Structural and Functional Consequences of Three Cancer-Associated Mutations of the Oncogenic Phosphatase SHP2.

The proto-oncogene PTPN11 encodes a cytoplasmic protein tyrosine phosphatase, SHP2, which is required for normal development and sustained activation of the Ras-MAPK signaling pathway. Germline mutations in SHP2 cause developmental disorders, and somatic mutations have been identified in childhood and adult cancers and drive leukemia in mice. Despite our knowledge of the PTPN11 variations associated with pathology, the structural and functional consequences of many disease-associated mutants remain poorly understood. Here, we combine X-ray crystallography, small-angle X-ray scattering, and biochemistry to elucidate structural and mechanistic features of three cancer-associated SHP2 variants harboring single point mutations within the N-SH2:PTP interdomain autoinhibitory interface. Our findings directly compare the impact of each mutation on autoinhibition of the phosphatase and advance the development of structure-guided and mutation-specific SHP2 therapies.

Enzyme-dependent lysine deprotonation in EZH2 catalysis.

Protein lysine methyltransferases (PKMTs) are key players in epigenetic regulation and have been associated with a variety of diseases, including cancers. The catalytic subunit of Polycomb Repressive Complex 2, EZH2 (EC 2.1.1.43), is a PKMT and a member of a family of SET domain lysine methyltransferases that catalyze the transfer of a methyl group from S-adenosyl-l-methionine to lysine 27 of histone 3 (H3K27). Wild-type (WT) EZH2 primarily catalyzes the mono- and dimethylation of H3K27; however, a clinically relevant active site mutation (Y641F) has been shown to alter the reaction specificity, dominantly catalyzing trimethylation of H3K27, and has been linked to tumor genesis and maintenance. Herein, we explore the chemical mechanism of methyl transfer by EZH2 and its Y641F mutant with pH-rate profiles and solvent kinetic isotope effects (sKIEs) using a short peptide derived from histone H3 [H3(21-44)]. A key component of the chemical reaction is the essential deprotonation of the ε-NH3(+) group of lysine to accommodate subsequent methylation. This deprotonation has been suggested by independent studies (1) to occur prior to binding to the enzyme (by bulk solvent) or (2) to be facilitated within the active site following binding, either (a) by the enzyme itself or (b) by a water molecule with access to the binding pocket. Our pH-rate and sKIE data best support a model in which lysine deprotonation is enzyme-dependent and at least partially rate-limiting. Furthermore, our experimental data are in agreement with prior computational models involving enzyme-dependent solvent deprotonation through a channel providing bulk solvent access to the active site. The mechanism of deprotonation and the rate-limiting catalytic steps appear to be unchanged between the WT and Y641F mutant enzymes, despite their activities being highly dependent on different substrate methylation states, suggesting determinants of substrate and product specificity in EZH2 are independent of catalytic events limiting the steady-state rate.

A transglutaminase homologue as a condensation catalyst in antibiotic assembly lines.

The unrelenting emergence of antibiotic-resistant bacterial pathogens demands the investigation of antibiotics with new modes of action. The pseudopeptide antibiotic andrimid is a nanomolar inhibitor of the bacterial acetyl-CoA carboxylase that catalyses the first committed step in prokaryotic fatty acid biosynthesis. Recently, the andrimid (adm) biosynthetic gene cluster was isolated and heterologously expressed in Escherichia coli. This establishes a heterologous biological host in which to rapidly probe features of andrimid formation and to use biosynthetic engineering to make unnatural variants of this important and promising new class of antibiotics. Bioinformatic analysis of the adm cluster revealed a dissociated biosynthetic assembly system lacking canonical amide synthases between the first three carrier protein domains. Here we report that AdmF, a transglutaminase (TGase) homologue, catalyses the formation of the first amide bond, an N-acyl-beta-peptide link, in andrimid biosynthesis. Hence, AdmF is a newly discovered biosynthetic enzyme that acts as a stand-alone amide synthase between protein-bound, thiotemplated substrates in an antibiotic enzymatic assembly line. TGases (enzyme class (EC) 2.3.2.13) normally catalyse the cross-linking of (poly)peptides by creating isopeptidic bonds between the gamma-carboxamide group of a glutamine side chain of one protein and various amine donors, including lysine side chains. To the best of our knowledge, the present study constitutes the first report of a TGase-like enzyme recruited for the assembly of an antibiotic. Moreover, genome mining using the AdmF sequence yielded additional TGases in unassigned natural product biosynthetic pathways. With many more microbial genomes being sequenced, such a strategy could potentially unearth biosynthetic pathways producing new classes of antibiotics.

