Interplay among nucleosomal DNA, histone tails, and corepressor CoREST underlies LSD1-mediated H3 demethylation.

With its noncatalytic domains, DNA-binding regions, and a catalytic core targeting the histone tails, LSD1-CoREST (lysine-specific demethylase 1; REST corepressor) is an ideal model system to study the interplay between DNA binding and histone modification in nucleosome recognition. To this end, we covalently associated LSD1-CoREST to semisynthetic nucleosomal particles. This enabled biochemical and biophysical characterizations of nucleosome binding and structural elucidation by small-angle X-ray scattering, which was extensively validated through binding assays and site-directed mutagenesis of functional interfaces. Our results suggest that LSD1-CoREST functions as an ergonomic clamp that induces the detachment of the H3 histone tail from the nucleosomal DNA to make it available for capture by the enzyme active site. The key notion emerging from these studies is the inherently competitive nature of the binding interactions because nucleosome tails, chromatin modifiers, transcription factors, and DNA represent sites for multiple and often mutually exclusive interactions.

Structure-Enabled Discovery of a Stapled Peptide Inhibitor to Target the Oncogenic Transcriptional Repressor TLE1.

TLE1 is an oncogenic transcriptional co-repressor that exerts its repressive effects through binding of transcription factors. Inhibition of this protein-protein interaction represents a putative cancer target, but no small-molecule inhibitors have been published for this challenging interface. Herein, the structure-enabled design and synthesis of a constrained peptide inhibitor of TLE1 is reported. The design features the introduction of a four-carbon-atom linker into the peptide epitope found in many TLE1 binding partners. A concise synthetic route to a proof-of-concept peptide, cycFWRPW, has been developed. Biophysical testing by isothermal titration calorimetry and thermal shift assays showed that, although the constrained peptide bound potently, it had an approximately five-fold higher Kd than that of the unconstrained peptide. The co-crystal structure suggested that the reduced affinity was likely to be due to a small shift of one side chain, relative to the otherwise well-conserved conformation of the acyclic peptide. This work describes a constrained peptide inhibitor that may serve as the basis for improved inhibitors.

Phosphorylation of neuronal Lysine-Specific Demethylase 1LSD1/KDM1A impairs transcriptional repression by regulating interaction with CoREST and histone deacetylases HDAC1/2.

Epigenetic mechanisms play important roles in brain development, orchestrating proliferation, differentiation, and morphogenesis. Lysine-Specific Demethylase 1 (LSD1 also known as KDM1A and AOF2) is a histone modifier involved in transcriptional repression, forming a stable core complex with the corepressors corepressor of REST (CoREST) and histone deacetylases (HDAC1/2). Importantly, in the mammalian CNS, neuronal LSD1-8a, an alternative splicing isoform of LSD1 including the mini-exon E8a, sets alongside LSD1 and is capable of enhancing neurite growth and morphogenesis. Here, we describe that the morphogenic properties of neuronal LSD1-8a require switching off repressive activity and this negative modulation is mediated in vivo by phosphorylation of the Thr369b residue coded by exon E8a. Three-dimensional crystal structure analysis using a phospho-mimetic mutant (Thr369bAsp), indicate that phosphorylation affects the residues surrounding the exon E8a-coded amino acids, causing a local conformational change. We suggest that phosphorylation, without affecting demethylase activity, causes in neurons CoREST and HDAC1/2 corepressors detachment from LSD1-8a and impairs neuronal LSD1-8a repressive activity. In neurons, Thr369b phosphorylation is required for morphogenic activity, converting neuronal LSD1-8a in a dominant-negative isoform, challenging LSD1-mediated transcriptional repression on target genes.

Expanding the druggable space of the LSD1/CoREST epigenetic target: new potential binding regions for drug-like molecules, peptides, protein partners, and chromatin.

