Search

BACKGROUND: Clinical trials of the KRAS inhibitors adagrasib and sotorasib have shown promising activity in cancers harboring KRAS glycine-to-cysteine amino acid substitutions at codon 12 (KRAS(G12C)). The mechanisms of acquired resistance to these therapies are currently unknown. METHODS: Among patients with KRAS(G12C)-mutant cancers treated with adagrasib monotherapy, we performed genomic and histologic analyses that compared pretreatment samples with those obtained after the development of resistance. Cell-based experiments were conducted to study mutations that confer resistance to KRAS(G12C) inhibitors. RESULTS: A total of 38 patients were included in this study: 27 with non–small-cell lung cancer, 10 with colorectal cancer, and 1 with appendiceal cancer. Putative mechanisms of resistance to adagrasib were detected in 17 patients (45% of the cohort), of whom 7 (18% of the cohort) had multiple coincident mechanisms. Acquired KRAS alterations included G12D/R/V/W, G13D, Q61H, R68S, H95D/Q/R, Y96C, and high-level amplification of the KRAS(G12C) allele. Acquired bypass mechanisms of resistance included MET amplification; activating mutations in NRAS, BRAF, MAP2K1, and RET; oncogenic fusions involving ALK, RET, BRAF, RAF1, and FGFR3; and loss-of-function mutations in NF1 and PTEN. In two of nine patients with lung adenocarcinoma for whom paired tissue-biopsy samples were available, histologic transformation to squamous-cell carcinoma was observed without identification of any other resistance mechanisms. Using an in vitro deep mutational scanning screen, we systematically defined the landscape of KRAS mutations that confer resistance to KRAS(G12C) inhibitors. CONCLUSIONS: Diverse genomic and histologic mechanisms impart resistance to covalent KRAS(G12C) inhibitors, and new therapeutic strategies are required to delay and overcome this drug resistance in patients with cancer. (Funded by Mirati Therapeutics and others; ClinicalTrials.gov number, NCT03785249.)

This publication details the successful use of FBDD (fragment-based drug discovery) principles in the invention of a novel covalent Bruton's tyrosine kinase inhibitor, which ultimately became the Takeda Pharmaceuticals clinical candidate TAK-020. Described herein are the discovery of the fragment 5-phenyl-2,4-dihydro-3H-1,2,4-triazol-3-one, the subsequent optimization of this hit molecule to the candidate, and synthesis and performance in pharmacodynamic and efficacy models along with direct biophysical comparison of TAK-020 with other clinical-level assets and the marketed drug Ibrutinib.

The MTAP gene is proximal to and co-deleted in nearly all CDKN2A-deleted cancers. This genetic alteration is present in an estimated 9% of all cancers and is especially prevalent in cancers with high unmet medical need (e.g. mesothelioma (32%), pancreatic (22%), lung squamous (20%)). Multiple independent research teams have demonstrated that tumor cell lines with homozygous MTAP deletions are hypersensitive to shRNA-mediated knock down of PRMT5. MTAP is required for the methionine salvage pathway and MTAP-del cells accumulate MTA, an inhibitory co-factor which competes for binding to the co-factor binding site of PRMT5 with the activating co-factor SAM. PRMT5 is a methyltransferase that adds symmetric dimethylarginine (SDMA) modification to proteins and is essential for mammalian cell survival. A small molecule that selectively binds and stabilizes the catalytically inactive PRMT5•MTA complex may represent a synthetic lethal-based precision medicine for the treatment of MTAP/CDKN2A—del tumors. Notably, 1st generation PRMT5 small molecule inhibitors do not target MTA-complexed PRMT5 and do not exhibit selective inhibition of MTAP-del cancer cells. Here we report a new series of compounds discovered via a fragment-based approach that selectively bind to the PRMT5•MTA complex. A fragment hit was identified in an SPR binding assay with PRMT5•MTA (KD 18 μM). The binding mode was determined by X-ray crystallography and revealed that the fragment makes productive interactions with K333, F327, E435, E444, E435, and W579 of PRMT5 as well as with the co-liganded MTA. Fragment growing aided by structure-based design identified a key interaction with the L312 backbone N-H that enhances binding to PRMT5•MTA (MRTX4646, SPR KD 57 nM). Further exploration highlighted an interaction with the F580 backbone N-H as important for cellular activity and selectivity. This interaction with F580 was illustrated by MRTX7512 which exhibits an IC50 value of 633 nM for inhibition of SDMA in engineered HCT116 MTAP-del cells and demonstrates 15-fold selectivity compared with HCT116 MTAP-WT cells (IC50 9763 nM). Further optimization of cellular potency and pharmacokinetic properties identified MRTX9768, a potent inhibitor of SDMA and cell proliferation in HCT116 MTAP-del cells (SDMA IC50 3 nM; prolif. IC50 11 nM) with marked selectivity over HCT116 MTAP-WT cells (SDMA IC50 544 nM; prolif. IC50 861 nM). In xenograft studies, oral administration of MRTX9768 demonstrates dose-dependent inhibition of SDMA in MTAP-del tumors, with less SDMA modulation observed in bone marrow. In summary, we have used a fragment-based approach to discover a new class of orally active PRMT5•MTA inhibitors that demonstrate selective antitumor activity in MTAP-del tumor cells while sparing MTAP-WT cells. Citation Format: Christopher R. Smith, Svitlana Kulyk, J. D. Lawson, Lars D. Engstrom, Ruth Aranda, David M. Briere, Robin Gunn, Krystal Moya, Lisa Rahbaek, Laura Waters, Anthony Ivetac, James G. Christensen, Peter Olson, Matthew A. Marx. Fragment based discovery of MRTX9768, a synthetic lethal-based inhibitor designed to bind the PRMT5-MTA complex and selectively target MTAP/CDKN2A-deleted tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB003.