Andrimid producers encode an acetyl-CoA carboxyltransferase subunit resistant to the action of the antibiotic.

Andrimid is a hybrid nonribosomal peptide-polyketide antibiotic that blocks the carboxyl-transfer reaction of bacterial acetyl-CoA carboxylase (ACC) and thereby inhibits fatty acid biosynthesis with submicromolar potency. The andrimid biosynthetic gene cluster from Pantoea agglomerans encodes an admT gene with homology to the acetyl-CoA carboxyltransferase (CT) beta-subunit gene accD. Escherichia coli cells overexpressing admT showed resistance to andrimid. Co-overproduction of AdmT with E. coli CT alpha-subunit AccA allowed for the in vitro reconstitution of an active heterologous tetrameric CT A(2)T(2) complex. A subsequent andrimid-inhibition assay revealed an IC(50) of 500 nM for this hybrid A(2)T(2) in contrast to that of 12 nM for E. coli CT A(2)D(2). These results validated that AdmT is an AccD homolog that confers resistance in the andrimid producer. Mutagenesis studies guided by the x-ray crystal structure of the E. coli A(2)D(2) complex disclosed a single amino acid mutation of AdmT (L203M) responsible for 5-fold andrimid sensitivity (IC(50) = 100 nM). Complementarily, the E. coli AccD mutant M203L became 5-fold more resistant in the CT assays. This observation allowed for bioinformatic identification of several Vibrio cholerae strains in which accD genes encode the Met<-->Leu switches, and their occurrences correlate predictively with sensitivities to andrimid in vivo.

Identification of TNO155, an Allosteric SHP2 Inhibitor for the Treatment of Cancer.

SHP2 is a nonreceptor protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also plays an important role in the programed cell death pathway (PD-1/PD-L1). As an oncoprotein as well as a potential immunomodulator, controlling SHP2 activity is of high therapeutic interest. As part of our comprehensive program targeting SHP2, we identified multiple allosteric binding modes of inhibition and optimized numerous chemical scaffolds in parallel. In this drug annotation report, we detail the identification and optimization of the pyrazine class of allosteric SHP2 inhibitors. Structure and property based drug design enabled the identification of protein-ligand interactions, potent cellular inhibition, control of physicochemical, pharmaceutical and selectivity properties, and potent in vivo antitumor activity. These studies culminated in the discovery of TNO155, (3S,4S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine (1), a highly potent, selective, orally efficacious, and first-in-class SHP2 inhibitor currently in clinical trials for cancer.

Getting Your Hands Dirty Outside the Lab: A Practical Primer for Conducting Wearable Vibrotactile Haptics Research.

As haptics have become an ingrained part of our wearable experience, particularly through phones, smartwatches, and fitness trackers, significant research effort has been conducted to find new ways of using wearable haptics to convey information, especially while we are on-the-go. In this paper, instead of focusing on aspects of haptic information design, such as tacton encoding methods, actuators, and technical fabrication of devices, we address the more general recurring issues and "gotchas" that arise when moving from core haptic perceptual studies and in-lab wearable experiments to real world testing of wearable vibrotactile haptic systems. We summarize key issues for practitioners to take into account when designing and carrying out in-the-wild wearable haptic user studies, as well as for user studies in a lab environment that seek to simulate real-world conditions. We include not only examples from published work and commercial sources, but also hard-won illustrative examples derived from issues and failures from our own haptic studies. By providing a broad-based, accessible overview of recurring issues, we expect that both novice and experienced haptic researchers will find suggestions that will improve their own mobile wearable haptic studies.

beta-Hydroxylation of the aspartyl residue in the phytotoxin syringomycin E: characterization of two candidate hydroxylases AspH and SyrP in Pseudomonas syringae.

