Conformational-Design-Driven Discovery of EZM0414: A Selective, Potent SETD2 Inhibitor for Clinical Studies.

SETD2, a lysine N-methyltransferase, is a histone methyltransferase that plays an important role in various cellular processes and was identified as a target of interest in multiple myeloma that features a t(4,14) translocation. We recently reported the discovery of a novel small-molecule SETD2 inhibitor tool compound that is suitable for preclinical studies. Herein we describe the conformational-design-driven evolution of the advanced chemistry lead, which resulted in compounds appropriate for clinical evaluation. Further optimization of this chemical series led to the discovery of EZM0414, which is a potent, selective, and orally bioavailable inhibitor of SETD2 with good pharmacokinetic properties and robust pharmacodynamic activity in a mouse xenograft model.

Discovery of a First-in-Class Inhibitor of the Histone Methyltransferase SETD2 Suitable for Preclinical Studies.

SET domain-containing protein 2 (SETD2), a histone methyltransferase, has been identified as a target of interest in certain hematological malignancies, including multiple myeloma. This account details the discovery of EPZ-719, a novel and potent SETD2 inhibitor with a high selectivity over other histone methyltransferases. A screening campaign of the Epizyme proprietary histone methyltransferase-biased library identified potential leads based on a 2-amidoindole core. Structure-based drug design (SBDD) and drug metabolism/pharmacokinetics (DMPK) optimization resulted in EPZ-719, an attractive tool compound for the interrogation of SETD2 biology that enables in vivo target validation studies.

Small molecule inhibitors and CRISPR/Cas9 mutagenesis demonstrate that SMYD2 and SMYD3 activity are dispensable for autonomous cancer cell proliferation.

A key challenge in the development of precision medicine is defining the phenotypic consequences of pharmacological modulation of specific target macromolecules. To address this issue, a variety of genetic, molecular and chemical tools can be used. All of these approaches can produce misleading results if the specificity of the tools is not well understood and the proper controls are not performed. In this paper we illustrate these general themes by providing detailed studies of small molecule inhibitors of the enzymatic activity of two members of the SMYD branch of the protein lysine methyltransferases, SMYD2 and SMYD3. We show that tool compounds as well as CRISPR/Cas9 fail to reproduce many of the cell proliferation findings associated with SMYD2 and SMYD3 inhibition previously obtained with RNAi based approaches and with early stage chemical probes.

Characterization of Inhibitor Binding Through Multiple Inhibitor Analysis: A Novel Local Fitting Method.

Understanding inhibitor binding modes is a key aspect of drug development. Early in a drug discovery effort these considerations often impact hit finding strategies and hit prioritization. Multiple inhibitor experiments, where enzyme inhibition is measured in the presence of two simultaneously varied inhibitors, can provide valuable information about inhibitor binding. These experiments utilize the inhibitor concentration dependence of the observed combined inhibition to determine the relationship between two compounds. In this way, it can be determined whether two inhibitors bind exclusively, independently, synergistically, or antagonistically. Novel inhibitors can be tested against each other or reference compounds to assist hit classification and characterization of inhibitor binding. In this chapter, we discuss the utility and design of multiple inhibitor experiments and present a new local curve fitting method for analyzing these data utilizing IC50 replots. The IC50 replot method is analogous to that used for determining mechanisms of inhibition with respect to substrate, as originally proposed by Cheng and Prusoff (Cheng and Prusoff Biochem Pharmacol 22: 3099-3108, 1973). The IC50 replot generated by this method reveals distinct patterns that are diagnostic of the nature of the interaction between two inhibitors. Multiple inhibition of the histone methyltransferase EZH2 by EPZ-5687 and the reaction product S-adenosylhomocysteine is presented as an example of the method.

Novel Oxindole Sulfonamides and Sulfamides: EPZ031686, the First Orally Bioavailable Small Molecule SMYD3 Inhibitor.

