNX proteins (RUNX1-3) and a CBFβ binding partner. CBFβ enhances DNA binding of RUNX subunits by relieving auto-inhibition. Both RUNX1 and CBFβ are frequently mutated in human leukemia. More recently, RUNX proteins have been shown to be key players in epithelial cancers, suggesting the targeting of this pathway could have broad utility. In order to test this, we developed small molecules which bind to CBFβ and inhibit its binding to RUNX. Treatment with these inhibitors reduces binding of RUNX1 to target genes, alters the expression of RUNX1 target genes, and impacts cell survival and differentiation. These inhibitors show efficacy against leukemia cells as well as basal-like (triple-negative) breast cancer cells. These inhibitors provide effective tools to probe the utility of targeting RUNX transcription factor function in other cancers.

Normal fibroblasts surrounding tumor cells play a crucial role in cancer progression through formation of the tumor microenvironment. Because factors secreted from normal fibroblasts can modulate the tumor microenvironment, it is necessary to identify key factors associated with regulation of secreted factors and to investigate the molecular mechanisms contributing to the tumor microenvironment formation process. In this study, we found that radiation induced the expression and K63-linkage poly-ubiquitination of TRAF4 in normal lung fibroblasts. The K63-linkage poly-ubiquitinated TRAF4 formed complexes with NOX2 or NOX4 by mediating phosphorylated p47-phox in normal lung fibroblasts. Moreover, we showed that TRAF4 stabilized NOX complexes by decreasing lysosomal degradation of NOX2 and NOX4 after irradiation. NOX complexes increased endosomal ROS levels that were permeable into cytoplasm, leading to NF-κB-mediated ICAM1 up-regulation. Soluble ICAM1 was subsequently secreted into conditioned media of radiation-activated normal lung fibroblasts. The conditioned media from irradiated normal fibroblasts enhanced proliferation and epithelial-mesenchymal transition of non-small cell lung cancer cells both in vitro and in vivo. These results demonstrate that TRAF4 in irradiated fibroblasts is positively associated with aggressiveness of adjacent cancer cells by altering the tumor microenvironment. Thus, we suggest that regulation of TRAF4 might be a promising strategy for cancer therapy.

A key functional event in eukaryotic gene activation is the formation of dynamic protein-protein interaction networks between transcriptional activators and transcriptional coactivators. Seemingly incongruent with the tight regulation of transcription, many biochemical and biophysical studies suggest that activators use nonspecific hydrophobic and/or electrostatic interactions to bind to coactivators, with few if any specific contacts. Here a mechanistic dissection of a set of representative dynamic activator•coactivator complexes, comprised of the ETV/PEA3 family of activators and the coactivator Med25, reveals a different molecular recognition model. The data demonstrate that small sequence variations within an activator family significantly redistribute the conformational ensemble of the complex while not affecting overall affinity, and distal residues within the activator-not often considered as contributing to binding-play a key role in mediating conformational redistribution. The ETV/PEA3•Med25 ensembles are directed by specific contacts between the disordered activator and the Med25 interface, which is facilitated by structural shifts of the coactivator binding surface. Taken together, these data highlight the critical role coactivator plasticity plays in recognition of disordered activators and indicate that molecular recognition models of disordered proteins must consider the ability of the binding partners to mediate specificity.