Histatin-5 (Hst-5) is an antimicrobial, salivary protein that is involved in the host defense system. Hst-5 has been proposed to bind functionally relevant zinc and copper but presents challenges in ...structural studies due to its disordered conformation in aqueous solution. Here, we used circular dichroism (CD) and UV resonance Raman (UVRR) spectroscopy to define metallo-Hst-5 interactions in aqueous solution. A zinc-containing Hst-5 sample exhibits shifted Raman bands, relative to bands observed in the absence of zinc. Based on comparison to model compounds and to a family of designed, zinc-binding beta hairpins, the alterations in the Hst-5 UVRR spectrum are attributed to zinc coordination by imidazole side chains. Zinc addition also shifted a tyrosine aromatic ring UVRR band through an electrostatic interaction. Copper addition did not have these effects. A sequence variant, H18A/H19A, was employed; this mutant has less potent antifungal activity, when compared to Hst-5. Zinc addition had only a small effect on the thermal stability of this mutant. Interestingly, both zinc and copper addition shifted histidine UVRR bands in a manner diagnostic for metal coordination. Results obtained with a K13E/R22G mutant were similar to those obtained with wildtype. These experiments show that H18 and H19 contribute to a zinc binding site. In the H18A/H19A mutant the specificity of the copper/zinc binding sites is lost. The experiments implicate specific zinc binding to be important in the antimicrobial activity of Hst-5.
RAS mutations account for >15% of all human tumors, and of these ~85% are due to mutations in a particular RAS gene: KRAS. Recent studies revealed that KRAS harbors four druggable allosteric sites. ...Here, we have (a) used molecular simulations to generate ensembles of wild type and four major oncogenic KRAS mutants (G12V, G12D, G13D, and Q61H); (b) characterized the druggability of each allosteric pocket in each protein; (c) conducted extensive ensemble‐based virtual screening using pocket‐tailored ligand libraries; (d) prioritized hits through hierarchical postdocking analysis; and (e) validated predicted hits with NMR. Of the 785 diverse potential hits identified by our in silico analysis, we tested 90 for their ability to bind KRAS using NMR and found that nine cause backbone amide chemical shift perturbations of residues near the functionally responsive switch loops, suggesting potential binding. We conducted detailed biophysical analyses on a novel indole‐based compound to demonstrate the potential of our workflow to yield lead compounds. We believe the detailed information documented in this work regarding the druggability profile of each allosteric site and the chemical fingerprints of compounds that target them will serve as vital resources for future structure‐based drug design efforts against KRAS, a high‐value target for cancer therapy.
An ensemble‐based drug discovery workflow to discover potential direct KRAS allosteric inhibitors at high success rate.
Approximately 15% of all human tumors harbor mutant KRAS, a membrane-associated small GTPase and notorious oncogene. Mutations that render KRAS constitutively active will lead to uncontrolled cell ...growth and cancer. However, despite aggressive efforts in recent years, there are no drugs on the market that directly target KRAS and inhibit its aberrant functions. In the current work, we combined structure-based design with a battery of cell and biophysical assays to discover a novel pyrazolopyrimidine-based allosteric KRAS inhibitor that binds to activated KRAS with sub-micromolar affinity and disrupts effector binding, thereby inhibiting KRAS signaling and cancer cell growth. These results show that pyrazolopyrimidine-based compounds may represent a first-in-class allosteric noncovalent inhibitors of KRAS. Moreover, by studying two of its analogues, we identified key chemical features of the compound that interact with a set of specific residues at the switch regions of KRAS and play critical roles for its high-affinity binding and unique mode of action, thus providing a blueprint for future optimization efforts.