SMYD3 has been implicated in a range of cancers; however, until now no potent selective small molecule inhibitors have been available for target validation studies. A novel oxindole series of SMYD3 inhibitors was identified through screening of the Epizyme proprietary histone methyltransferase-biased library. Potency optimization afforded two tool compounds, sulfonamide EPZ031686 and sulfamide EPZ030456, with cellular potency at a level sufficient to probe the in vitro biology of SMYD3 inhibition. EPZ031686 shows good bioavailability following oral dosing in mice making it a suitable tool for potential in vivo target validation studies.

CARM1 Preferentially Methylates H3R17 over H3R26 through a Random Kinetic Mechanism.

CARM1 is a type I arginine methyltransferase involved in the regulation of transcription, pre-mRNA splicing, cell cycle progression, and the DNA damage response. CARM1 overexpression has been implicated in breast, prostate, and liver cancers and therefore is an attractive target for cancer therapy. To date, little about the kinetic properties of CARM1 is known. In this study, substrate specificity and the kinetic mechanism of the human enzyme were determined. Substrate specificity was examined by testing CARM1 activity with several histone H3-based peptides in a radiometric assay. Comparison of kcat/KM values reveals that methylation of H3R17 is preferred over that of H3R26. These effects are KM-driven as kcat values remain relatively constant for the peptides tested. Shortening the peptide at the C-terminus by five amino acid residues greatly reduced binding affinity, indicating distal residues may contribute to substrate binding. CARM1 appears to bind monomethylated peptides with an affinity similar to that of unmethylated peptides. Monitoring of the CARM1-dependent production of monomethylated and dimethylated peptides over time by self-assembled monolayer and matrix-assisted laser desorption ionization mass spectrometry revealed that methylation by CARM1 is distributive. Additionally, dead-end and product inhibition studies suggest CARM1 conforms to a random sequential kinetic mechanism. By defining the kinetic properties and mechanism of CARM1, these studies may aid in the development of small molecule CARM1 inhibitors.

Characterization of the Enzymatic Activity of SETDB1 and Its 1:1 Complex with ATF7IP.

The protein methyltransferase (PMT) SETDB1 is a strong candidate oncogene in melanoma and lung carcinomas. SETDB1 methylates lysine 9 of histone 3 (H3K9), utilizing S-adenosylmethionine (SAM) as the methyl donor and its catalytic activity, has been reported to be regulated by a partner protein ATF7IP. Here, we examine the contribution of ATF7IP to the in vitro activity and substrate specificity of SETDB1. SETDB1 and ATF7IP were co-expressed and 1:1 stoichiometric complexes were purified for comparison against SETDB1 enzyme alone. We employed both radiometric flashplate-based and SAMDI mass spectrometry assays to follow methylation on histone H3 15-mer peptides, where lysine 9 was either unmodified, monomethylated, or dimethylated. Results show that SETDB1 and the SETDB1:ATF7IP complex efficiently catalyze both monomethylation and dimethylation of H3K9 peptide substrates. The activity of the binary complex was 4-fold lower than SETDB1 alone. This difference was due to a decrease in the value of kcat as the substrate KM values were comparable between SETDB1 and the SETDB1:ATF7IP complex. H3K9 methylation by SETDB1 occurred in a distributive manner, and this too was unaffected by the presence of ATF7IP. This finding is important as H3K9 can be methylated by HMTs other than SETDB1 and a distributive mechanism would allow for interplay between multiple HMTs on H3K9. Our results indicate that ATF7IP does not directly modulate SETDB1 catalytic activity, suggesting alternate roles, such as affecting cellular localization or mediating interaction with additional binding partners.

Crystallographic structure of a small molecule SIRT1 activator-enzyme complex.

SIRT1, the founding member of the mammalian family of seven NAD(+)-dependent sirtuins, is composed of 747 amino acids forming a catalytic domain and extended N- and C-terminal regions. We report the design and characterization of an engineered human SIRT1 construct (mini-hSIRT1) containing the minimal structural elements required for lysine deacetylation and catalytic activation by small molecule sirtuin-activating compounds (STACs). Using this construct, we solved the crystal structure of a mini-hSIRT1-STAC complex, which revealed the STAC-binding site within the N-terminal domain of hSIRT1. Together with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis using full-length hSIRT1, these data establish a specific STAC-binding site and identify key intermolecular interactions with hSIRT1. The determination of the interface governing the binding of STACs with human SIRT1 facilitates greater understanding of STAC activation of this enzyme, which holds significant promise as a therapeutic target for multiple human diseases.

