KRAS G12C fragment screening renders new binding pockets.

KRAS genes belong to the most frequently mutated family of oncogenes in cancer. The G12C mutation, found in a third of lung, half of colorectal and pancreatic cancer cases, is believed to be responsible for a substantial number of cancer deaths. For 30 years, KRAS has been the subject of extensive drug-targeting efforts aimed at targeting KRAS protein itself, but also its post-translational modifications, membrane localization, protein-protein interactions and downstream signalling pathways. So far, most KRAS targeting strategies have failed, and there are no KRAS-specific drugs available. However, clinical candidates targeting the KRAS G12C protein have recently been developed. MRTX849 and recently approved Sotorasib are covalent binders targeting the mutated cysteine 12, occupying Switch II pocket.Herein, we describe two fragment screening drug discovery campaigns that led to the identification of binding pockets on the KRAS G12C surface that have not previously been described. One screen focused on non-covalent binders to KRAS G12C, the other on covalent binders.

Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation.

Despite the significant therapeutic advances provided by immune-checkpoint blockade and chimeric antigen receptor T cell treatments, many malignancies remain unresponsive to immunotherapy. Bispecific antibodies targeting tumor antigens and activating T cell receptor signaling have shown some clinical efficacy; however, providing co-stimulatory signals may improve T cell responses against tumors. Here, we developed a trispecific antibody that interacts with CD38, CD3 and CD28 to enhance both T cell activation and tumor targeting. The engagement of both CD3 and CD28 affords efficient T cell stimulation, whereas the anti-CD38 domain directs T cells to myeloma cells, as well as to certain lymphomas and leukemias. In vivo administration of this antibody suppressed myeloma growth in a humanized mouse model and also stimulated memory/effector T cell proliferation and reduced regulatory T cells in non-human primates at well-tolerated doses. Collectively, trispecific antibodies represent a promising platform for cancer immunotherapy.

A single T cell epitope drives the neutralizing anti-drug antibody response to natalizumab in multiple sclerosis patients.

Natalizumab (NZM), a humanized monoclonal IgG4 antibody to α4 integrins, is used to treat patients with relapsing-remitting multiple sclerosis (MS)1,2, but in about 6% of the cases persistent neutralizing anti-drug antibodies (ADAs) are induced leading to therapy discontinuation3,4. To understand the basis of the ADA response and the mechanism of ADA-mediated neutralization, we performed an in-depth analysis of the B and T cell responses in two patients. By characterizing a large panel of NZM-specific monoclonal antibodies, we found that, in both patients, the response was polyclonal and targeted different epitopes of the NZM idiotype. The neutralizing activity was acquired through somatic mutations and correlated with a slow dissociation rate, a finding that was supported by structural data. Interestingly, in both patients, the analysis of the CD4+ T cell response, combined with mass spectrometry-based peptidomics, revealed a single immunodominant T cell epitope spanning the FR2-CDR2 region of the NZM light chain. Moreover, a CDR2-modified version of NZM was not recognized by T cells, while retaining binding to α4 integrins. Collectively, our integrated analysis identifies the basis of T-B collaboration that leads to ADA-mediated therapeutic resistance and delineates an approach to design novel deimmunized antibodies for autoimmune disease and cancer treatment.

The Cost and Cost-utility of Three Public Health HIV Case-finding Strategies: Evidence from Rhode Island, 2012-2014.

To evaluate three testing strategies to identify new HIV diagnoses in Rhode Island (RI). RI deployed three testing strategies, by using rapid HIV tests at clinical settings, community-based organization (CBO) settings, and the Partner Notification Services (PNS) program from 2012 to 2014. We reviewed the rapid HIV test results and confirmatory test results to identify new diagnoses, and conducted a cost-utility analysis. The average cost per new diagnosis was $33,015 at CBO settings, $5446 at clinical settings, and $33,818 at the PNS program. The cost-utility analysis showed the state-wide program was cost-saving; testing was cost-saving at clinical settings, and cost-effective at CBO settings and the PNS program. Further analyses showed that cost-effectiveness varied widely across CBOs. The HIV testing expansion program in RI was cost-saving overall. The heterogeneity of cost-effectiveness across settings should provide guidance to officials for allocation of future resources to HIV testing.