The pseudomonal phytotoxin syringomycin E and related nonribosomal peptides contain an L- threo-beta-hydroxyaspartyl residue at the eighth position of the lipodepsipeptide backbone as part of a conserved nonproteinogenic tripeptide motif. Informatic analysis of the P. syringae genome suggests only one putative non-heme iron hydroxylase, AspH. On heterologous expression in Escherichia coli AspH shows robust catalytic activity with free L-Asp and L-Asp thioesters to make beta-OH-Asp but yields the erythro diastereomer rather than the threo configuration that is found in syringomycin. Further analysis of the Syr gene cluster indicated that SyrP, previously annotated as the gene regulatory protein for the five-gene Syr cluster, is actually homologous to the known non-heme mononuclear iron hydroxylase TauD. Indeed, purified SyrP acts on Asp tethered as the protein-bound S-pantetheinyl thioester on the eighth module of the SyrE megasynthetase. The hydroxylation gives the anticipated L- threo-3-OH-Asp diastereomer found in syringomycin. The knockout of syrP abolishes the production of the mature syringomycin E, while knockout of aspH has no effect on syringomycin production.

Allosteric Mutant IDH1 Inhibitors Reveal Mechanisms for IDH1 Mutant and Isoform Selectivity.

Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1R132H mutation destabilizes an IDH1 "regulatory segment," which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.

Optimization of 3-Pyrimidin-4-yl-oxazolidin-2-ones as Allosteric and Mutant Specific Inhibitors of IDH1.

High throughput screening and subsequent hit validation identified 4-isopropyl-3-(2-((1-phenylethyl)amino)pyrimidin-4-yl)oxazolidin-2-one as a potent inhibitor of IDH1R132H. Synthesis of the four separate stereoisomers identified the (S,S)-diastereomer (IDH125, 1f) as the most potent isomer. This also showed reasonable cellular activity and excellent selectivity vs IDH1wt. Initial structure-activity relationship exploration identified the key tolerances and potential for optimization. X-ray crystallography identified a functionally relevant allosteric binding site amenable to inhibitors, which can penetrate the blood-brain barrier, and aided rational optimization. Potency improvement and modulation of the physicochemical properties identified (S,S)-oxazolidinone IDH889 (5x) with good exposure and 2-HG inhibitory activity in a mutant IDH1 xenograft mouse model.

Use of an Enactive Insole for Reducing the Risk of Falling on Different Types of Soil Using Vibrotactile Cueing for the Elderly.

BACKGROUND: Our daily activities imply displacements on various types of soil. For persons with gait disorder or losing functional autonomy, walking on some types of soil could be challenging because of the risk of falling it represents. METHODS: In this paper, we present, in a first part, the use of an enactive shoe for an automatic differentiation of several types of soil. In a second part, using a second improved prototype (an enactive insole), twelve participants with Parkinson's disease (PD) and nine age-matched controls have performed the Timed Up and Go (TUG) test on six types of soil with and without cueing. The frequency of the cueing was set at 10% above the cadence computed at the lower risk of falling (walking over the concrete). Depending on the cadence computed at the lower risk, the enactive insole activates a vibrotactile cueing aiming to improve gait and balance control. Finally, a risk index is computed using gait parameters in relation to given type of soil. RESULTS: The frequency analysis of the heel strike vibration allows the differentiation of various types of soil. The risk computed is associated to an appropriate rhythmic cueing in order to improve balance and gait impairment. The results show that a vibrotactile cueing could help to reduce the risk of falling. CONCLUSIONS: Firstly, this paper demonstrates the feasibility of reducing the risk of falling while walking on different types of soil using vibrotactile cueing. We found a significant difference and a significant decrease in the computed risks of falling for most of types of soil especially for deformable soils which can lead to fall. Secondly, heel strike provides an approximation of the impulse response of the soil that can be analyzed with time and frequency-domain modeling. From these analyses, an index is computed enabling differentiation the types of soil.