Quantitative analysis of receptor tyrosine kinase-effector coupling at functionally relevant stimulus levels.

A major goal of current signaling research is to develop a quantitative understanding of how receptor activation is coupled to downstream signaling events and to functional cellular responses. Here, we measure how activation of the RET receptor tyrosine kinase on mouse neuroblastoma cells by the neurotrophin artemin (ART) is quantitatively coupled to key downstream effectors. We show that the efficiency of RET coupling to ERK and Akt depends strongly on ART concentration, and it is highest at the low (∼100 pM) ART levels required for neurite outgrowth. Quantitative discrimination between ERK and Akt pathway signaling similarly is highest at this low ART concentration. Stimulation of the cells with 100 pM ART activated RET at the rate of ∼10 molecules/cell/min, leading at 5-10 min to a transient peak of ∼150 phospho-ERK (pERK) molecules and ∼50 pAkt molecules per pRET, after which time the levels of these two signaling effectors fell by 25-50% while the pRET levels continued to slowly rise. Kinetic experiments showed that signaling effectors in different pathways respond to RET activation with different lag times, such that the balance of signal flux among the different pathways evolves over time. Our results illustrate that measurements using high, super-physiological growth factor levels can be misleading about quantitative features of receptor signaling. We propose a quantitative model describing how receptor-effector coupling efficiency links signal amplification to signal sensitization between receptor and effector, thereby providing insight into design principles underlying how receptors and their associated signaling machinery decode an extracellular signal to trigger a functional cellular outcome.

Evidence for a common mechanism of SIRT1 regulation by allosteric activators.

A molecule that treats multiple age-related diseases would have a major impact on global health and economics. The SIRT1 deacetylase has drawn attention in this regard as a target for drug design. Yet controversy exists around the mechanism of sirtuin-activating compounds (STACs). We found that specific hydrophobic motifs found in SIRT1 substrates such as PGC-1α and FOXO3a facilitate SIRT1 activation by STACs. A single amino acid in SIRT1, Glu(230), located in a structured N-terminal domain, was critical for activation by all previously reported STAC scaffolds and a new class of chemically distinct activators. In primary cells reconstituted with activation-defective SIRT1, the metabolic effects of STACs were blocked. Thus, SIRT1 can be directly activated through an allosteric mechanism common to chemically diverse STACs.

Allosteric activation via kinetic control: potassium accelerates a conformational change in IMP dehydrogenase.

Allosteric activators are generally believed to shift the equilibrium distribution of enzyme conformations to favor a catalytically productive structure; the kinetics of conformational exchange is seldom addressed. Several observations suggested that the usual allosteric mechanism might not apply to the activation of IMP dehydrogenase (IMPDH) by monovalent cations. Therefore, we investigated the mechanism of K(+) activation in IMPDH by delineating the kinetic mechanism in the absence of monovalent cations. Surprisingly, the K(+) dependence of k(cat) derives from the rate of flap closure, which increases by ≥65-fold in the presence of K(+). We performed both alchemical free energy simulations and potential of mean force calculations using the orthogonal space random walk strategy to computationally analyze how K(+) accelerates this conformational change. The simulations recapitulate the preference of IMPDH for K(+), validating the computational models. When K(+) is replaced with a dummy ion, the residues of the K(+) binding site relax into ordered secondary structure, creating a barrier to conformational exchange. K(+) mobilizes these residues by providing alternate interactions for the main chain carbonyls. Potential of mean force calculations indicate that K(+) changes the shape of the energy well, shrinking the reaction coordinate by shifting the closed conformation toward the open state. This work suggests that allosteric regulation can be under kinetic as well as thermodynamic control.

SIRT1 activation by small molecules: kinetic and biophysical evidence for direct interaction of enzyme and activator.