S-adenosylhomocysteine hydrolase (AHCY) catalyzes the reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine and l-homocysteine. This enzyme is frequently overexpressed in many tumor types and is considered to be a validated anti-tumor target. In order to enable the development of small molecule AHCY inhibitors as targeted cancer therapeutics we developed an assay based on a RapidFire high-throughput mass spectrometry detection system, which allows the direct measurement of AHCY enzymatic activity. This technique avoids many of the problems associate with the previously reported method of using a thiol-reactive fluorescence probes to measure AHCY activity. Screening of a ∼500,000 compound library using this technique identified multiple SAH competitive hits. Co-crystal structures of the hit compounds complexed with AHCY were obtained showing that the compounds indeed bind in the SAH site of the enzyme. In addition, some hit compounds increased the SAH levels in HCT116 cells and showed growth inhibition. These compounds could be promising starting points for the optimization of cancer treatments.

A series of potent ALK5 inhibitors were designed using a SBDD approach and subsequently optimized to improve drug likeness. Starting with a 4-substituted quinoline screening hit, SAR was conducted using a ALK5 binding model to understand the binding site and optimize activity. The resulting inhibitors displayed excellent potency but were limited by high in vitro clearance in rat and human microsomes. Using a scaffold morphing strategy, these analogs were transformed into a related pyrazolo[4,3-b]pyridine series with improved ADME properties.

Using SBDD, a series of 4-amino-7-azaindoles were discovered as a novel class of Alk5 inhibitors that are potent in both Alk5 enzymatic and cellular assays. Subsequently a ring cyclization strategy was utilized to improve ADME properties leading to the discovery of a series of 1H-imidazo[4,5-c]pyridin-2(3H)-one drug like Alk5 inhibitors.

Methionine aminopeptidase 2 (MetAP2) is an enzyme that cleaves an N-terminal methionine residue from a number of newly synthesized proteins. Pre-clinical and clinical studies suggest that MetAP2 inhibitors could be used as a novel treatment for obesity. Herein we describe our use of fragment screening methods and structural biology to quickly identify and elaborate an indazole fragment into a series of reversible MetAP2 inhibitors with

The discovery and optimization of a series of 4-aminocinnoline-3-carboxamide inhibitors of Bruton's tyrosine kinase are reported. A fragment-based screening approach incorporating X-ray co-crystallography was used to identify a cinnoline fragment and characterize its binding mode in the ATP binding site of Btk. Optimization of the fragment hit resulted in the identification of a lead compound which reduced paw swelling in a dose- and exposure-dependent fashion in a rat model of collagen-induced arthritis.

Axl has been a target of interest in the oncology field for several years based on its role in various oncogenic processes. To date, no wild-type Axl crystal structure has been reported. Herein, we describe the structure-based optimization of a novel chemotype of Axl inhibitors, 1H-imidazole-2-carboxamide, using a mutated kinase homolog, Mer(I650M), as a crystallographic surrogate. Iterative optimization of the initial lead compound (1) led to compound (21), a selective and potent inhibitor of wild-type Axl. Compound (21) will serve as a useful compound for further in vivo studies.