Allosteric Inhibition of SHP2: Identification of a Potent, Selective, and Orally Efficacious Phosphatase Inhibitor.

SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also purportedly plays an important role in the programmed cell death pathway (PD-1/PD-L1). Because it is an oncoprotein associated with multiple cancer-related diseases, as well as a potential immunomodulator, controlling SHP2 activity is of significant therapeutic interest. Recently in our laboratories, a small molecule inhibitor of SHP2 was identified as an allosteric modulator that stabilizes the autoinhibited conformation of SHP2. A high throughput screen was performed to identify progressable chemical matter, and X-ray crystallography revealed the location of binding in a previously undisclosed allosteric binding pocket. Structure-based drug design was employed to optimize for SHP2 inhibition, and several new protein-ligand interactions were characterized. These studies culminated in the discovery of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine (SHP099, 1), a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor.

High-throughput screening identifies inhibitors of the SARS coronavirus main proteinase.

The causative agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus, SARS-CoV. The main proteinase of SARS-CoV, 3CLpro, is an attractive target for therapeutics against SARS owing to its fundamental role in viral replication. We sought to identify novel inhibitors of 3CLpro to advance the development of appropriate therapies in the treatment of SARS. 3CLpro was cloned, expressed, and purified from the Tor2 isolate. A quenched fluorescence resonance energy transfer assay was developed for 3CLpro to screen the proteinase against 50,000 drug-like small molecules on a fully automated system. The primary screen identified 572 hits; through a series of virtual and experimental filters, this number was reduced to five novel small molecules that show potent inhibitory activity (IC50 = 0.5-7 microM) toward SARS-CoV 3CLpro.

The novolactone natural product disrupts the allosteric regulation of Hsp70.

The highly conserved 70 kDa heat shock proteins (Hsp70) play an integral role in proteostasis such that dysregulation has been implicated in numerous diseases. Elucidating the precise role of Hsp70 family members in the cellular context, however, has been hampered by the redundancy and intricate regulation of the chaperone network, and relatively few selective and potent tools. We have characterized a natural product, novolactone, that targets cytosolic and ER-localized isoforms of Hsp70 through a highly conserved covalent interaction at the interface between the substrate-binding and ATPase domains. Biochemical and structural analyses indicate that novolactone disrupts interdomain communication by allosterically inducing a conformational change in the Hsp70 protein to block ATP-induced substrate release and inhibit refolding activities. Thus, novolactone is a valuable tool for exploring the requirements of Hsp70 chaperones in diverse cellular contexts.

Synthesis and evaluation of keto-glutamine analogues as potent inhibitors of severe acute respiratory syndrome 3CLpro.

The 3C-like proteinase (3CL(pro)) of severe acute respiratory syndrome (SARS) coronavirus is a key target for structure-based drug design against this viral infection. The enzyme recognizes peptide substrates with a glutamine residue at the P1 site. A series of keto-glutamine analogues with a phthalhydrazido group at the alpha-position were synthesized and tested as reversible inhibitiors against SARS 3CL(pro). Attachment of tripeptide (Ac-Val-Thr-Leu) to these glutamine-based "warheads" generated significantly better inhibitors (4a-c, 8a-d) with IC(50) values ranging from 0.60 to 70 microM.

Structure-efficiency relationship of [1,2,4]triazol-3-ylamines as novel nicotinamide isosteres that inhibit tankyrases.

