Discovery of novel, dual mechanism ERK inhibitors by affinity selection screening of an inactive kinase.

An affinity-based mass spectrometry screening technology was used to identify novel binders to both nonphosphorylated and phosphorylated ERK2. Screening of inactive ERK2 identified a pyrrolidine analogue 1 that bound to both nonphosphorylated and phosphorylated ERK2 and inhibited ERK2 kinase activity. Chemical optimization identified compound 4 as a novel, potent, and highly selective ERK1,2 inhibitor which not only demonstrated inhibition of phosphorylation of ERK substrate p90RSK but also demonstrated inhibition of ERK1,2 phosphorylation on the activation loop. X-ray cocrystallography revealed that upon binding of compound 4 to ERK2, Tyr34 undergoes a rotation (flip) along with a shift in the poly-Gly rich loop to create a new binding pocket into which 4 can bind. This new binding mode represents a novel mechanism by which high affinity ATP-competitive compounds may achieve excellent kinase selectivity.

Affinity characterization-mass spectrometry methodology for quantitative analyses of small molecule protein binding in solution.

Affinity characterization by mass spectrometry (AC-MS) is a novel LC-MS methodology for quantitative determination of small molecule ligand binding to macromolecules. Its most distinguishing feature is the direct determination of all three concentration terms of the equilibrium binding equation, i.e., (M), (L), and (ML), which denote the macromolecule, ligand, and the corresponding complex, respectively. Although it is possible to obtain the dissociation constant from a single mixing experiment, saturation analyses are still valuable for assessing the overall binding phenomenon based on an established formalism. In addition to providing the prerequisite dissociation constant and binding stoichiometry, the technique also provides valuable information about the actual solubility of both macromolecule and ligand upon dilution and mixing in binding buffers. The dissociation constants and binding mode for interactions of DNA primase and thymidylate synthetase (TS) with high and low affinity small molecule ligands were obtained using the AC-MS method. The data were consistent with the expected affinity of TS for these ligands based on dissociation constants determined by alternative thermal-denaturation techniques: TdF or TdCD, and also consistent enzyme inhibition constants reported in the literature. The validity of AC-MS was likewise extended to a larger set of soluble protein-ligand systems. It was established as a valuable resource for counter screen and structure-activity relationship studies in drug discovery, especially when other classical techniques could only provide ambiguous results.

Small-molecule inhibitors of FABP4/5 ameliorate dyslipidemia but not insulin resistance in mice with diet-induced obesity.

Fatty acid binding protein-4 (FABP4) and FABP5 are two closely related FA binding proteins expressed primarily in adipose tissue and/or macrophages. The small-molecule FABP4 inhibitor BMS309403 was previously reported to improve insulin sensitivity in leptin-deficient Lep(ob)/Lep(ob) (ob/ob) mice. However, this compound was not extensively characterized in the more physiologically relevant animal model of mice with diet-induced obesity (DIO). Here, we report the discovery and characterization of a novel series of FABP4/5 dual inhibitors represented by Compounds 1-3. Compared with BMS309403, the compounds had significant in vitro potency toward both FABP4 and FABP5. In cell-based assays, Compounds 2 and 3 were more potent than BMS309403 to inhibit lipolysis in 3T3-L1 adipocytes and in primary human adipocytes. They also inhibited MCP-1 release from THP-1 macrophages as well as from primary human macrophages. When chronically administered to DIO mice, BMS309403 and Compound 3 reduced plasma triglyceride and free FA levels. Compound 3 reduced plasma free FAs at a lower dose level than BMS309403. However, no significant change was observed in insulin, glucose, or glucose tolerance. Our results indicate that the FABP4/5 inhibitors ameliorate dyslipidemia but not insulin resistance in DIO mice.

Novel benzimidazole inhibitors bind to a unique site in the kinesin spindle protein motor domain.