Mutations in KRAS account for about 20% of human cancers. Despite the major progress in recent years toward the development of KRAS inhibitors, including the discovery of covalent inhibitors of the ...G12C KRAS variant for the treatment of non-small-cell lung cancer, much work remains to be done to discover broad-acting inhibitors to treat many other KRAS-driven cancers. In a previous report, we showed that a 308.4 Da small-molecule ligand (2R)-2-(N’-(1H-indole-3-carbonyl)hydrazino)-2-phenyl-acetamide binds to KRAS with low micro-molar affinity Chem. Biol. Drug Des. 2019; 94(2):1441–1456. Binding of this ligand, which we call ACA22, to the p1 pocket of KRAS and its interactions with residues at beta-strand 1 and the switch loops have been supported by data from nuclear magnetic resonance spectroscopy and microscale thermophoresis experiments. However, the inhibitory potential of the compound was not demonstrated. Here, we show that ACA22 inhibits KRAS-mediated signal transduction in cells expressing wild type (WT) and G12D mutant KRAS and reduces levels of guanosine triphosphate-loaded WT KRAS more effectively than G12D KRAS. We ruled out the direct effect on nucleotide exchange or effector binding as possible mechanisms of inhibition using a variety of biophysical assays. Combining these observations with binding data that show comparable affinities of the compound for the active and inactive forms of the mutant but not the WT, we propose conformational selection as a possible mechanism of action of ACA22.
We describe six compounds as early hits for the development of direct inhibitors of KRAS, an important anticancer drug target. We show that these compounds bind to KRAS with affinities in the low ...micromolar range and exert different effects on its interactions with binding partners. Some of the compounds exhibit selective binding to the activated form of KRAS and inhibit signal transduction through both the MAPK or the phosphatidylinositide 3-kinase PI3K-protein kinase B (AKT) pathway in cells expressing mutant KRAS. Most inhibit intrinsic and/or SOS-mediated KRAS activation while others inhibit RAS-effector interaction. We propose these compounds as starting points for the development of non-covalent allosteric KRAS inhibitors.
Long-distance electron transfer (ET) plays a critical role in solar energy conversion, DNA synthesis, and mitochondrial respiration. Tyrosine (Y) side chains can function as intermediates in these ...reactions. The oxidized form of tyrosine deprotonates to form a neutral tyrosyl radical, Y•, a powerful oxidant. In photosystem II (PSII) and ribonucleotide reductase, redox-active tyrosines are involved in the proton-coupled electron transfer (PCET) reactions, which are key in catalysis. In these proteins, redox-linked structural dynamics may play a role in controlling the radical’s extraordinary oxidizing power. To define these dynamics in a structurally tractable system, we have constructed biomimetic peptide maquettes, which are inspired by PSII. UV resonance Raman studies were conducted of ET and PCET reactions in these β-hairpins, which contain a single tyrosine residue. At pH 11, UV photolysis induces ET from the deprotonated phenolate side chain to solvent. At pH 8.5, interstrand proton transfer to a π-stacked histidine accompanies the Y oxidation reaction. The UV resonance Raman difference spectrum, associated with Y oxidation, was obtained from the peptide maquettes in D2O buffers. The difference spectra exhibited bands at 1441 and 1472 cm–1, which are assigned to the amide II′ (CN) vibration of the β-hairpin. This amide II′ spectral change was attributed to substantial alterations in amide hydrogen bonding, which are coupled with the Y/Y• redox reaction and are reversible. These experiments show that ET and PCET reactions can create new minima in the protein conformational landscape. This work suggests that charge-coupled conformational changes can occur in complex proteins that contain redox-active tyrosines. These redox-linked dynamics could play an important role in control of PCET in biological oxygen evolution, respiration, and DNA synthesis.
In class 1a ribonucleotide reductase (RNR), a substrate-based radical is generated in the α2 subunit by long-distance electron transfer involving an essential tyrosyl radical (Y122O·) in the β2 ...subunit. The conserved W48 β2 is ∼10 Å from Y122OH; mutations at W48 inactivate RNR. Here, we design a beta hairpin peptide, which contains such an interacting tyrosine-tryptophan dyad. The NMR structure of the peptide establishes that there is no direct hydrogen bond between the phenol and the indole rings. However, electronic coupling between the tyrosine and tryptophan occurs in the peptide. In addition, downshifted ultraviolet resonance Raman (UVRR) frequencies are observed for the radical state, reproducing spectral downshifts observed for β2. The frequency downshifts of the ring and CO bands are consistent with charge transfer from YO· to W or another residue. Such a charge transfer mechanism implies a role for the β2 Y-W dyad in electron transfer.