Differential Water Thermodynamics Determine PI3K-Beta/Delta Selectivity for Solvent-Exposed Ligand Modifications.

Phosphoinositide 3-kinases (PI3Ks) are involved in important cellular functions and represent desirable targets for drug discovery efforts, especially related to oncology; however, the four PI3K subtypes (α, ß, γ, and δ) have highly similar binding sites, making the design of selective inhibitors challenging. A series of inhibitors with selectivity toward the ß subtype over δ resulted in compound 3(S), which has entered a phase I/Ib clinical trial for patients with advanced PTEN-deficient cancer. Interestingly, X-ray crystallography revealed that the modifications making inhibitor 3(S) and related compounds selective toward the ß-isoform do not interact directly with either PI3Kß or PI3Kδ, thereby confounding rationalization of the SAR. Here, we apply explicit solvent molecular dynamics and solvent thermodynamic analysis using WaterMap in an effort to understand the unusual affinity and selectivity trends. We find that differences in solvent energetics and water networks, which are modulated upon binding of different ligands, explain the experimental affinity and selectivity trends. This study highlights the critical role of water molecules in molecular recognition and the importance of considering water networks in drug discovery efforts to rationalize and improve selectivity.

SAR156497, an exquisitely selective inhibitor of aurora kinases.

The Aurora family of serine/threonine kinases is essential for mitosis. Their crucial role in cell cycle regulation and aberrant expression in a broad range of malignancies have been demonstrated and have prompted intensive search for small molecule Aurora inhibitors. Indeed, over 10 of them have reached the clinic as potential anticancer therapies. We report herein the discovery and optimization of a novel series of tricyclic molecules that has led to SAR156497, an exquisitely selective Aurora A, B, and C inhibitor with in vitro and in vivo efficacy. We also provide insights into its mode of binding to its target proteins, which could explain its selectivity.

Discovery of (2S)-8-[(3R)-3-methylmorpholin-4-yl]-1-(3-methyl-2-oxobutyl)-2-(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one: a novel potent and selective inhibitor of Vps34 for the treatment of solid tumors.

Vps34 (the human class III phosphoinositide 3-kinase) is a lipid kinase involved in vesicle trafficking and autophagy and therefore constitutes an interesting target for cancer treatment. Because of the lack of specific Vps34 kinase inhibitors, we aimed to identify such compounds to further validate the role of this lipid kinase in cancer maintenance and progression. Herein, we report the discovery of a series of tetrahydropyrimidopyrimidinone derivatives. Starting with hit compound 1a, medicinal chemistry optimization led to compound 31. This molecule displays potent activity, an exquisite selectivity for Vps34 with excellent properties. The X-ray crystal structure of compound 31 in human Vps34 illustrates how the unique molecular features of the morpholine synthon bestows selectivity against class I PI3Ks. This molecule exhibits suitable in vivo mouse PK parameters and induces a sustained inhibition of Vps34 upon acute administration. Compound 31 constitutes an optimized Vps34 inhibitor that could be used to investigate human cancer biology.

A highly potent and selective Vps34 inhibitor alters vesicle trafficking and autophagy.

Vps34 is a phosphoinositide 3-kinase (PI3K) class III isoform that has attracted major attention over the recent years because of its role in autophagy. Herein we describe the biological characterization of SAR405, which is a low-molecular-mass kinase inhibitor of Vps34 (KD 1.5 nM). This compound has an exquisite protein and lipid kinase selectivity profile that is explained by its unique binding mode and molecular interactions within the ATP binding cleft of human Vps34. To the best of our knowledge, this is the first potent and specific Vps34 inhibitor described so far. Our results demonstrate that inhibition of Vps34 kinase activity by SAR405 affects both late endosome-lysosome compartments and prevents autophagy. Moreover, we show that the concomitant inhibition of Vps34 and mTOR, with SAR405 and the US Food and Drug Administration-approved mTOR inhibitor everolimus, results in synergistic antiproliferative activity in renal tumor cell lines, indicating a potential clinical application in cancer.

Discovery and optimization of pyrimidone indoline amide PI3Kß inhibitors for the treatment of phosphatase and tensin homologue (PTEN)-deficient cancers.

