KRASG12C has emerged as a promising target in the treatment of solid tumors. Covalent inhibitors targeting the mutant cysteine-12 residue have been shown to disrupt signaling by this long-“undruggable” target; however clinically viable inhibitors have yet to be identified. Here, we report efforts to exploit a cryptic pocket (H95/Y96/Q99) we identified in KRASG12C to identify inhibitors suitable for clinical development. Structure-based design efforts leading to the identification of a novel quinazolinone scaffold are described, along with optimization efforts that overcame a configurational stability issue arising from restricted rotation about an axially chiral biaryl bond. Biopharmaceutical optimization of the resulting leads culminated in the identification of AMG 510, a highly potent, selective, and well-tolerated KRASG12C inhibitor currently in phase I clinical trials (NCT03600883).

Thrombus (blood clot) is implicated in a number of life threatening diseases, e.g., heart attack, stroke, pulmonary embolism. EP-2104R is an MRI contrast agent designed to detect thrombus by binding to the protein fibrin, present in all thrombi. EP-2104R comprises an 11 amino acid peptide derivatized with 2 GdDOTA-like moieties at both the C- and N-terminus of the peptide (4 Gd in total). EP-2104R was synthesized by a mixture of solid phase and solution techniques. The La(III) analogue was characterized by and 1D and 2D NMR spectroscopy and was found to have the expected structure. EP-2104R was found to be significantly more inert to Gd(III) loss than commercial contrast agents. At the most extreme conditions tested (pH 3, 60 degrees C, 96 hrs), less than 10% of Gd was removed from EP-2104R by a challenge with a DTPA based ligand, while the commercial contrast agents equilibrated within minutes to hours. EP-2104R binds equally to two sites on human fibrin (Kd = 1.7 +/- 0.5 microM) and has a similar affinity to mouse, rat, rabbit, pig, and dog fibrin. EP-2104R has excellent specificity for fibrin over fibrinogen (over 100-fold) and for fibrin over serum albumin (over 1000-fold). The relaxivity of EP-2104R bound to fibrin at 37 degrees C and 1.4 T was 71.4 mM(-1) s(-1) per molecule of EP-2104R (17.4 per Gd), about 25 times higher than that of GdDOTA measured under the same conditions. Strong fibrin binding, fibrin selectivity, and high molecular relaxivity enable EP-2104R to detect blood clots in vivo.

High-resolution solid-state NMR spectroscopy has become a promising method for the determination of three-dimensional protein structures for systems which are difficult to crystallize or exhibit low solubility. Here we describe the structure determination of microcrystalline ubiquitin using 2D (13)C-(13)C correlation spectroscopy under magic angle spinning conditions. High-resolution (13)C spectra have been acquired from hydrated microcrystals of site-directed (13)C-enriched ubiquitin. Inter-residue carbon-carbon distance constraints defining the global protein structure have been evaluated from 'dipolar-assisted rotational resonance' experiments recorded at various mixing times. Additional constraints on the backbone torsion angles have been derived from chemical shift analysis. Using both distance and dihedral angle constraints, the structure of microcrystalline ubiquitin has been refined to a root-mean-square deviation of about 1 A. The structure determination strategies for solid samples described herein are likely to be generally applicable to many proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy.

The spin-polarized radical pairs in protonated and deuterated Zn-substituted reaction centers from two different mutants of the photosynthetic bacteria Rhodobacter sphaeroides and in plant photosystem I from Synechococcus elongatus are investigated by pulsed EPR spectroscopy. Spin-polarized radical pairs give rise to a characteristic out-of-phase electron spin echo. This echo shows a deep envelope modulation with a frequency governed by the spin−spin interaction. The known distance dependence of the magnetic dipolar interaction allows the determination of the distance between the cofactors carrying the unpaired electron spins. For the bacterial reaction centers this distance is known for the electronic ground state from crystal structures and is compared here with the distance of the radical pair spins, i.e. the charge-separated state. In photosystem I the location of the acceptor A1 is not known yet. A distance of 25.4 ± 0.3 Å between and is obtained here and gives new structural information on photosystem I.

Huang is an employee and shareholder of Theseus Pharmaceuticals and a former employee of ARIAD. Y. Hu is an employee of Takeda. F. Li is a former employee of ARIAD. M.T. Greenfield is a former employee of ARIAD. S.G. Zech is an employee and shareholder of Amgen Inc. and a former employee of ARIAD. B. Das is a former employee of ARIAD. N.I. Narasimhan is a former employee of ARIAD. T. Clackson is an employee and shareholder of Xilio Therapeutics and is a former employee of ARIAD. D. Dalgarno is an employee and shareholder of Theseus Pharmaceuticals and a former employee of ARIAD. W.C. Shakespeare is an employee and shareholder of Theseus Pharmaceuticals and a former employee of ARIAD. M. Fitzgerald is a former employee Research.