Affinity selection-mass spectrometry (AS-MS) screening of kinesin spindle protein (KSP) followed by enzyme inhibition studies and temperature-dependent circular dichroism (TdCD) characterization was utilized to identify a series of benzimidazole compounds. This series also binds in the presence of Ispinesib, a known anticancer KSP inhibitor in phase I/II clinical trials for breast cancer. TdCD and AS-MS analyses support simultaneous binding implying existence of a novel non-Ispinesib binding pocket within KSP. Additional TdCD analyses demonstrate direct binding of these compounds to Ispinesib-resistant mutants (D130V, A133D, and A133D + D130V double mutant), further strengthening the hypothesis that the compounds bind to a distinct binding pocket. Also importantly, binding to this pocket causes uncompetitive inhibition of KSP ATPase activity. The uncompetitive inhibition with respect to ATP is also confirmed by the requirement of nucleotide for binding of the compounds. After preliminary affinity optimization, the benzimidazole series exhibited distinctive antimitotic activity as evidenced by blockade of bipolar spindle formation and appearance of monoasters. Cancer cell growth inhibition was also demonstrated either as a single agent or in combination with Ispinesib. The combination was additive as predicted by the binding studies using TdCD and AS-MS analyses. The available data support the existence of a KSP inhibitory site hitherto unknown in the literature. The data also suggest that targeting this novel site could be a productive strategy for eluding Ispinesib-resistant tumors. Finally, AS-MS and TdCD techniques are general in scope and may enable screening other targets in the presence of known drugs, clinical candidates, or tool compounds that bind to the protein of interest in an effort to identify potency-enhancing small molecules that increase efficacy and impede resistance in combination therapy.

Inhibitors of the lipid phosphatase SHIP2 discovered by high-throughput affinity selection-mass spectrometry screening of combinatorial libraries.

This manuscript describes the discovery and characterization of inhibitors of the lipid phosphatase SHIP2, an important target for the treatment of Type 2 diabetes, using the Automated Ligand Identification System. ALIS is an affinity selection-mass spectrometry platform for label-free, high throughput screening of mixture-based combinatorial libraries. We detail the mass-encoded synthesis of a library that yielded NGD-61338, a pyrazole-based SHIP2 inhibitor. Quantitative ALIS affinity measurements and inhibition of SHIP2 enzymatic activity indicate that this compound has micromolar binding affinity and inhibitory activity for this target. This inhibitor, which does not contain a phosphatase "warhead," binds the active site of SHIP2 as determined by ALIS-based competition experiments with the enzyme's natural substrate, phosphatidylinositol 3,4,5-triphosphate (PIP3). Structure-activity relationships for NGD-61338 and two other ligand classes discovered by ALIS screening were explored using a combination of combinatorial library synthesis and ALIS-enabled affinity ranking in compound mixtures.

Rovibrational spectra of the N2-HF complex at the vHF=3 level.

We report the analyses of the three intermolecular combination bands of the hydrogen-bonded N2-HF complex at vHF=3, observed by molecular beam intracavity laser induced fluorescence. The origin of the HF intermolecular bending combination band, (3001(1)0)<--(00000), is 11 548.45(3) cm(-1), 328.2 cm(-1) higher than that of the (30000)<--(00000) transition with an origin at 11 220.250(1) cm(-1). The average rotational constant of the (3001(1)0) level is 0.103 63(1) cm(-1), a 4.8% reduction from B(30000)=0.109 21(1) cm(-1). Perturbations are observed as line splittings, increased line widths, and reduced peak intensities of a number of lines of the e and f components of (3001(1)0). In addition, the centrifugal distortion coefficients of both components are large, negative, and different. The N2 intermolecular bend transition (30001(1))<--(00000) has an origin at 11 288.706(1) cm(-1), 68.456(2) cm(-1) above that of the (30000)<--(00000) transition. This is the lowest combination state at v(HF)=3 level. It is unperturbed, yielding B(30001(1))=0.110.10(1) cm(-1). The transition to the intermolecular stretching state, (30100)<--(00000), has an origin at 11 318.858(1) cm(-1) with B(30100)=0.105 84(1) cm(-1). Both the (30100) and (30000) levels show an isolated perturbation at J=4. The Lorentzian component of the line widths, which show considerable variation with soft mode, are GammaL(30000)=490(30) MHz, GammaL(30100)=630(30) MHz, GammaL(3001(1)0)=250(30) MHz, and GammaL(30001(1))=500(50) MHz.

Effect of ionic size on the deposition of charge-regulated particles to a charged surface.