Compelling molecular biology publications have reported the implication of phosphoinositide kinase PI3Kß in PTEN-deficient cell line growth and proliferation. These findings supported a scientific rationale for the development of PI3Kß-specific inhibitors for the treatment of PTEN-deficient cancers. This paper describes the discovery of 2-[2-(2,3-dihydro-indol-1-yl)-2-oxo-ethyl]-6-morpholin-4-yl-3H-pyrimidin-4-one (7) and the optimization of this new series of active and selective pyrimidone indoline amide PI3Kß inhibitors. 2-[2-(2-Methyl-2,3-dihydro-indol-1-yl)-2-oxo-ethyl]-6-morpholin-4-yl-3H-pyrimidin-4-one (28), identified following a carefully designed methyl scan, displayed improved physicochemical and in vitro pharmacokinetic properties. Structural biology efforts enabled the acquisition of the first X-ray cocrystal structure of p110ß with the selective inhibitor compound 28 bound to the ATP site. The nonplanar binding mode described herein is consistent with observed structure-activity relationship for the series. Compound 28 demonstrated significant in vivo activity in a UACC-62 xenograft model in mice, warranting further preclinical investigation. Following successful development, compound 28 entered phase I/Ib clinical trial in patients with advanced cancer.

Discovery and optimization of new benzimidazole- and benzoxazole-pyrimidone selective PI3Kß inhibitors for the treatment of phosphatase and TENsin homologue (PTEN)-deficient cancers.

Most of the phosphoinositide-3 kinase (PI3K) kinase inhibitors currently in clinical trials for cancer treatment exhibit pan PI3K isoform profiles. Single PI3K isoforms differentially control tumorigenesis, and PI3Kß has emerged as the isoform involved in the tumorigenicity of PTEN-deficient tumors. Herein we describe the discovery and optimization of a new series of benzimidazole- and benzoxazole-pyrimidones as small molecular mass PI3Kß-selective inhibitors. Starting with compound 5 obtained from a one-pot reaction via a novel intermediate 1, medicinal chemistry optimization led to the discovery of compound 8, which showed a significant activity and selectivity for PI3Kß and adequate in vitro pharmacokinetic properties. The X-ray costructure of compound 8 in PI3Kδ showed key interactions and structural features supporting the observed PI3Kß isoform selectivity. Compound 8 achieved sustained target modulation and tumor growth delay at well tolerated doses when administered orally to SCID mice implanted with PTEN-deficient human tumor xenografts.

Four distinct structural domains in Clostridium difficile toxin B visualized using SAXS.

Clostridium difficile is a nosocomial bacterial pathogen causing antibiotic-associated diarrhea and fatal pseudomembranous colitis. Key virulence factors are toxin A and toxin B (TcdB), two highly related toxins that are members of the large clostridial toxin family. These large multifunctional proteins disrupt cell function using a glucosyltransferase domain that is translocated into the cytosol after vesicular internalization of intact holotoxin. Although substantial information about the biochemical mechanisms of intoxication exists, research has been hampered by limited structural information, particularly of intact holotoxin. Here, we used small-angle X-ray scattering (SAXS) methods to obtain an ab initio low-resolution structure of native TcdB, which demonstrated that this molecule is monomeric in solution and possesses a highly asymmetric shape with a maximum dimension of approximately 275 A. Combining this SAXS information with crystallographic or modeled structures of individual functional domains of TcdB reveals for the first time that the three-dimensional structure of TcdB is organized into four distinct structural domains. Structures of the N-terminal glucosyltransferase, the cysteine protease, and the C-terminal repeat region can be aligned within three domains of the SAXS envelope. A fourth domain, predicted to be involved in the translocation of the glucosyltransferase, appears as a large solvent-exposed protrusion. Knowledge of the shapes and relative orientations of toxin domains provides new insight into defining functional domain boundaries and provides a framework for understanding how potential intra-domain interactions enable conformational changes to propagate between domains to facilitate intoxication processes.

Partner notification after STD and HIV exposures and infections: knowledge, attitudes, and experiences of Massachusetts men who have sex with men.

