Antibody-Drug Conjugates (ADCs) Derived from Interchain Cysteine Cross-Linking Demonstrate Improved Homogeneity and Other Pharmacological Properties over Conventional Heterogeneous ADCs.

Conventional antibody-drug conjugates (ADCs) are heterogeneous mixtures of chemically distinct molecules that vary in both drugs/antibody (DAR) and conjugation sites. Suboptimal properties of heterogeneous ADCs have led to new site-specific conjugation methods for improving ADC homogeneity. Most site-specific methods require extensive antibody engineering to identify optimal conjugation sites and introduce unique functional groups for conjugation with appropriately modified linkers. Alternative nonrecombinant methods have emerged in which bifunctional linkers are utilized to cross-link antibody interchain cysteines and afford ADCs containing four drugs/antibody. Although these methods have been shown to improve ADC homogeneity and stability in vitro, their effect on the pharmacological properties of ADCs in vivo is unknown. In order to determine the relative impact of interchain cysteine cross-linking on the therapeutic window and other properties of ADCs in vivo, we synthesized a derivative of the known ADC payload, MC-MMAF, that contains a bifunctional dibromomaleimide (DBM) linker instead of a conventional maleimide (MC) linker. The DBM-MMAF derivative was conjugated to trastuzumab and a novel anti-CD98 antibody to afford ADCs containing predominantly four drugs/antibody. The pharmacological properties of the resulting cross-linked ADCs were compared with analogous heterogeneous ADCs derived from conventional linkers. The results demonstrate that DBM linkers can be applied directly to native antibodies, without antibody engineering, to yield highly homogeneous ADCs via cysteine cross-linking. The resulting ADCs demonstrate improved pharmacokinetics, superior efficacy, and reduced toxicity in vivo compared to analogous conventional heterogeneous ADCs.

A mutation in TGFB3 associated with a syndrome of low muscle mass, growth retardation, distal arthrogryposis and clinical features overlapping with Marfan and Loeys-Dietz syndrome.

The transforming growth factor ß (TGF-ß) family of growth factors are key regulators of mammalian development and their dysregulation is implicated in human disease, notably, heritable vasculopathies including Marfan (MFS, OMIM #154700) and Loeys-Dietz syndromes (LDS, OMIM #609192). We described a syndrome presenting at birth with distal arthrogryposis, hypotonia, bifid uvula, a failure of normal post-natal muscle development but no evidence of vascular disease; some of these features overlap with MFS and LDS. A de novo mutation in TGFB3 was identified by exome sequencing. Several lines of evidence indicate the mutation is hypomorphic suggesting that decreased TGF-ß signaling from a loss of TGFB3 activity is likely responsible for the clinical phenotype. This is the first example of a mutation in the coding portion of TGFB3 implicated in a clinical syndrome suggesting TGFB3 is essential for both human palatogenesis and normal muscle growth.

On the chalcogenophilicity of mercury: evidence for a strong Hg-Se bond in [Tm(Bu(t))]HgSePh and its relevance to the toxicity of mercury.

One of the reasons for the toxic effects of mercury has been attributed to its influence on the biochemical roles of selenium. For this reason, it is important to understand details pertaining to the nature of Hg-Se interactions and this has been achieved by comparison of a series of mercury chalcogenolate complexes that are supported by tris(2-mercapto-1-t-butyl-imidazolyl)hydroborato ligation, namely [Tm(Bu(t))]HgEPh (E = S, Se, Te). In particular, X-ray diffraction studies on [Tm(Bu(t))]HgEPh demonstrate that although the Hg-S bonds involving the [Tm(Bu(t))] ligand are longer than the corresponding Cd-S bonds of [Tm(Bu(t))]CdEPh, the Hg-EPh bonds are actually shorter than the corresponding Cd-EPh bonds, an observation which indicates that the apparent covalent radii of the metals in these compounds are dependent on the nature of the bonds. Furthermore, the difference in Hg-EPh and Cd-EPh bond lengths is a function of the chalcogen and increases in the sequence S (0.010 A) < Se (0.035 A) < Te (0.057 A). This trend indicates that the chalcogenophilicity of mercury increases in the sequence S < Se < Te. Thus, while mercury is often described as being thiophilic, it is evident that it actually has a greater selenophilicity, a notion that is supported by the observation of facile selenolate transfer from zinc to mercury upon treatment of [Tm(Bu(t))]HgSCH(2)C(O)N(H)Ph with [Tm(Bu(t))]ZnSePh. The significant selenophilicity of mercury is in accord with the aforementioned proposal that one reason for the toxicity of mercury is associated with it reducing the bioavailability of selenium.