The MAPK signaling cascade, comprised of several linear and intersecting pathways, propagates signaling into the nucleus resulting in cytokine and chemokine release. The Map Kinase Kinase isoforms 3 and 6 (MKK3 and MKK6) are responsible for the phosphorylation and activation of p38, and are hypothesized to play a key role in regulating this pathway without the redundancy seen in downstream effectors. Using FBDD, we have discovered efficient and selective inhibitors of MKK3 and MKK6 that can serve as tool molecules to help further understand the role of these kinases in MAPK signaling, and the potential impact of inhibiting kinases upstream of p38.

Comparisons were made among four categories of protein flexibility: (1) low-B-factor ordered regions, (2) high-B-factor ordered regions, (3) short disordered regions, and (4) long disordered regions. Amino acid compositions of the four categories were found to be significantly different from each other, with high-B-factor ordered and short disordered regions being the most similar pair. The high-B-factor (flexible) ordered regions are characterized by a higher average flexibility index, higher average hydrophilicity, higher average absolute net charge, and higher total charge than disordered regions. The low-B-factor regions are significantly enriched in hydrophobic residues and depleted in the total number of charged residues compared to the other three categories. We examined the predictability of the high-B-factor regions and developed a predictor that discriminates between regions of low and high B-factors. This predictor achieved an accuracy of 70% and a correlation of 0.43 with experimental data, outperforming the 64% accuracy and 0.32 correlation of predictors based solely on flexibility indices. To further clarify the differences between short disordered regions and ordered regions, a predictor of short disordered regions was developed. Its relatively high accuracy of 81% indicates considerable differences between ordered and disordered regions. The distinctive amino acid biases of high-B-factor ordered regions, short disordered regions, and long disordered regions indicate that the sequence determinants for these flexibility categories differ from one another, whereas the significantly-greater-than-chance predictability of these categories from sequence suggest that flexible ordered regions, short disorder, and long disorder are, to a significant degree, encoded at the primary structure level., published_or_final_version

The RAS/RAF/MEK/ERK signal transduction cascade plays an important role in the regulation of critical cellular processes such as cell proliferation, migration, and differentiation. The up-regulation of this pathway can negatively affect cell homeostasis and is responsible for the development of various forms of cancer and inflammation processes. Therefore, there is a strong interest in pursuing drug programs targeting some of the enzymes involved in this pathway. In addition to the determination of Ki, Kd, IC50, and/or EC50, a more thorough kinetic analysis can provide useful information for the selection of the best lead series during the early stage of the drug discovery process. This study describes a medium-throughput fluorescent probe displacement assay for the rapid determination of the k(off) constant, residence time, and kinetic efficiency for ERK (extracellular signal-regulated kinase) inhibitors. Using this method, we have identified several inhibitors that we have subjected to further kinetic analysis by comparing k(off) constants determined for these time-dependent inhibitors using either the active or inactive form of ERK2.

The back door has been proposed to be an exit pathway from the myosin active site for phosphate (P(i)) generated by adenosine 5'-triphosphate hydrolysis. We used molecular dynamics simulations to investigate the interaction of P(i) with the back door and the plausibility of P(i) release via this route. Molecular dynamics simulations were performed on the Dictyostelium motor domain with bound Mg.adenosine 5'-diphosphate (ADP) and P(i), modeled upon the Mg.ADP.BeF(x) and Mg.ADP.V(i) structures. Simulations revealed that the relaxation of ADP and free P(i) from their initial positions reduced the diameter of the back door via motions of switch 1 and switch 2 located in the upper and lower 50-kDa subdomains, respectively. In neither simulation could P(i) freely diffuse out the back door. Water molecules, however, could flux through the back door in the Mg.ADP.BeF(x)-based simulation but not in the Mg.ADP.V(i)-based simulation. In neither structure was water observed fluxing through the main (front door) entrance. These observations suggest that the ability of P(i) to leave via the back door is linked tightly to conformational changes between the upper and lower 50-kDa subdomains. The simulations offer structural explanations for (18)O-exchange with P(i) at the active site, and P(i) release being the rate-limiting step in the myosin adenosine 5'-triphosphatase.