Lysine specific demethylase-1 (LSD1/KDM1A) in complex with its corepressor protein CoREST is a promising target for epigenetic drugs. No therapeutic that targets LSD1/CoREST, however, has been reported to date. Recently, extended molecular dynamics (MD) simulations indicated that LSD1/CoREST nanoscale clamp dynamics is regulated by substrate binding and highlighted key hinge points of this large-scale motion as well as the relevance of local residue dynamics. Prompted by the urgent need for new molecular probes and inhibitors to understand LSD1/CoREST interactions with small-molecules, peptides, protein partners, and chromatin, we undertake here a configurational ensemble approach to expand LSD1/CoREST druggability. The independent algorithms FTMap and SiteMap and our newly developed Druggable Site Visualizer (DSV) software tool were used to predict and inspect favorable binding sites. We find that the hinge points revealed by MD simulations at the SANT2/Tower interface, at the SWIRM/AOD interface, and at the AOD/Tower interface are new targets for the discovery of molecular probes to block association of LSD1/CoREST with chromatin or protein partners. A fourth region was also predicted from simulated configurational ensembles and was experimentally validated to have strong binding propensity. The observation that this prediction would be prevented when using only the X-ray structures available (including the X-ray structure bound to the same peptide) underscores the relevance of protein dynamics in protein interactions. A fifth region was highlighted corresponding to a small pocket on the AOD domain. This study sets the basis for future virtual screening campaigns targeting the five novel regions reported herein and for the design of LSD1/CoREST mutants to probe LSD1/CoREST binding with chromatin and various protein partners.

Crystal structure of the catalytic domain of Haspin, an atypical kinase implicated in chromatin organization.

Haspin, a nuclear and chromosome-associated serine/threonine (S/T) kinase, is responsible for mitotic phosphorylation of Thr-3 of histone H3. Haspin bears recognizable similarity to the eukaryotic protein kinase (ePK) fold, but its sequence is highly divergent and there is therefore considerable interest in its structural organization. We report the 2.15-A crystal structure of the kinase domain of human Haspin. The ePK fold of Haspin contains an array of insertions and deletions. The structure illustrates how Haspin escapes the classical activation scheme of most other kinases. The alphaC helix, which bears a conserved glutamate that is essential for catalysis, adopts its final active conformation within the small lobe of the kinase. It is sandwiched between an alpha-helical insertion that precedes the kinase domain, and the activation segment, which adopts an unprecedented conformation. The activation segment, which does not contain phosphorylatable residues, packs against an unusually structured alphaEF helix. Significantly extruded from the core of the fold, it forms an extensive plateau, hosting several residues implicated in substrate binding. Overall, the structure of the Haspin kinase domain reveals an active conformation that is poised for substrate recognition and phosphorylation in the absence of external regulators.

Evaluation of APOBEC3B Recognition Motifs by NMR Reveals Preferred Substrates.

APOBEC3B (A3B) deamination activity on ssDNA is considered a contributing factor to tumor heterogeneity and drug resistance in a number of human cancers. Despite its clinical impact, little is known about A3B ssDNA substrate preference. We have used nuclear magnetic resonance to monitor the catalytic turnover of A3B substrates in real-time. This study reports preferred nucleotide sequences for A3B substrates, including optimized 4-mer oligonucleotides, and reveals a breadth of substrate recognition that includes DNA sequences known to be mutated in drug-resistant cancer clones. Our results are consistent with available clinical and structural data and may inform the design of substrate-based A3B inhibitors.

Structure of the Epigenetic Oncogene MMSET and Inhibition by N-Alkyl Sinefungin Derivatives.

The members of the NSD subfamily of lysine methyl transferases are compelling oncology targets due to the recent characterization of gain-of-function mutations and translocations in several hematological cancers. To date, these proteins have proven intractable to small molecule inhibition. Here, we present initial efforts to identify inhibitors of MMSET (aka NSD2 or WHSC1) using solution phase and crystal structural methods. On the basis of 2D NMR experiments comparing NSD1 and MMSET structural mobility, we designed an MMSET construct with five point mutations in the N-terminal helix of its SET domain for crystallization experiments and elucidated the structure of the mutant MMSET SET domain at 2.1 Å resolution. Both NSD1 and MMSET crystal systems proved resistant to soaking or cocrystallography with inhibitors. However, use of the close homologue SETD2 as a structural surrogate supported the design and characterization of N-alkyl sinefungin derivatives, which showed low micromolar inhibition against both SETD2 and MMSET.

Pan-histone demethylase inhibitors simultaneously targeting Jumonji C and lysine-specific demethylases display high anticancer activities.

In prostate cancer, two different types of histone lysine demethylases (KDM), LSD1/KDM1 and JMJD2/KDM4, are coexpressed and colocalize with the androgen receptor. We designed and synthesized hybrid LSD1/JmjC or "pan-KDM" inhibitors 1-6 by coupling the skeleton of tranylcypromine 7, a known LSD1 inhibitor, with 4-carboxy-4'-carbomethoxy-2,2'-bipyridine 8 or 5-carboxy-8-hydroxyquinoline 9, two 2-oxoglutarate competitive templates developed for JmjC inhibition. Hybrid compounds 1-6 are able to simultaneously target both KDM families and have been validated as potential antitumor agents in cells. Among them, 2 and 3 increase H3K4 and H3K9 methylation levels in cells and cause growth arrest and substantial apoptosis in LNCaP prostate and HCT116 colon cancer cells. When tested in noncancer mesenchymal progenitor (MePR) cells, 2 and 3 induced little and no apoptosis, respectively, thus showing cancer-selective inhibiting action.