Synthesis, structure, and reactivity of two-coordinate mercury alkyl compounds with sulfur ligands: relevance to mercury detoxification.

The susceptibility of two-coordinate mercury alkyl compounds of the type X-Hg-R (where X is a monodentate sulfur donor) towards protolytic cleavage has been investigated as part of ongoing efforts to obtain information relevant to understanding the mechanism of action of the organomercurial lyase, MerB. Specifically, the reactivity of the two-coordinate mercury alkyl compounds PhSHgR, [mim(Bu(t))]HgR and {[Hmim(Bu(t))]HgR}(+) (Hmim(Bu(t)) = 2-mercapto-1-t-butylimidazole; R = Me, Et) towards PhSH was investigated, thereby demonstrating that the ability to cleave the Hg-C bond is very dependent on the nature of the system. For example, whereas the reaction of PhSHgMe with PhSH requires heating at 145 degrees C for several weeks to liberate CH(4), the analogous reaction of PhSHgEt with PhSH leads to evolution of C(2)H(6) over the course of 2 days at 100 degrees C. Furthermore, protolytic cleavage of the Hg-C bond by PhSH is promoted by Hmim(Bu(t)). For example, whereas the reaction of {[Hmim(Bu(t))]HgEt}(+) with PhSH eliminates C(2)H(6) at elevated temperatures, the protolytic cleavage occurs over a period of 2 days at room temperature in the presence of Hmim(Bu(t)). The ability of Hmim(Bu(t)) to promote the protolytic cleavage is interpreted in terms of the formation of a higher coordinate species {[Hmim(Bu(t))](n)HgR}(+) that is more susceptible to Hg-C bond cleavage than is two-coordinate {[Hmim(Bu(t))]HgR}(+). These observations support the notion that access to a species with a coordination number greater than two is essential for efficient activity of MerB.

Ligand reactivity in diarylamido/bis(phosphine) PNP complexes of Mn(CO)3 and Re(CO)3.

The syntheses of meridionally ligated tricarbonyl complexes (PNP)Mn(CO)(3) and (PNP)Re(CO)(3) are described (PNP = [2-P(CHMe(2))(2)-4-MeC(6)H(3)](2)N(-)). Cyclic voltammograms show reversible one-electron redox couples for both parent compounds (-0.34 V vs Cp(2)Fe(0/+) for (PNP)Mn(CO)(3), -0.25 V vs Cp(2)Fe(0/+) for (PNP)Re(CO)(3)), and chemical oxidation with AgOTf results in formation of the corresponding paramagnetic triflate salts [(PNP)Mn(CO)(3)]OTf and [(PNP)Re(CO)(3)]OTf. Electron paramagnetic resonance spectra and computational results indicate that this event is primarily ligand centered; allylation of the organic ligand moiety of [(PNP)Mn(CO)(3)]OTf with allyltributylstannane is consistent with this assignment. The oxidation (PNP)Mn(CO)(3) to [(PNP)Mn(CO)(3)]OTf results in a shift in average CO stretching frequency of 30 cm(-1); protonation of (PNP)Mn(CO)(3) with TfOH to form [(PNHP)Mn(CO)(3)]OTf results in a similar magnitude shift.

Monovalent indium in a sulfur-rich coordination environment: synthesis, structure and reactivity of tris(2-mercapto-1-tert-butylimidazolyl)hydroborato indium, [TmBut]In.

[Tm(Bu(t))]In, the first structurally-characterized monovalent indium compound that features a sulfur-rich coordination environment, has been synthesized via treatment of InCl with [Tm(Bu(t))]K; in contrast to the thallium counterpart, the lone pair of [Tm(Bu(t))]In is a site of reactivity, thereby allowing formation of [Tm(Bu(t))]In-->B(C(6)F(5))(3) and [Tm(Bu(t))]In(kappa(2)-S(4)) upon treatment with B(C(6)F(5))(3) and S(8), respectively.

