Wastewater sequencing reveals early cryptic SARS-CoV-2 variant transmission.

As SARS-CoV-2 continues to spread and evolve, detecting emerging variants early is critical for public health interventions. Inferring lineage prevalence by clinical testing is infeasible at scale, especially in areas with limited resources, participation, or testing and/or sequencing capacity, which can also introduce biases1-3. SARS-CoV-2 RNA concentration in wastewater successfully tracks regional infection dynamics and provides less biased abundance estimates than clinical testing4,5. Tracking virus genomic sequences in wastewater would improve community prevalence estimates and detect emerging variants. However, two factors limit wastewater-based genomic surveillance: low-quality sequence data and inability to estimate relative lineage abundance in mixed samples. Here we resolve these critical issues to perform a high-resolution, 295-day wastewater and clinical sequencing effort, in the controlled environment of a large university campus and the broader context of the surrounding county. We developed and deployed improved virus concentration protocols and deconvolution software that fully resolve multiple virus strains from wastewater. We detected emerging variants of concern up to 14 days earlier in wastewater samples, and identified multiple instances of virus spread not captured by clinical genomic surveillance. Our study provides a scalable solution for wastewater genomic surveillance that allows early detection of SARS-CoV-2 variants and identification of cryptic transmission.

Crossing the Halfway Point: Aptamer-Based, Highly Multiplexed Assay for the Assessment of the Proteome.

Measuring responses in the proteome to various perturbations improves our understanding of biological systems. The value of information gained from such studies is directly proportional to the number of proteins measured. To overcome technical challenges associated with highly multiplexed measurements, we developed an affinity reagent-based method that uses aptamers with protein-like side chains along with an assay that takes advantage of their unique properties. As hybrid affinity reagents, modified aptamers are fully comparable to antibodies in terms of binding characteristics toward proteins, including epitope size, shape complementarity, affinity and specificity. Our assay combines these intrinsic binding properties with serial kinetic proofreading steps to allow highly effective partitioning of stable specific complexes from unstable nonspecific complexes. The use of these orthogonal methods to enhance specificity effectively overcomes the severe limitation to multiplexing inherent to the use of sandwich-based methods. Our assay currently measures half of the unique proteins encoded in the human genome with femtomolar sensitivity, broad dynamic range and exceptionally high reproducibility. Using machine learning to identify patterns of change, we have developed tests based on measurement of multiple proteins predictive of current health states and future disease risk to guide a holistic approach to precision medicine.

COVID-19 and the Motorcycle Taxi Sector in Sub-Saharan African Cities: A Key Stakeholders' Perspective.

This article assesses the impact of the COVID-19 outbreak on the urban motorcycle taxi (MCT) sector in Sub-Saharan Africa (SSA). MCT operators in SSA provide essential transport services and have shown ingenuity and an ability to adapt and innovate when responding to different challenges, including health challenges. However, policymakers and regulators often remain somewhat hostile toward the sector. The article discusses the measures and restrictions put in place to reduce the spread of COVID-19 and key stakeholders' perspectives on these and on the sector's level of compliance. Primary data were collected in six SSA countries during the last quarter of 2020. Between 10 and 15 qualitative interviews with key stakeholders relevant to the urban MCT sector were conducted in each country. These interviews were conducted with stakeholders based in the capital city and a secondary city, to ensure a geographically broader understanding of the measures, restrictions, and perspectives. The impact of COVID-19 measures on the MCT and motor-tricycle taxi sector was significant and overwhelmingly negative. Lockdowns, restrictions on the maximum number of passengers allowed to be carried at once, and more generally, a COVID-19-induced reduction in demand, resulted in a drop in income for operators, according to the key stakeholders. However, some key stakeholders indicated an increase in MCT activity and income because of the motorcycles' ability to bypass police and army controls. In most study countries measures were formulated in a non-consultative manner. This, we argue, is symptomatic of governments' unwillingness to seriously engage with the sector.

Multiple conformational states of the HPK1 kinase domain in complex with sunitinib reveal the structural changes accompanying HPK1 trans-regulation.

Hematopoietic progenitor kinase 1 (HPK1 or MAP4K1) is a Ser/Thr kinase that operates via the c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways to dampen the T-cell response and antitumor immunity. Accordingly, selective HPK1 inhibition is considered a means to enhance antitumor immunity. Sunitinib, a multi-receptor tyrosine kinase (RTK) inhibitor approved for the management of gastrointestinal stromal tumors (GISTs), renal cell carcinoma (RCC), and pancreatic cancer, has been reported to inhibit HPK1 in vitro In this report, we describe the crystal structures of the native HPK1 kinase domain in both nonphosphorylated and doubly phosphorylated states, in addition to a double phosphomimetic mutant (T165E,S171E), each complexed with sunitinib at 2.17-3.00-Å resolutions. The native nonphosphorylated cocrystal structure revealed an inactive dimer in which the activation loop of each monomer partially occupies the ATP- and substrate-binding sites of the partner monomer. In contrast, the structure of the protein with a doubly phosphorylated activation loop exhibited an active kinase conformation with a greatly reduced monomer-monomer interface. Conversely, the phosphomimetic mutant cocrystal structure disclosed an alternative arrangement in which the activation loops are in an extended domain-swapped configuration. These structural results indicate that HPK1 is a highly dynamic kinase that undergoes trans-regulation via dimer formation and extensive intramolecular and intermolecular remodeling of the activation segment.