Synthesis, biological activity and mechanistic insights of 1-substituted cyclopropylamine derivatives: a novel class of irreversible inhibitors of histone demethylase KDM1A.

Histone demethylase KDM1A (also known as LSD1) has become an attractive therapeutic target for the treatment of cancer as well as other disorders such as viral infections. We report on the synthesis of compounds derived from the expansion of tranylcypromine as a chemical scaffold for the design of novel demethylase inhibitors. These compounds, which are substituted on the cyclopropyl core moiety, were evaluated for their ability to inhibit KDM1A in vitro as well as to function in cells by modulating the expression of Gfi-1b, a well recognized KDM1A target gene. The molecules were all found to covalently inhibit KDM1A and to become increasingly selective against human monoamine oxidases MAO A and MAO B through the introduction of bulkier substituents on the cyclopropylamine ring. Structural and biochemical analysis of selected trans isomers showed that the two stereoisomers are endowed with similar inhibitory activities against KDM1A, but form different covalent adducts with the FAD co-enzyme.

Protein recognition by short peptide reversible inhibitors of the chromatin-modifying LSD1/CoREST lysine demethylase.

The combinatorial assembly of protein complexes is at the heart of chromatin biology. Lysine demethylase LSD1(KDM1A)/CoREST beautifully exemplifies this concept. The active site of the enzyme tightly associates to the N-terminal domain of transcription factors of the SNAIL1 family, which therefore can competitively inhibit the binding of the N-terminal tail of the histone substrate. Our enzymatic, crystallographic, spectroscopic, and computational studies reveal that LSD1/CoREST can bind to a hexapeptide derived from the SNAIL sequence through recognition of a positively charged α-helical turn that forms upon binding to the enzyme. Variations in sequence and length of this six amino acid ligand modulate affinities enabling the same binding site to differentially interact with proteins that exert distinct biological functions. The discovered short peptide inhibitors exhibit antiproliferative activities and lay the foundation for the development of peptidomimetic small molecule inhibitors of LSD1.

Molecular mimicry and ligand recognition in binding and catalysis by the histone demethylase LSD1-CoREST complex.

Histone demethylases LSD1 and LSD2 (KDM1A/B) catalyze the oxidative demethylation of Lys4 of histone H3. We used molecular dynamics simulations to probe the diffusion of the oxygen substrate. Oxygen can reach the catalytic center independently from the presence of a bound histone peptide, implying that LSD1 can complete subsequent demethylation cycles without detaching from the nucleosomal particle. The simulations highlight the role of a strictly conserved active-site Lys residue providing general insight into the enzymatic mechanism of oxygen-reacting flavoenzymes. The crystal structure of LSD1-CoREST bound to a peptide of the transcription factor SNAIL1 unravels a fascinating example of molecular mimicry. The SNAIL1 N-terminal residues bind to the enzyme active-site cleft, effectively mimicking the H3 tail. This finding predicts that other members of the SNAIL/Scratch transcription factor family might associate to LSD1/2. The combination of selective histone-modifying activity with the distinct recognition mechanisms underlies the biological complexity of LSD1/2.

Molecular Insights into Human Monoamine Oxidase B Inhibition by the Glitazone Anti-Diabetes Drugs.

The widely employed anti-diabetic drug pioglitazone (Actos) is shown to be a specific and reversible inhibitor of human monoamine oxidase B (MAO B). The crystal structure of the enzyme-inhibitor complex shows the R-enantiomer is bound with the thiazolidinedione ring near the flavin. The molecule occupies both substrate and entrance cavities of the active site establishing non-covalent interactions with the surrounding amino acids. These binding properties differentiate pioglitazone from the clinically used MAO inhibitors, which act through covalent inhibition mechanisms and do not exhibit a high degree of MAO A versus B selectivity. Rosiglitazone (Avandia) and troglitazone, other members of the glitazone class, are less selective in that they are weaker inhibitors of both MAO A and MAO B These results suggest that pioglitazone may have utility as a "re-purposed" neuro-protectant drug in retarding the progression of disease in Parkinson's patients. They also provide new insights for the development of reversible isoenzyme-specific MAO inhibitors.