Molecular structures of thimerosal (Merthiolate) and other arylthiolate mercury alkyl compounds.

The molecular structure of sodium ethylmercury thiosalicylate (also known as thimerosal and Merthiolate) and related arylthiolate mercury alkyl compounds, namely PhSHgMe and PhSHgEt, have been determined by single crystal X-ray diffraction. (1)H NMR spectroscopic studies indicate that the appearance of the (199)Hg mercury satellites of the ethyl group of thimerosal is highly dependent on the magnetic field and the viscosity of the solvent as a consequence of relaxation due to chemical shift anisotropy.

An ImmunoSignature test distinguishes Trypanosoma cruzi, hepatitis B, hepatitis C and West Nile virus seropositivity among asymptomatic blood donors.

BACKGROUND: The complexity of the eukaryotic parasite Trypanosoma (T.) cruzi manifests in its highly dynamic genome, multi-host life cycle, progressive morphologies and immune-evasion mechanisms. Accurate determination of infection or Chagas' disease activity and prognosis continues to challenge researchers. We hypothesized that a diagnostic platform with higher ligand complexity than previously employed may hold value. METHODOLOGY: We applied the ImmunoSignature Technology (IST) for the detection of T. cruzi-specific antibodies among healthy blood donors. IST is based on capturing the information in an individual's antibody repertoire by exposing their peripheral blood to a library of >100,000 position-addressable, chemically-diverse peptides. PRINCIPAL FINDINGS: Initially, samples from two Chagas cohorts declared positive or negative by bank testing were studied. With the first cohort, library-peptides displaying differential binding signals between T. cruzi sero-states were used to train an algorithm. A classifier was fixed and tested against the training-independent second cohort to determine assay performance. Next, samples from a mixed cohort of donors declared positive for Chagas, hepatitis B, hepatitis C or West Nile virus were assayed on the same library. Signals were used to train a single algorithm that distinguished all four disease states. As a binary test, the accuracy of predicting T. cruzi seropositivity by IST was similar, perhaps modestly reduced, relative to conventional ELISAs. However, the results indicate that information beyond determination of seropositivity may have been captured. These include the identification of cohort subclasses, the simultaneous detection and discerning of other diseases, and the discovery of putative new antigens. CONCLUSIONS & SIGNIFICANCE: The central outcome of this study established IST as a reliable approach for specific determination of T. cruzi seropositivity versus disease-free individuals or those with other diseases. Its potential contribution for monitoring and controlling Chagas lies in IST's delivery of higher resolution immune-state readouts than obtained with currently-used technologies. Despite the complexity of the ligand presentation and large quantitative readouts, performing an IST test is simple, scalable and reproducible.

Synthesis and structural characterization of tris(2-seleno-1-mesitylimidazolyl) hydroborato complexes: a new type of strongly electron donating tripodal selenium ligand.

A new tripodal ligand that features three selenium donors, namely the tris(2-seleno-1-mesitylimidazolyl)hydroborato ligand, [Tse(Mes)], has been constructed via the reaction of KBH(4) with 1-mesitylimidazole-2-selone; comparison of the IR spectroscopic data of [Tse(Mes)]Re(CO)(3) with those of a variety of related LRe(CO)(3) complexes demonstrates that the [Tse(Mes)] ligand is more strongly electron donating than Cp, Cp*, [Tp], [Tp(Me(2))] and [Tm(Mes)] ligands.

Thiolate exchange in [TmR]ZnSR' complexes and relevance to the mechanisms of thiolate alkylation reactions involving zinc enzymes and proteins.