The goal of this study was to provide structural information about the regulatory domains of double-headed smooth muscle heavy meromyosin, including the N terminus of the regulatory light chain, in both the phosphorylated and unphosphorylated states. We extended our previous photo-cross-linking studies (Wu, X., Clack, B. A., Zhi, G., Stull, J. T., and Cremo, C. R. (1999) J. Biol. Chem. 274, 20328-20335) to determine regions of the regulatory light chain that are cross-linked by a cross-linker attached to Cys(108) on the partner regulatory light chain. For this purpose, we have synthesized two new biotinylated sulfhydryl reactive photo-cross-linking reagents, benzophenone, 4-(N-iodoacetamido)-4'-(N-biotinylamido) and benzophenone, 4-(N-maleimido)-4'-(N-biotinylamido). Cross-linked peptides were purified by avidin affinity chromatography and characterized by Edman sequencing and mass spectrometry. Labeled Cys(108) from one regulatory light chain cross-linked to (71)GMMSEAPGPIN(81), a loop in the N-terminal half of the regulatory light chain, and to (4)RAKAKTTKKRPQR(16), a region for which there is no atomic resolution data. Both cross-links were to the partner regulatory light chain and occurred in unphosphorylated but not phosphorylated heavy meromyosin. Using these data, data from our previous study, and atomic coordinates from various myosin isoforms, we have constructed a structural model of the regulatory domain in an unphosphorylated double-headed molecule that predicts the general location of the N terminus. The implications for the structural basis of the phosphorylation-mediated regulatory mechanism are discussed.

The photoaffinity spin-labeled non-nucleoside ATP analogue, 2-(4-azido-2-nitrophenyl)amino-2,2-(1-oxyl-2,2,6,6-tetramethyl-4-piperidylidene)di(oxymethylene)ethyl triphosphate (SSL-NANTP), has been shown to be a substrate for skeletal mysoin subfragment 1 (S1) that can be photoincorporated at the active site of S1 [Chen, X., et al. (2000) Bioconjugate Chem. 11, 725-733]. Electron paramagnetic resonance spectroscopy shows that the probe undergoes restricted motion with respect to the protein. The parent compound, NANTP {2-[(4-azido-2-nitrophenyl)amino]ethyl triphosphate}, is specifically photoincorporated at Trp-130 on the amino-terminal 23 kDa tryptic fragment in rabbit skeletal myosin. Surprisingly, amino acid sequence analysis shows that SSL-NANTP is photoincorporated on the carboxy-terminal 20 kDa tryptic fragment at Lys-681 on the side opposite Trp-130 in the nucleotide pocket. This is the first direct evidence showing that this residue in the 20 kDa tryptic fragment is close enough to the active site to be photolabeled by trapped ATP analogues. After actin treatment in the presence of MgATP, SSL-NANDP-labeled myosin S1 had normal ATPase activity, indicating that photolabeling did not significantly alter the enzymatic properties of S1. Photoincorporated SSL-NANDP was bound inside the nucleotide site of S1, with an effective concentration of ∼20 mM as judged by the concentration of MgADP needed to displace it. Molecular dynamics simulations suggest that the ability of NANTP and SSL-NANTP to photolabel different sites results from different orientations of the phenyl ring in the active site. For SSL-NANTP, the p-azido group on the phenyl ring points toward Lys-681. For NANTP, it points in the opposite direction toward Trp-130.

Interaction of fd or M13 filamentous phage with a chloroform/water interface induces morphological change, contracting the filaments sequentially into shortened rods (I-forms), and then into spheroidal particles (S-forms). To further investigate this phage contraction, 34 and 26 chloroform-resistant isolates of fd and M13, respectively, were selected after chloroform treatment of wild-type phages at pH 8. 2 and 4 degrees C. DNA sequencing of gene VIII of the 34 fd isolates revealed five different mutants: these were D5H, M28L, V31L, I37T, and S50T. All 26 M13 isolates were I37T. These mutants exhibited variable sensitivity to chloroform, but all contracted much more slowly than wild-type phage during treatment at 4 degrees C. They all contracted like wild-type phage at 37 degrees C. Site-directed mutagenesis showed that the indicated single mutations carried the chloroform resistance. In structural models of the phage, the D5H locus is on the outside and the S50T locus is on the inside. The M28L and I37T loci are buried in a mostly hydrophobic region in the middle. Although these four mutants are spread out radially, they are localized in the axial direction into a thin disk in the model. The last mutant locus, V31L, is out of this disk, but this locus is proximal to the M28L and I37T loci and also in contact with the surface via a deep hydrophobic hole or depression. These five mutants, their locations, and their variable affects on contraction suggest that chloroform-induced contraction involves a specific mechanism rather than a generalized solvent-induced denaturation and that the critical structural changes occur in a localized level in the phage. These results add weight to suggestions that the sequential contraction of filaments-->I-forms-->S-forms mimic corresponding steps in phage penetration, and, in the reverse order, for phage assembly.