In proteins, proton-coupled electron transfer (PCET) can involve the transient oxidation and reduction of the aromatic amino acid tyrosine. Due to the short life time of tyrosyl radical ...intermediates, transient absorption spectroscopy provides an important tool in deciphering electron-transfer mechanisms. In this report, the photoionization of solution tyrosine and tyrosinate was investigated using transient, picosecond absorption spectroscopy. The results were compared to data acquired from a tyrosine-containing β-hairpin peptide. This maquette, peptide A, is an 18-mer that exhibits π–π interaction between tyrosine (Y5) and histidine (H14). Y5 and H14 carry out an orthogonal PCET reaction when Y5 is oxidized in the mid-pH range. Photolysis of all samples (280 nm, instrument response: 360 fs) generated a solvated electron signal within 3 ps. A signal from the S1 state and a 410 nm signal from the neutral tyrosyl radical were also formed in 3 ps. Fits to S1 and tyrosyl radical decay profiles revealed biphasic kinetics with time constants of 10–50 and 400–1300 ps. The PCET reaction at pH 9 was associated with a significant decrease in the rate of tyrosyl radical and S1 decay compared to electron transfer (ET) alone (pH 11). This pH dependence was observed both in solution and peptide samples. The pH 9 reaction may occur with a sequential electron-transfer, proton-transfer (ETPT) mechanism. Alternatively, the pH 9 reaction may occur by coupled proton and electron transfer (CPET). CPET would be associated with a reorganization energy larger than that of the pH 11 reaction. Significantly, the decay kinetics of S1 and the tyrosyl radical were accelerated in peptide A compared to solution samples at both pH values. These data suggest either an increase in electronic coupling or a specific, sequence-dependent interaction, which facilitates ET and PCET in the β hairpin.
We describe an aptamer-based surface enhanced resonance Raman scattering (SERRS) sensor with high sensitivity, specificity, and stability for the detection of a coagulation protein, human α-thrombin. ...The sensor achieves high sensitivity and a limit of detection of 100 pM by monitoring the SERRS signal change upon the single-step of thrombin binding to immobilized thrombin binding aptamer. The selectivity of the sensor is demonstrated by the specific discrimination of thrombin from other protein analytes. The specific recognition and binding of thrombin by the thrombin binding aptamer is essential to the mechanism of the aptamer-based sensor, as shown through measurements using negative control oligonucleotides. In addition, the sensor can detect 1 nM thrombin in the presence of complex biofluids, such as 10% fetal calf serum, demonstrating that the immobilized, 5′-capped, 3′-capped aptamer is sufficiently robust for clinical diagnostic applications. Furthermore, the proposed sensor may be implemented for multiplexed detection using different aptamer-Raman probe complexes.
Strong dependence of the quantum yield of electron injection, Φinjection, on the excess vibrational energy of the short-lived S1 excited state of the donor was observed for a carboxyazulene ...chromophore, 6-MeAz-2-COOH, bound to the surface of colloidal TiO2. The oxidation state of 6-MeAz-2-COOH was aligned with the CB of TiO2 in such manner that the lowest vibrational levels of the S1 state were below the band edge. Two distinct electron injection regimes were observed: a long wavelength one which was attributed to a low yield direct injection into trap sites and a short wavelength one corresponding to a much higher yield injection into the bulk CB of the TiO2 nanoparticles. There is a 9-fold increase of Φinjection as λexcitation decreases from 690 to 525 nm, with the steepest, 4-fold rise occurring between 585 and 550 nm, that is, when the excess energy is approximately equivalent to 3 quanta of skeletal vibrations of the donor. The charge recombination kinetics in the 6-MeAz-2-COOH@TiO2 system is also different for the low energy (λ > 585 nm) and high energy (λ < 585 nm) excitation of the donor. This behavior is consistent with excess energy dependent spatial range of injection and different trapping sites that are accessible to the “cold” and “hot” electrons, with the latter exhibiting a broader distribution of lifetimes and 24-times higher long-term yield at 30 ps after the excitation. Through demonstrating that it is possible to harvest electrons from unrelaxed, vibrationally hot donor states under ambient conditions in solution, these results open interesting directions for new developments in photovoltaics and photocatalysis.
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