SIRT1 is a protein deacetylase that has emerged as a therapeutic target for the development of activators to treat diseases of aging. SIRT1-activating compounds (STACs) have been developed that produce biological effects consistent with direct SIRT1 activation. At the molecular level, the mechanism by which STACs activate SIRT1 remains elusive. In the studies reported herein, the mechanism of SIRT1 activation is examined using representative compounds chosen from a collection of STACs. These studies reveal that activation of SIRT1 by STACs is strongly dependent on structural features of the peptide substrate. Significantly, and in contrast to studies reporting that peptides must bear a fluorophore for their deacetylation to be accelerated, we find that some STACs can accelerate the SIRT1-catalyzed deacetylation of specific unlabeled peptides composed only of natural amino acids. These results, together with others of this study, are at odds with a recent claim that complex formation between STACs and fluorophore-labeled peptides plays a role in the activation of SIRT1 (Pacholec, M., Chrunyk, B., Cunningham, D., Flynn, D., Griffith, D., Griffor, M., Loulakis, P., Pabst, B., Qiu, X., Stockman, B., Thanabal, V., Varghese, A., Ward, J., Withka, J., and Ahn, K. (2010) J. Biol. Chem. 285, 8340-8351). Rather, the data suggest that STACs interact directly with SIRT1 and activate SIRT1-catalyzed deacetylation through an allosteric mechanism.

The structural basis of Cryptosporidium -specific IMP dehydrogenase inhibitor selectivity.

Cryptosporidium parvum is a potential biowarfare agent, an important AIDS pathogen, and a major cause of diarrhea and malnutrition. No vaccines or effective drug treatment exist to combat Cryptosporidium infection. This parasite relies on inosine 5'-monophosphate dehydrogenase (IMPDH) to obtain guanine nucleotides, and inhibition of this enzyme blocks parasite proliferation. Here, we report the first crystal structures of CpIMPDH. These structures reveal the structural basis of inhibitor selectivity and suggest a strategy for further optimization. Using this information, we have synthesized low-nanomolar inhibitors that display 10(3) selectivity for the parasite enzyme over human IMPDH2.

A kinetic alignment of orthologous inosine-5'-monophosphate dehydrogenases.

IMP dehydrogenase (IMPDH) catalyzes two very different chemical transformations, a dehydrogenase reaction and a hydrolysis reaction. The enzyme toggles between the open conformation required for the dehydrogenase reaction and the closed conformation of the hydrolase reaction by moving a mobile flap into the NAD site. Despite these multiple functional constraints, the residues of the flap and NAD site are highly diverged, and the equilibrium between open and closed conformations ( K c ) varies widely. In order to understand how differences in the dynamic properties of the flap influence the catalytic cycle, we have delineated the kinetic mechanism of IMPDH from the pathogenic protozoan parasite Cryptosporidium parvum ( CpIMPDH), which was obtained from a bacterial source through horizontal gene transfer, and its host counterpart, human IMPDH type 2 (hIMPDH2). Interestingly, the intrinsic binding energy of NAD (+) differentially distributes across the dinucleotide binding sites of these two enzymes as well as in the previously characterized IMPDH from Tritrichomonas foetus ( TfIMPDH). Both the dehydrogenase and hydrolase reactions display significant differences in the host and parasite enzymes, in keeping with the phylogenetic and structural divergence of their active sites. Despite large differences in K c , the catalytic power of both the dehydrogenase and hydrolase conformations are similar in CpIMPDH and TfIMPDH. This observation suggests that the closure of the flap simply sets the stage for catalysis rather than plays a more active role in the chemical transformation. This work provides the essential mechanistic framework for drug discovery.

New ways to target old receptors.

Numerous important drugs target cytokines and growth factors or their receptors. Our understanding of the molecular mechanisms governing receptor activation and signaling has lagged in key areas, however, limiting drug discovery efforts to relatively few basic strategies. Recently, substantial progress has been made on several aspects of this problem. These include improved methods for establishing the mechanism of receptor activation, a clearer understanding of the biochemical basis for differential signaling by ligands that act through a common receptor, new methods for measuring the affinities of steps in receptor activation on live cells, and progress toward a systems level understanding of receptor signaling. These advances are providing a new understanding of the function of these receptors that presents opportunities for the development of improved drugs.