OBJECTIVES: We assessed Boston-area men who have sex with men (MSM) in terms of their knowledge of partner notification (PN)/partner counseling and referral services (PCRS) and intentions to use such services if exposed to/infected with a sexually transmitted disease (STD) or human immunodeficiency virus (HIV) in the future. METHODS: The study used a convenience sample of STD clinic patients (n=48) and a modified respondent-driven sampling method (n=70) to reach a diverse sample of MSM (total sample n=118) in Massachusetts. Participants completed a one-on-one, open-ended, semistructured qualitative interview and quantitative survey. RESULTS: Overall, white, HIV-infected MSM had the highest level of knowledge about PN activities. MSM who were unfamiliar with PN were disproportionately nonwhite and HIV-uninfected. Participants were more likely to notify past partners of HIV exposure than STD exposure. The preferred method of PN for the majority of MSM was direct person-to-person notification. Notably, nonwhite participants were more likely to endorse Massachusetts Department of Public Health PN services than white MSM, who preferred involvement of primary care providers. CONCLUSIONS: PN is an important public health strategy for treating and preventing STDs and HIV among at-risk populations, especially MSM who engage in sexual behavior with anonymous or otherwise non-notifiable sexual partners. Although many MSM had an understanding of the ethical desirability of informing exposed partners and recognized the value of preventative behaviors, they require further education to overcome barriers to PN as well as to gain knowledge of the various methods of both traditional and nontraditional notification, such as Internet PN.

Health care access and sexually transmitted infection screening frequency among at-risk Massachusetts men who have sex with men.

OBJECTIVES: We sought to assess risk exposures, health care access, and screening rates for HIV and sexually transmitted infections (STIs) among men who have sex with men (MSM) in Massachusetts. METHODS: We used a modified respondent-driven sampling method to collect data between March 2006 and May 2007. Overall, 126 MSM completed a survey. RESULTS: Seventy percent of participants reported unprotected receptive anal intercourse with at least 1 nonmonogamous male partner; 50% reported having had a previous STI. Although 98% had visited a health care provider in the previous year, 39% had not been screened for STIs during the previous 2 years. Bisexual respondents were less likely to have told their health care providers that they engage in male-to-male sexual contact (OR = 4.66; P < .001), less likely to have been tested for STIs during in the previous 2 years (OR = 6.91; P < .001), and more likely to engage in insertive anal intercourse without a condom with an HIV-infected partner (OR = 5.04; P < .005) than were non-bisexual respondents. CONCLUSIONS: Clinicians need to assess sexual risk-taking behaviors and more routinely screen for STIs among sexually active men regardless of disclosure of a history of having sex with men.

Asymptomatic gonorrhea and chlamydial infections detected by nucleic acid amplification tests among Boston area men who have sex with men.

BACKGROUND: The purpose of this project was to determine the prevalence of asymptomatic sexually transmitted diseases (STDs) among men who have sex with men (MSM) in the Boston area who had been sexually active (oral and/or anal sex) with another male within the past year. METHODS: Over a 1-month period (March 2007), asymptomatic MSM in care at a Boston community health center (n = 114) were screened for gonorrhea and chlamydia using the BD ProbeTec technique. Deidentified medical record data were analyzed and linked to prevalence monitoring results. RESULTS: Eleven percent of the sample tested positive for one of the 2 STDs (gonorrhea or chlamydia) from at least one mucosal site. Individuals who were infected with an STD were considerably more likely to have a prior history of one or more STD infections when compared with those without an STD history (OR = 3.69; P <0.02). There were no significant differences observed in psychosocial and other behavioral risk factors between patients with or without an STD. CONCLUSIONS: Screening asymptomatic MSM using nucleic acid amplification tests (NAATs) revealed a substantial STD burden that might not have been diagnosed using traditional assays. These data are critical for the design of effective public health interventions for this population.

HIV and STD status among MSM and attitudes about Internet partner notification for STD exposure.