Tankyrases 1 and 2 are members of the poly(ADP-ribose) polymerase (PARP) family of enzymes that modulate Wnt pathway signaling. While amide- and lactam-based nicotinamide mimetics that inhibit tankyrase activity, such as XAV939, are well-known, herein we report the discovery and evaluation of a novel nicotinamide isostere that demonstrates selectivity over other PARP family members. We demonstrate the utilization of lipophilic efficiency-based structure-efficiency relationships (SER) to rapidly drive the evaluation of this series. These efforts led to a series of selective, cell-active compounds with solubility, physicochemical, and in vitro properties suitable for further optimization.

Identification of NVP-TNKS656: the use of structure-efficiency relationships to generate a highly potent, selective, and orally active tankyrase inhibitor.

Tankyrase 1 and 2 have been shown to be redundant, druggable nodes in the Wnt pathway. As such, there has been intense interest in developing agents suitable for modulating the Wnt pathway in vivo by targeting this enzyme pair. By utilizing a combination of structure-based design and LipE-based structure efficiency relationships, the core of XAV939 was optimized into a more stable, more efficient, but less potent dihydropyran motif 7. This core was combined with elements of screening hits 2, 19, and 33 and resulted in highly potent, selective tankyrase inhibitors that are novel three pocket binders. NVP-TNKS656 (43) was identified as an orally active antagonist of Wnt pathway activity in the MMTV-Wnt1 mouse xenograft model. With an enthalpy-driven thermodynamic signature of binding, highly favorable physicochemical properties, and high lipophilic efficiency, NVP-TNKS656 is a novel tankyrase inhibitor that is well suited for further in vivo validation studies.

[1,2,4]triazol-3-ylsulfanylmethyl)-3-phenyl-[1,2,4]oxadiazoles: antagonists of the Wnt pathway that inhibit tankyrases 1 and 2 via novel adenosine pocket binding.

The Wnt signaling pathway is critical to the regulation of key cellular processes. When deregulated, it has been shown to play a crucial role in the growth and progression of multiple human cancers. The identification of small molecule modulators of Wnt signaling has proven challenging, largely due to the relative paucity of druggable nodes in this pathway. Several recent publications have identified small molecule inhibitors of the Wnt pathway, and tankyrase (TNKS) inhibition has been demonstrated to antagonize Wnt signaling via axin stabilization. Herein, we report the early hit assessment of a series of compounds previously reported to antagonize Wnt signaling. We report the biophysical, computational characterization, structure-activity relationship, and physicochemical properties of a novel series of [1,2,4]triazol-3-ylsulfanylmethyl)-3-phenyl-[1,2,4]oxadiazole inhibitors of TNKS1 and 2. Furthermore, a cocrystal structure of compound 24 complexed to TNKS1 demonstrates an alternate binding mode for PARP family member proteins that does not involve interactions with the nicotinamide binding pocket.

Gatekeeping versus promiscuity in the early stages of the andrimid biosynthetic assembly line.

The antibiotic andrimid, a nanomolar inhibitor of bacterial acetyl coenzyme A carboxylase, is generated on an unusual polyketide/nonribosomal peptide enzyme assembly line in that all thiolation (T) domains/small-molecule building stations are on separate proteins. In addition, a transglutaminase homologue is used to condense andrimid building blocks together on the andrimid assembly line. The first two modules of the andrimid assembly line yields an octatrienoyl-beta-Phe-thioester tethered to the AdmI T domain, with amide bond formation carried out by a free-standing transglutaminase homologue AdmF. Analysis of the aminomutase AdmH reveals its specific conversion from l-Phe to (S)-beta-Phe, which in turn is activated by AdmJ and ATP to form (S)-beta-Phe-aminoacyl-AMP. AdmJ then transfers the (S)-beta-Phe moiety to one of the free-standing T domains, AdmI, but not AdmA, which instead gets loaded with an octatrienoyl group by other enzymes. AdmF, the amide synthase, will accept a variety of acyl groups in place of the octatrienoyl donor if presented on either AdmA or AdmI. AdmF will also use either stereoisomer of phenylalanine or beta-Phe when presented on AdmA and AdmI, but not when placed on noncognate T domains. Further, we show the polyketide synthase proteins responsible for the polyunsaturated acyl cap can be bypassed in vitro with N-acetylcysteamine as a low-molecular-weight acyl donor to AdmF and also in vivo in an Escherichia coli strain bearing the andrimid biosynthetic gene cluster with a knockout in admA.