The zinc and cadmium thiolate complexes [TmBut]MSCH2C(O)N(H)Ph (M = Zn, Cd) may be obtained via treatment of the respective methyl complex [TmBut]MMe with PhN(H)C(O)CH2SH. The molecular structure of [TmBut]ZnSCH2C(O)N(H)Ph has been determined by X-ray diffraction, thereby demonstrating the presence of an intramolecular N-H S hydrogen bond between the amide N-H group and thiolate sulfur atom. [TmBut]ZnSCH2C(O)N(H)Ph mimics the function of the Ada DNA repair protein by undergoing alkylation with MeI to give [TmBut]ZnI and MeSCH2C(O)N(H)Ph. A series of crossover experiments and 1H NMR magnetization transfer studies establish that thiolate exchange between [TmR]ZnSR' derivatives is facile in this system, an observation that supports the previous suggestion that the alkylation of [TmPh]ZnSCH2C(O)N(H)Ph by MeI may proceed via a sequence that involves dissociation of [PhN(H)C(O)CH2S]-.

Methyl, hydrochalcogenido, and phenylchalcogenolate complexes of zinc in a sulfur rich coordination environment: syntheses and structural characterization of the tris(2-mercapto-1-tert-butylimidazolyl)hydroboratozinc complexes [TmBut]ZnMe, [TmBut]ZnEH (E = S, Se) and [TmBut]ZnEPh (E = O, S, Se, Te).

A series of hydrochalcogenido and phenylchalcogenolate complexes of zinc supported by tris(2-mercapto-1-tert-butylimidazolyl)hydroborato ligation, [Tm(Bu(t))]ZnEH (E = S, Se) and [Tm(Bu(t))]ZnEPh (E = O, S, Se, Te) have been synthesized from [Tm(Bu(t))]ZnMe; structural characterization by X-ray diffraction indicates that the variation in Zn-E bonding as a function of the chalcogen is significantly different from that in other series of metal-chalcogenolate compounds.

Defining estrogenic mechanisms of bisphenol A analogs through high throughput microscopy-based contextual assays.

Environmental exposures to chemically heterogeneous endocrine-disrupting chemicals (EDCs) mimic or interfere with hormone actions and negatively affect human health. Despite public interest and the prevalence of EDCs in the environment, methods to mechanistically classify these diverse chemicals in a high throughput (HT) manner have not been actively explored. Here, we describe the use of multiparametric, HT microscopy-based platforms to examine how a prototypical EDC, bisphenol A (BPA), and 18 poorly studied BPA analogs (BPXs), affect estrogen receptor (ER). We show that short exposure to BPA and most BPXs induces ERα and/or ERß loading to DNA changing target gene transcription. Many BPXs exhibit higher affinity for ERß and act as ERß antagonists, while they act largely as agonists or mixed agonists and antagonists on ERα. Finally, despite binding to ERs, some BPXs exhibit lower levels of activity. Our comprehensive view of BPXs activities allows their classification and the evaluation of potential harmful effects. The strategy described here used on a large-scale basis likely offers a faster, more cost-effective way to identify safer BPA alternatives.

Decarbonylation of ethanol to methane, carbon monoxide and hydrogen by a [PNP]Ir complex.

The putative three-coordinate Ir(i) PNP(Pr(i)) (PNP(Pr(i)) = [N{2-P(CHMe(2))(2)-4-MeC(6)H(3)}(2)](-)) pincer complex decarbonylates ethanol to yield methane, hydrogen and [PNP(Pr(i))]Ir(CO). The mechanism involves the isolable trans-[PNP(Pr(i))]Ir(H)(Me)(CO), which is susceptible to photochemical reductive elimination of methane.

Cleaving mercury-alkyl bonds: a functional model for mercury detoxification by MerB.

The extreme toxicity of organomercury compounds that are found in the environment has focused attention on the mechanisms of action of bacterial remediating enzymes. We describe facile room-temperature protolytic cleavage by a thiol of the Hg-C bond in mercury-alkyl compounds that emulate the structure and function of the organomercurial lyase MerB. Specifically, the tris(2-mercapto-1-t-butylimidazolyl)hydroborato ligand [Tm(Bu(t))], which features three sulfur donors, has been used to synthesize [Tm(Bu(t))]HgR alkyl compounds (R = methyl or ethyl) that react with phenylthiol (PhSH) to yield [Tm(Bu(t))]HgSPh and RH. Although [Tm(Bu(t))]HgR compounds exist as linear two-coordinate complexes in the solid state, 1H nuclear magnetic resonance spectroscopy indicates that the complexes exist in rapid equilibrium with their higher-coordinate [kappa2-Tm(Bu(t))]HgR and [kappa3-Tm(Bu(t))]HgR isomers in solution. Facile access to a higher-coordinate species is proposed to account for the exceptional reactivity of [Tm(Bu(t))]HgR relative to that of other two-coordinate mercury-alkyl compounds.