OBJECTIVES: This study assessed the acceptability and perceived utility of Internet-based partner notification (PN) of sexually transmitted disease (STD) exposure for men who have sex with men (MSM) by human immunodeficiency virus (HIV) serostatus. STUDY DESIGN: We recruited 1848 US MSM via a banner advertisement posted on an MSM website for meeting sexual partners between October and November 2005. RESULTS: Even though there was broad acceptance of a PN e-mail across HIV serostatus groups, HIV-infected men rated the importance of each component (e.g., information about where to get tested/treated, additional education regarding the STD exposed to, a mechanism for verifying the authenticity of the PN e-mail) lower than HIV-uninfected or status-unknown participants (all P's <0.01). Additionally, HIV-infected participants were less likely to use the services offered within a PN e-mail (if they were to receive an e-mail notifying them of possible STD exposure in the future), and were less likely to inform their partners of possible STD exposure via an Internet notification system in the future (all P's <0.01). A similar trend emerged about men who reported not having a previous STD compared with those who did. Men who reported no previous STD found Internet PN more acceptable. CONCLUSIONS: Overall, this study documents broad acceptance of Internet PN by at-risk MSM, regardless of HIV serostatus, including a willingness to receive or initiate PN-related e-mail. If public health officials consider using Internet notification services, they may need to anticipate and address concerns of HIV-infected MSM, and will need to use a culturally-sensitive, social marketing campaign to ensure that those who may benefit from these services are willing to use this modality for PN. Internet PN should be considered as a tool to decrease rising STD and HIV rates among MSM who use the Internet to meet sexual partners.

Structural and functional consequences of single amino acid substitutions in the pyrimidine base binding pocket of Escherichia coli CMP kinase.

Bacterial CMP kinases are specific for CMP and dCMP, whereas the related eukaryotic NMP kinase phosphorylates CMP and UMP with similar efficiency. To explain these differences in structural terms, we investigated the contribution of four key amino acids interacting with the pyrimidine ring of CMP (Ser36, Asp132, Arg110 and Arg188) to the stability, catalysis and substrate specificity of Escherichia coli CMP kinase. In contrast to eukaryotic UMP/CMP kinases, which interact with the nucleobase via one or two water molecules, bacterial CMP kinase has a narrower NMP-binding pocket and a hydrogen-bonding network involving the pyrimidine moiety specific for the cytosine nucleobase. The side chains of Arg110 and Ser36 cannot establish hydrogen bonds with UMP, and their substitution by hydrophobic amino acids simultaneously affects the K(m) of CMP/dCMP and the k(cat) value. Substitution of Ser for Asp132 results in a moderate decrease in stability without significant changes in K(m) value for CMP and dCMP. Replacement of Arg188 with Met does not affect enzyme stability but dramatically decreases the k(cat)/K(m) ratio compared with wild-type enzyme. This effect might be explained by opening of the enzyme/nucleotide complex, so that the sugar no longer interacts with Asp185. The reaction rate for different modified CMP kinases with ATP as a variable substrate indicated that none of changes induced by these amino acid substitutions was 'propagated' to the ATP subsite. This 'modular' behavior of E. coli CMP kinase is unique in comparison with other NMP kinases.

Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics.

Laccases are multicopper oxidases that catalyze the oxidation of a wide range of phenols or arylamines, and their use in industrial oxidative processes is increasing. We purified from the white rot fungus Trametes versicolor a laccase that exists as five different isozymes, depending on glycosylation. The 2.4 A resolution structure of the most abundant isozyme of the glycosylated enzyme was solved. The four copper atoms are present, and it is the first crystal structure of a laccase in its active form. The crystallized enzyme binds 2,5-xylidine, which was used as a laccase inducer in the fungus culture. This arylamine is a very weak reducing substrate of the enzyme. The cavity enclosing 2,5-xylidine is rather wide, allowing the accommodation of substrates of various sizes. Several amino acid residues make hydrophobic interactions with the aromatic ring of the ligand. In addition, two charged or polar residues interact with its amino group. The first one is an histidine that also coordinates the copper that functions as the primary electron acceptor. The second is an aspartate conserved among fungal laccases. The purified enzyme can oxidize various hydroxylated compounds of the phenylurea family of herbicides that we synthesized. These phenolic substrates have better affinities at pH 5 than at pH 3, which could be related to the 2,5-xylidine binding by the aspartate. This is the first high-resolution structure of a multicopper oxidase complexed to a reducing substrate. It provides a model for engineering laccases that are either more efficient or with a wider substrate specificity.