Characterization of a C-C bond hydrolase from Sphingomonas wittichii RW1 with novel specificities towards polychlorinated biphenyl metabolites.

Sphingomonas wittichii RW1 degrades chlorinated dibenzofurans and dibenzo-p-dioxins via meta cleavage. We used inverse PCR to amplify dxnB2, a gene encoding one of three meta-cleavage product (MCP) hydrolases identified in the organism that are homologues of BphD involved in biphenyl catabolism. Purified DxnB2 catalyzed the hydrolysis of 8-OH 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) approximately six times faster than for HOPDA at saturating substrate concentrations. Moreover, the specificity of DxnB2 for HOPDA (k(cat)/K(m) = 1.2 x 10(7) M(-1) s(-1)) was about half that of the BphDs of Burkholderia xenovorans LB400 and Rhodococcus globerulus P6, two potent polychlorinated biphenyl (PCB)-degrading strains. Interestingly, DxnB2 transformed 3-Cl and 4-OH HOPDAs, compounds that inhibit the BphDs and limit PCB degradation. DxnB2 had a higher specificity for 9-Cl HOPDA than for HOPDA but a lower specificity for 8-Cl HOPDA (k(cat)/K(m) = 1.7 x 10(6) M(-1) s(-1)), the chlorinated analog of 8-OH HOPDA produced during dibenzofuran catabolism. Phylogenetic analyses based on structure-guided sequence alignment revealed that DxnB2 belongs to a previously unrecognized class of MCP hydrolases, evolutionarily divergent from the BphDs although the physiological substrates of both enzyme types are HOPDAs. However, both classes of enzymes have mainly small hydrophobic residues lining the subsite that binds the C-6 phenyl of HOPDA, in contrast to the bulky hydrophobic residues (Phe106, Phe135, Trp150, and Phe197) found in the class II enzymes that prefer substrates possessing a C-6 alkyl. Thr196 and/or Asn203 appears to be an important determinant of specificity for DxnB2, potentially forming hydrogen bonds with the 8-OH substituent. This study demonstrates that the substrate specificities of evolutionarily divergent hydrolases may be useful for degrading mixtures of pollutants, such as PCBs.

Structures of ternary complexes of BphK, a bacterial glutathione S-transferase that reductively dechlorinates polychlorinated biphenyl metabolites.

Prokaryotic glutathione S-transferases are as diverse as their eukaryotic counterparts but are much less well characterized. BphK from Burkholderia xenovorans LB400 consumes two GSH molecules to reductively dehalogenate chlorinated 2-hydroxy-6-oxo-6-phenyl-2,4-dienoates (HOPDAs), inhibitory polychlorinated biphenyl metabolites. Crystallographic structures of two ternary complexes of BphK were solved to a resolution of 2.1A. In the BphK-GSH-HOPDA complex, GSH and HOPDA molecules occupy the G- and H-subsites, respectively. The thiol nucleophile of the GSH molecule is positioned for SN2 attack at carbon 3 of the bound HOPDA. The respective sulfur atoms of conserved Cys-10 and the bound GSH are within 3.0A, consistent with product release and the formation of a mixed disulfide intermediate. In the BphK-(GSH)2 complex, a GSH molecule occupies each of the two subsites. The three sulfur atoms of the two GSH molecules and Cys-10 are aligned suitably for a disulfide exchange reaction that would regenerate the resting enzyme and yield disulfide-linked GSH molecules. A second conserved residue, His-106, is adjacent to the thiols of Cys-10 and the GSH bound to the G-subsite and thus may stabilize a transition state in the disulfide exchange reaction. Overall, the structures support and elaborate a proposed dehalogenation mechanism for BphK and provide insight into the plasticity of the H-subsite.