A novel application of electron paramagnetic resonance (EPR) is reported to gain three dimensional structural information on cofactors in proteins. The method is applied here to determine the unknown position of the electron acceptor QK, a phylloquinone (vitamin K1), in the electron transfer chain in photosystem I of oxygenic photosynthesis. The unusual electron spin echo (out-of-phase echo) observed for the light induced radical pair P700.+QK.- in PS I allows the measurement of the dipolar coupling between the two radical pair spins which yields directly the distance between these two radicals. Full advantage of the information in the out-of-phase echo modulation can be taken if measurements using single crystals are performed. With such samples, the orientation of the principal axis of the dipolar interaction, i.e., the axis connecting P700.+QK.-, can be determined with respect to the crystal axes system. An angle of theta = (27 +/- 5)degrees between the dipolar coupling axis and the crystallographic c-axis has been derived from the modulation of the out-of-phase echo. Furthermore, the projection of the dipolar axis into the crystallographic a,b-plane, is found to be parallel to the a-axis. The results allow for the determination of two possible locations of QK within the electron transfer chain of photosystem I. These two positions are related to each other by the pseudo C2 symmetry of the chlorophyll cofactors.

The charge separated state P 700•+ A 1•-(P 700 ) primary electron donor, A 1 ) phylloquinone electron acceptor) in photosystem I of oxygenic photosynthesis has been investigated by EPR spectroscopy in frozen solution and single crystals. The transient EPR spectra of P 700•+ A 1 •recorded in frozen solution of fully deuterated samples at X-, Q-, and W-band frequencies are shown to contain sufficient information to yield the orientation of the g-tensors of both A 1•and P 700•+ with respect to the axis connecting both spins. So far incomplete information on the orientation of A 1•relative to the membrane plane has been complemented by data from time-resolved EPR on single crystals measured at Q-band. The phylloquinone headgroup orientation evaluated from the EPR data in the charge-separated state P 700•+ A 1 •is compared with the presently available X-ray structural model. The g-tensor of P 700 •+ has also been determined from cw-EPR experiments at W-band on single crystals, independent of the orientational data of the P 700 •+ g-tensor from the time-resolved EPR experiments.The direction of the principal axes of g(P 700•+ ) differ from the molecular axes system of the chlorophylls comprising P 700 as found previously in the case of P 865•+ in bacterial reaction centers. The implications of the complete structural model from the A 1 •and P 700•+ molecular magnetic interaction tensors in the active charge separated state P 700•+ A 1 •in PS I are discussed.

The Photosystem I (PS I) reaction center contains two branches of nearly symmetric cofactors bound to the PsaA and PsaB heterodimer. From the x-ray crystal structure it is known that Trp A 1؊ radical pair show that none of the mutations causes a significant change in the orientation of the measured phylloquinone. Pulsed ENDOR spectra reveal that the W697F PsaA mutation leads to about a 5% increase in the hyperfine coupling of the methyl group on the phylloquinone ring, whereas the S692C PsaA mutation causes a similar decrease in this coupling. The changes in the methyl hyperfine coupling are also reflected in the transient EPR spectra of P 700 ؉ A 1 ؊ and the CW EPR spectra of photoaccumulated A 1 ؊ . We conclude that: (i) the transient EPR spectra at room temperature are predominantly from radical pairs in the PsaA branch of cofactors; (ii) at low temperature the electron cycle involving P 700 and A 1 similarly occurs along the PsaA branch of cofactors; and (iii) mutation of amino acids in close contact with the PsaA side quinone leads to changes in the spin density distribution of the reduced quinone observed by EPR. Photosynthetic reaction centers (RCs)1 are classified into two general types depending on the identity and function of the terminal electron acceptors. Those RCs that incorporate ironsulfur clusters are classified as "Type I," and those that incorporate a mobile (secondary) quinone are classified as "Type II." Type I RCs include Photosystem I (PS I) of cyanobacteria and plants and those in heliobacteria and green sulfur bacteria. Type II RCs include Photosystem II of cyanobacteria and plants and those in green non-sulfur bacteria and purple bacteria. Despite the difference in the identity of the terminal electron acceptors, Type I and Type II RCs share a common motif in terms of polypeptide arrangement and cofactor composition (1).The primary cofactors are bound to proteins that are present as dimers in the membrane. This results in a set of electron transfer cofactors that are arranged (pseudo)symmetrically (2). In PS I, these cofactors include a special pair of chlorophyll a/aЈ molecules as the primary donor, two bridging chlorophyll a molecules, and two chlorophyll a molecules, at least one of which functions as the primary acceptor (3). In the purple bacterial reaction center, the cofactors include a special pair of bacteriochlorophyll a molecules as the primary donor, two bridging bacteriochlorophyll a molecules, and two pheophytin molecules, one of which functions as the primary acceptor (4). PS I and the purple bacterial reaction center contain two quinones; in the latter, one quinone is rather immobile (Q A ) and the other is mobile (Q B ), whereas in PS I both quinones (Q K -A and Q K -B) are, to the best of our knowledge, immobile in their normal function.In Type II reaction centers, a single turnover results in the reduction of Q B , to a semiquinone and a second turnover results in the further reduction (and protonation) of Q B to a hydroquinone. The stability of Q B Ϫ requires that t...