Several methods for predicting pressure losses for upward, concurrent, continuous, multiphase flow in vertical oilwell tubing were tested against measured pressure losses from 726 well tests. This comparison of well known pressure-loss correlations reveals the relative strengths and weaknesses of each correlation and should be useful in selecting satisfactory methods for various applications. Introduction Several correlations have been published that can be used to predict pressure losses in vertical oilwell tubing for the simultaneous, upward, concurrent, continuous flow of oil, water, and gas. Because of the extreme complexity of multiphase flow, the proposed correlations are by necessity highly empirical. The validity of the correlations is then somewhat limited to the quality and scope of the data upon which they are based. Therefore, some correlations perform quite well for cases in the range of the data used in developing the correlation but fail for other applications.The purpose of this study is to ascertain the accuracy of several pressure-loss prediction methods in terms of flow variables familiar to the practicing engineer. The results of the study should assist the petroleum production engineer in selecting the most petroleum production engineer in selecting the most accurate method for his problem and also should indicate the general accuracy to be expected from the methods.The correlations included in the study are those of Poettmann and Carpenter, Baxendell and Thomas, Poettmann and Carpenter, Baxendell and Thomas, Duns and Ros, Fancher and Brown, Hagedorn and Brown, and Orkiszewski. Each of these correlations was proposed specifically for predicting pressure losses in vertical oilwell tubing for the upward flow of multi-phase well fluids.The pressure-loss prediction methods were programmed for the IBM 360 computer and tested programmed for the IBM 360 computer and tested against 726 well tests from field and experimental wells. A statistical analysis was made to find the most acceptable method for different ranges of flow variables and also the method having the best over-all performance for predicting the measured pressure performance for predicting the measured pressure losses. Pressure-Loss Prediction Correlations Pressure-Loss Prediction Correlations The six correlations chosen for study will be discussed briefly. A review of the history and content of multi-phase flow literature and a more detailed discussion of individual correlations may be found in Brown.The correlation of Poettmann and Carpenter is a relatively simple and practical one to predict pressure losses in flowing wells. However, its very pressure losses in flowing wells. However, its very simplicity limits its accuracy. The method was included in this study for comparison, and because it served as a starting point for later correlations.Poettmann and Carpenter correlated the irreversible energy losses of 49 well tests with a Fanning-type friction term. The friction term was related to the numerator of the Reynolds number for the well fluid mixture. No attempt was made to account for liquid holdup (volume fraction liquid in a pipe section), but rather an average density of produced fluids corrected for down-hole conditions was used. The correlation reproduced the measured pressure gradients to an average deviation of 1.8 percent and a standard deviation from the average of 8.3 percent. JPT P. 903

A study has been made on the penetration values in vitro of antibiotics through hog gastric mucin at pH 7·4. Determinations have also been done on the binding of antibiotics to mucus and human plasma. The mucolytic agent N-acetyl cysteine has been studied in regard to its effect on antibiotics penetrating mucus and for any possible inactivation of antibiotics.

The attraction of bacteria commonly found in the respiratory tract in cystic fibrosis to glycoprotein at pH 7 has been studied. The effect of washing on the removal of bacteria from the glycoprotein has also been investigated, and the value of doing both washed and unwashed sputum cultures is discussed.

The differential attraction in vitro of antibiotics to blood lymphocytes and salivary glycoprotein was studied in an attempt to determine the distribution of antibiotics in sputum. Results from studies in vivo on sputum antibiotic levels are also presented.

THE solubility parameter, δ, of a polymer or compound is a measure of intermolecular forces and is useful for the studies of many polymer characteristics, particularly glass transition temperatures1, solubilities and diffusion or transport properties.2 It is therefore highly desirable to have a convenient method for quickly estimating δ values of polymers. It was suggested by Hildebrand and Scott, and later by Scatchard, that solubility parameters could be calculated from optical data such as refractive index measurements3,4. In this article we report some semiempirical correlations between solubility parameters and refractive indices for a series of model compounds and polymers. This work refers only to hydrocarbon and other organic materials; further work would be required to determine if an extension to inorganic polymers is possible.

It was considered desirable to prepare a summary of procedures for the preparation of derivatives of the sulfenyl chloride reagent and to tabulate its recorded derivatives.