Targeting a prokaryotic protein in a eukaryotic pathogen: identification of lead compounds against cryptosporidiosis.

Cryptosporidium parvum is an important human pathogen and potential bioterrorism agent. No vaccines exist against C. parvum, the drugs currently approved to treat cryptosporidiosis are ineffective, and drug discovery is challenging because the parasite cannot be maintained continuously in cell culture. Mining the sequence of the C. parvum genome has revealed that the only route to guanine nucleotides is via inosine-5'-monophosphate dehydrogenase (IMPDH). Moreover, phylogenetic analysis suggests that the IMPDH gene was obtained from bacteria by lateral gene transfer. Here we exploit the unexpected evolutionary divergence of parasite and host enzymes by designing a high-throughput screen to target the most diverged portion of the IMPDH active site. We have identified four parasite-selective IMPDH inhibitors that display antiparasitic activity with greater potency than paromomycin, the current gold standard for anticryptosporidial activity.

Analogs of 1-phosphonooxy-2,2-dihydroxy-3-oxo-5-(methylthio)pentane, an acyclic intermediate in the methionine salvage pathway: a new preparation and characterization of activity with E1 enolase/phosphatase from Klebsiella oxytoca.

The methionine salvage pathway allows the in vivo recovery of the methylthio moiety of methionine upon the formation of methylthioadenosine (MTA) from S-adenosylmethionine (SAM). The Fe(II)-containing form of acireductone dioxygenase (ARD) catalyzes the penultimate step in the pathway in Klebsiella oxytoca, the oxidative cleavage of the acireductone 1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene (2) by dioxygen to give formate and 2-oxo-4-(methylthio)butyrate (3). The Ni(II)-bound form (Ni-ARD) catalyzes an off-pathway shunt, forming 3-(methylthio)propionate (4), carbon monoxide, and formate. Acireductone 2 is formed by the action of another enzyme, E1 enolase/phosphatase, on precursor 1-phosphonooxy-2,2-dihydroxy-3-oxo-5-methylthiopentane (1). Simple syntheses of several analogs of 1 are described, and their activity as substrates for E1 enolase/phosphatase characterized. A new bacterial overexpression system and purification procedure for E1, a member of the haloacid dehalogenase (HAD) superfamily, is described, and further characterization of the enzyme presented.

Substitution of the conserved Arg-Tyr dyad selectively disrupts the hydrolysis phase of the IMP dehydrogenase reaction.

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP via the covalent E-XMP* intermediate (E-XMP*), with the concomitant reduction of NAD(+). Hydrolysis of E-XMP* is rate-limiting, and the catalytic base required for this step has not been identified. An X-ray crystal structure of Tritrichomonas foetus IMPDH with mizoribine monophosphate (MZP) reveals a novel closed conformation in which a mobile flap occupies the NAD(+)/NADH site [Gan, L., Seyedsayamdost, M. R., Shuto, S., Matsuda, A., Petsko, G. A., and Hedstrom, L. (2003) Biochemistry 42, 857-863]. In this complex, a water molecule is coordinated between flap residues Arg418 and Tyr419 and MZP in a geometry that resembles the transition state for hydrolysis of E-XMP*, which suggests that the Arg418-Tyr419 dyad activates water. We constructed and characterized two point mutants, Arg418Ala and Tyr419Phe, to probe the role of the Arg418-Tyr419 dyad in the IMPDH reaction. Arg418Ala and Tyr419Phe decrease k(cat) by factors of 500 and 10, respectively, but have no effect on hydride transfer or NADH release. In addition, the mutants display increased solvent isotope effects and increased levels of steady-state accumulation of E-XMP*. Inhibitor analysis indicates that the mutations destabilize the closed conformation, but this effect can account for a decrease in k(cat) of no more than a factor of 2. These observations demonstrate that both the Arg418Ala and Tyr419Phe mutations selectively impair hydrolysis of E-XMP* by disrupting the chemical transformation. Moreover, since the effects of the Tyr419Phe mutation are comparatively small, these experiments suggest that Arg418 acts as the base to activate water.