The deposition of charge-regulated particles to a rigid, planar charged surface is modeled theoretically, taking the effects of the excluded area arising from deposited particles and finite ionic sizes into account. Here, a particle comprises a rigid core and an ion-penetrable charged membrane layer, which represents a general type of particle. If the membrane layer has a negligible thickness, the particle simulates a regular inorganic particle, and if the membrane layer has a finite thickness, it simulates biocolloids such as cells. The results of numerical simulation reveal that the rate of particle deposition is faster under the following conditions: (1) lower potential of the planar surface, (2) thicker membrane, (3) higher counterion valance, (4) lower fixed charge density, (5) smaller counterions, (6) larger co-ions, (7) larger functional group, and (8) lower pH. Neglecting the sizes of ionic species may lead to an appreciable deviation in both the electrical repulsive force between particle and surface and the rate of deposition. Typical deviation for the former is approximately 20%, and that for the latter is approximately -75%.

A general technique to rank protein-ligand binding affinities and determine allosteric versus direct binding site competition in compound mixtures.

To realize the full potential of combinatorial chemistry-based drug discovery, generic and efficient tools must be developed that apply the strengths of diversity-oriented chemical synthesis to the identification and optimization of lead compounds for disease-associated protein targets. We report an affinity selection-mass spectrometry (AS-MS) method for protein-ligand affinity ranking and the classification of ligands by binding site. The method incorporates the following steps: (1) an affinity selection stage, where protein-binding compounds are selected from pools of ligands in the presence of varying concentrations of a competitor ligand, (2) a first chromatography stage to separate unbound ligands from protein-ligand complexes, and (3) a second chromatography stage to dissociate the ligands from the complexes for identification and quantification by MS. The ability of the competitor ligand to displace a target-bound library member, as measured by MS, reveals the binding site classification and affinity ranking of the mixture components. The technique requires no radiolabel incorporation or direct biochemical assay, no modification or immobilization of the compounds or target protein, and all reaction components, including any buffers or cofactors required for protein stability, are free in solution. We demonstrate the method for several compounds of wide structural variety against representatives of the most important protein classes in contemporary drug discovery, including novel ATP-competitive and allosteric inhibitors of the Akt-1 (PKB) and Zap-70 kinases, and previously undisclosed antagonists of the M(2) muscarinic acetylcholine receptor, a G-protein coupled receptor (GPCR). The theoretical basis of the technique is analyzed mathematically, allowing quantitative estimation of binding affinities and, in the case of allosteric interaction, absolute determination of binding cooperativity. The method is readily applicable to high-throughput screening hit triage, combinatorial library-based affinity optimization, and developing structure-activity relationships among multiple ligands to a given receptor.

Spectroscopy of the OC-HF hydrogen-bonded complex at vHF=3.

The v(HF)=3 levels of the linear OC-HF complex are observed in the range of 10,800-11,500 cm(-1) using intracavity Ti-sapphire laser-induced fluorescence. The vibrational predissociation linewidths of both (30000) and (3001(1)0) states exceed 5 GHz; thus, the measured spectra are not rotationally resolvable. Under the assumption that these levels are not strongly perturbed, the rotational constants of the two levels are determined to be 0.1100(1) cm(-1) for (30000), 0.1081(1), and 0.1065(1) cm(-1) for f and e sublevels of (3001(1)0), respectively, through band contour fitting. The (30000)<--(00000) band origin is at 10,894.46(1) cm(-1), showing a HF wave number redshift of 478.3 cm(-1). The 4.07 redshift ratio of v(HF)=3 to that of v(HF)=1 indicates a significantly nonlinear increase of the intermolecular interaction energy through HF valence excitation. An ab initio interaction potential surface for HF valence coordinates varying from 0.8 to 1.25 A is used to examine vibrational dynamics. The HF valence vibration v(1) is treated perturbatively, showing that the vibrational redshifts are determined essentially in first order with only a very small second-order contribution. The (3001(1)0)<--(00000) combination transition is observed with the band origin at 11,432.66(1) cm(-1), giving the HF intermolecular bending mode to be 538.2 cm(-1). The high frequency of this vibration, compared to that in similar HF complexes, shows the strong angular anisotropy of the intermolecular interaction potential of OC-HF with respect to the HF subunit. The lifetime of the (3001(1)0) level increases to 28 ps from 14 ps for (30000).