Resensitization to Crizotinib by the Lorlatinib ALK Resistance Mutation L1198F.

In a patient who had metastatic anaplastic lymphoma kinase (ALK)-rearranged lung cancer, resistance to crizotinib developed because of a mutation in the ALK kinase domain. This mutation is predicted to result in a substitution of cysteine by tyrosine at amino acid residue 1156 (C1156Y). Her tumor did not respond to a second-generation ALK inhibitor, but it did respond to lorlatinib (PF-06463922), a third-generation inhibitor. When her tumor relapsed, sequencing of the resistant tumor revealed an ALK L1198F mutation in addition to the C1156Y mutation. The L1198F substitution confers resistance to lorlatinib through steric interference with drug binding. However, L1198F paradoxically enhances binding to crizotinib, negating the effect of C1156Y and resensitizing resistant cancers to crizotinib. The patient received crizotinib again, and her cancer-related symptoms and liver failure resolved. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT01970865.).

The Axl kinase domain in complex with a macrocyclic inhibitor offers first structural insights into an active TAM receptor kinase.

The receptor tyrosine kinase family consisting of Tyro3, Axl, and Mer (TAM) is one of the most recently identified receptor tyrosine kinase families. TAM receptors are up-regulated postnatally and maintained at high levels in adults. They all play an important role in immunity, but Axl has also been implicated in cancer and therefore is a target in the discovery and development of novel therapeutics. However, of the three members of the TAM family, the Axl kinase domain is the only one that has so far eluded structure determination. To this end, using differential scanning fluorimetry and hydrogen-deuterium exchange mass spectrometry, we show here that a lower stability and greater dynamic nature of the Axl kinase domain may account for its poor crystallizability. We present the first structural characterization of the Axl kinase domain in complex with a small-molecule macrocyclic inhibitor. The Axl crystal structure revealed two distinct conformational states of the enzyme, providing a first glimpse of what an active TAM receptor kinase may look like and suggesting a potential role for the juxtamembrane region in enzyme activity. We noted that the ATP/inhibitor-binding sites of the TAM members closely resemble each other, posing a challenge for the design of a selective inhibitor. We propose that the differences in the conformational dynamics among the TAM family members could potentially be exploited to achieve inhibitor selectivity for targeted receptors.

PF-06463922 is a potent and selective next-generation ROS1/ALK inhibitor capable of blocking crizotinib-resistant ROS1 mutations.

Oncogenic c-ros oncogene1 (ROS1) fusion kinases have been identified in a variety of human cancers and are attractive targets for cancer therapy. The MET/ALK/ROS1 inhibitor crizotinib (Xalkori, PF-02341066) has demonstrated promising clinical activity in ROS1 fusion-positive non-small cell lung cancer. However, emerging clinical evidence has shown that patients can develop resistance by acquiring secondary point mutations in ROS1 kinase. In this study we characterized the ROS1 activity of PF-06463922, a novel, orally available, CNS-penetrant, ATP-competitive small-molecule inhibitor of ALK/ROS1. In vitro, PF-06463922 exhibited subnanomolar cellular potency against oncogenic ROS1 fusions and inhibited the crizotinib-refractory ROS1(G2032R) mutation and the ROS1(G2026M) gatekeeper mutation. Compared with crizotinib and the second-generation ALK/ROS1 inhibitors ceritinib and alectinib, PF-06463922 showed significantly improved inhibitory activity against ROS1 kinase. A crystal structure of the PF-06463922-ROS1 kinase complex revealed favorable interactions contributing to the high-affinity binding. In vivo, PF-06463922 showed marked antitumor activity in tumor models expressing FIG-ROS1, CD74-ROS1, and the CD74-ROS1(G2032R) mutation. Furthermore, PF-06463922 demonstrated antitumor activity in a genetically engineered mouse model of FIG-ROS1 glioblastoma. Taken together, our results indicate that PF-06463922 has potential for treating ROS1 fusion-positive cancers, including those requiring agents with CNS-penetrating properties, as well as for overcoming crizotinib resistance driven by ROS1 mutation.

Conformational Studies and Atropisomerism Kinetics of the ALK Clinical Candidate Lorlatinib (PF-06463922) and Desmethyl Congeners.