Applications of bis(1-R-imidazol-2-yl)disulfides and diselenides as ligands for main-group and transition metals: kappa2-(N,N) coordination, S-S bond cleavage, and S-S/E-E (E = S, Se) bond metathesis reactions.

Bis(1-R-imidazol-2-yl)disulfides, (mim(R))2 (R = Ph, Bu(t)), and diselenides, (seim(Mes))2, serve as bidentate N,N-donor ligands for main-group and transition metals. For example, [kappa2-(mim(Bu)(t))2]MCl2 (M = Fe, Co, Ni, Zn), [kappa2-(mim(Ph))2]MCl2 (M = Co, Zn), [kappa2-(mim(Bu)(t))2]CuX (X = Cl, I), and [kappa2-(seim(Mes))2]MCl2 (M = Fe, Co, Ni) are obtained by treatment of (mim(Bu)(t))2 or (seim(Mes))2 with the respective metal halide and have been structurally characterized by X-ray diffraction. On the other hand, the zerovalent nickel complex Ni(PMe3)4 effects cleavage of the disulfide bond of (mim(Bu)(t))2 to give square-planar trans-Ni(PMe3)2(mim(Bu)(t))2 in which the (mim(Bu)(t)) ligands coordinate via nitrogen rather than sulfur, a most uncommon coordination mode for this class of ligands. Although [kappa2-(mim(R))2]MCl2 (M = Fe, Co, Ni, Zn) are not subject to homolytic cleavage of the S-S bond because the tetravalent state is not readily accessible, the observation that [kappa2-(mimPh)2]CoCl2 and [kappa2-(mim(Bu)(t))2]CoCl2 form an equilibrium mixture with the asymmetric disulfide [kappa2-(mim(Ph))(mim(Bu)(t))]CoCl2 indicates that S-S bond cleavage via another mechanism is possible. Likewise, metathesis between disulfide and diselenide ligands is observed in the formation of [kappa2-(mim(Bu)(t))(seim(Mes))]CoCl2 upon treatment of [kappa2-(mim(Bu)(t))2]CoCl2 with [kappa2-(seim(Mes))2]CoCl2.

Identification of NVP-TAE684, a potent, selective, and efficacious inhibitor of NPM-ALK.

Constitutive overexpression and activation of NPM-ALK fusion protein [t(2:5)(p23;q35)] is a key oncogenic event that drives the survival and proliferation of anaplastic large-cell lymphomas (ALCLs). We have identified a highly potent and selective small-molecule ALK inhibitor, NVP-TAE684, which blocked the growth of ALCL-derived and ALK-dependent cell lines with IC(50) values between 2 and 10 nM. NVP-TAE684 treatment resulted in a rapid and sustained inhibition of phosphorylation of NPM-ALK and its downstream effectors and subsequent induction of apoptosis and cell cycle arrest. In vivo, NVP-TAE684 suppressed lymphomagenesis in two independent models of ALK-positive ALCL and induced regression of established Karpas-299 lymphomas. NVP-TAE684 also induced down-regulation of CD30 expression, suggesting that CD30 may be used as a biomarker of therapeutic NPM-ALK kinase activity inhibition.

Methyl and arylchalcogenolate complexes of cadmium in a sulfur rich coordination environment: syntheses and structural characterization of the tris(2-mercapto-1-tert-butylimidazolyl)hydroborato cadmium complexes [Tm(Bu(t))]CdMe, and [Tm(Bu(t))]CdEAr (E = O, S, Se, Te) and analysis of the bonding in chalcogenolate compounds.

A series of arylchalcogenolate complexes of cadmium supported by tris(2-mercapto-1-tert-butylimidazolyl)hydroborato ligation, namely [Tm(Bu(t))]CdEAr (EAr = OC(6)H(3)Ph(2), SPh, SePh, TePh), has been synthesized from [Tm(Bu(t))]CdMe; structural characterization by X-ray diffraction indicates that the variation in Cd-EAr bond lengths is similar to that of Zn-EAr and correlates closely with the covalent radius of the chalcogen, in marked contrast to the large variation in M-OAr and M-SAr bond lengths observed for other metals (Zr and Sm).

Reactivity of the metal-->BX3 dative sigma-bond: 1,2-addition reactions of the Fe-->BX3 moiety of the ferraboratrane complex [kappa4-B(mimBu t)3]Fe(CO)2.

The ferraboratrane [kappa4-B(mim(Bu)t)3]Fe(CO)2 (mim(Bu)t = 2-mercapto-1-tert-butylimidazolyl), the first example of a complex which possesses a retrodative (i.e., metal-to-ligand) Fe-->B bond, is obtained via reaction of the tris(2-mercapto-1-tert-butylimidazolyl)hydroborato complex [Tm(Bu)t]FeCl with LiCH2SiMe3 followed by treatment with CO. Significantly, [kappa4-B(mim(Bu)t)3]Fe(CO)2 exhibits novel reactivity towards a variety of reagents that results in eradication of the Fe-->B bond via a formal 1,2-addition process and the formation of B-functionalized tris(mercaptoimidazolyl)borate derivatives, [XTm(Bu)t]FeY.

Synthesis and structural characterization of [kappa3-B,S,S-B(mimR)3]Ir(CO)(PPh3)H (R = Bu(t), Ph) and [kappa4-B(mim(Bu(t))3]M(PPh3)Cl (M = Rh, Ir): analysis of the bonding in metal borane compounds.

A series of iridium and rhodium complexes that feature M-->B dative bonds, namely [kappa(3)-B,S,S-B(mim(R))3]Ir(CO)(PPh3)H (R = But, Ph) and [kappa4-B(mim(Bu)t)3]M(PPh3)Cl (M = Rh, Ir), has been synthesized via (i) the reactions of Ir(PPh3)2(CO)Cl with [Tm(Bu)t]Tl and [Tm(Ph)]Li and (ii) the reactions of (COD)M(PPh3)Cl with [Tm(Bu)t]K. The complexes have been structurally characterized by X-ray diffraction, thereby demonstrating the presence of a M-->B dative bond in each complex. The nature of the M-->B interaction in these complexes has been addressed by computational methods which indicate that the metal centers possess a d(6) configuration. The d(6) configuration is in accord with the value predicted by using a method that employs the valence to determine d(n)(), but is not in accord with the d8 configuration that is predicted using the oxidation number. Thus, even though B(mim(R))3 may be regarded as a neutral closed-shell ligand, coordination to a d(n) transition metal via the boron results in the formation of a complex in which the metal center possesses a d(n-2) configuration.

EPR spectroscopic and computational characterization of the hydroxyethylidene-thiamine pyrophosphate radical intermediate of pyruvate:ferredoxin oxidoreductase.

The radical intermediate of pyruvate:ferredoxin oxidoreductase (PFOR) from Moorella thermoacetica was characterized using electron paramagnetic resonance (EPR) spectroscopy at X-band and D-band microwave frequencies. EPR spectra, obtained with various combinations of isotopically labeled substrate (pyruvate) and coenzyme (thiamine pyrophosphate (TPP)), were analyzed by spectral simulations. Parameters obtained from the simulations were compared with those predicted from electronic structure calculations on various radical structures. The g-values and 14N/15N-hyperfine splittings obtained from the spectra are consistent with a planar, hydroxyethylidene-thiamine pyrophosphate (HE-TPP) pi-radical, in which spin is delocalized onto the thiazolium sulfur and nitrogen atoms. The 1H-hyperfine splittings from the methyl group of pyruvate and the 13C-hyperfine splittings from C2 of both pyruvate and TPP are consistent with a model in which the pyruvate-derived oxygen atom of the HE-TPP radical forms a hydrogen bond. The hyperfine splitting constants and g-values are not compatible with those predicted for a nonplanar, sigma/n-type cation radical.