Lorlatinib (PF-06463922) is an ALK/ROS1 inhibitor and is in clinical trials for the treatment of ALK positive or ROS1 positive NSCLC (i.e. specific subsets of NSCLC). One of the laboratory objectives for this molecule indicated that it would be desirable to advance a molecule which was CNS penetrant in order to treat brain metastases. From this perspective, a macrocyclic template was attractive for a number of reasons. In particular, this template reduces the number of rotatable bonds, provides the potential to shield polar surface area and reinforces binding through a restricted conformation. All of these features led to better permeability for the molecules of interest and thus increased the chance for better blood brain barrier penetration. With a CNS penetrant molecule, kinase selectivity is a key consideration particularly with regard to proteins such as TrkB, which are believed to influence cognitive function. Removal of the chiral benzylic methyl substituent from lorlatinib was perceived as not only a means to simplify synthetic complexity, but also as a strategy to further truncate the molecule of interest. Examination of the NMR of the desmethyl analogues revealed that the compound existed as a mixture of atropisomers, which proved separable by chiral SFC. The individual atropisomers were evaluated through a series of in vitro assays, and shown to have a favorable selectivity profile when compared to lorlatinib. The challenge to develop such a molecule lies in the rate at which the atropisomers interchange dictated by the energy barrier required to do this. Here, we describe the synthesis of the desmethyl macrocycles, conformational studies on the atropisomers, and the kinetics of the interconversion. In addition, the corresponding conformational studies on lorlatinib are reported providing a hypothesis for why a single diastereomer is observed when the chiral benzylic methyl group is introduced.

Effect of water solvation on the lipophilicity of isomeric pyrimidine-carboxamides.

Incorporation of nitrogen is a common medicinal chemistry tactic to reduce logD values. Neighboring group participation influences logD, so the results are isomer dependent. The logD and logP differences observed between isomeric pyrimidines 1, 2 and 3 presumably result when the carbonyl or ether lone pairs are in close proximity to a heterocyclic nitrogen lone pair, recruiting water to bridge between the electron rich atoms. Various lipophilicity calculators did not discriminate between 1 (logD=2.6) and 3 (logD=1.0), but solvation energies using Poisson-Boltzmann and 3D-RISM methods rationalize the observed differences in lipophilicity among pyrimidine carboxamide isomers.

Measurement of microenvironmental ozone concentrations in Durham, North Carolina, using a 2B Technologies 205 Federal Equivalent Method monitor and an interference-free 2B Technologies 211 monitor.

UNLABELLED: During August and September of 2012, researchers conducted a microenvironmental (ME) monitoring study in Durham, North Carolina, using two 2B Technologies O3 monitors: a dual-beam model 205 Federal Equivalent Method (FEM) 254 nm photometer and a newly developed model 211 interference-free dual-beam photometer. The two monitors were mounted in a wheeled, fan-cooled suitcase together with a battery, a disposable N2O cartridge for the model 211 monitor and filtered sample lines. A scripted technician made paired O3 measurements in a variety of MEs within 2 miles of a fixed-site FEM O3 photometer at the Durham National GuardArmory. The ratio of the 211 to Armory O3 concentrations tended to be lowest (<0.3) for 45 indoor MEs and highest (>0.8) for 104 outdoor MEs. The mean values of the ratio for in-vehicle MEs tended to fall between 0.2 and 0.7--the mean for all 27 in-car tests was 0.3. The ratio values for indoor MEs tended to be higher when the enclosure was well ventilated. The outdoor ratios tended to be lower when the measurement was made downwind of nearby roadways, likely due to exhaust NO. The in-vehicle ratios tended to be larger with windows open than closed; the smallest occurred with closed windows, active air conditioning, and vent recirculation. The 205 - 211 measurement differences were generally small, with 94% of the 176 sample differences below 5 ppb. Five differences were above 10 ppb with the largest values (173.9 and 63.6 ppb) occurring inside a violin repair shop. Roadway proximity tended to increase the differences for outdoor locations. The largest in-vehicle difference (6 ppb) occurred at a convenience store service station. As addressed in regulatory models, such differences may reduce estimated population O3 exposure by 30-50% in indoor and in-vehicle MEs where individuals spend more than 80% of their time. IMPLICATIONS: Computer models used to estimate exposures of human populations-such as the Air Pollution Exposure Model (APEX) developed by the U.S. Environmental Protection Agency-can be improved by use of direct microenvironmental (ME) measurement comparisons to nearby fixed-site monitors used for determining regulatory compliance. Simultaneous measurements made by model 211 and model 205 ozone monitors in a variety of MEs indicated that Federal Equivalent Method photometers similar to the model 205 may read high in the presence of various interferences associated with indoor sources and motor vehicles, increasing modeled exposures in such environments by 20-100%.

Concentrations of mobile source air pollutants in urban microenvironments.

Human exposures to criteria and hazardous air pollutants (HAPs) in urban areas vary greatly due to temporal-spatial variations in emissions, changing meteorology, varying proximity to sources, as well as due to building, vehicle, and other environmental characteristics that influence the amounts of ambient pollutants that penetrate or infiltrate into these microenvironments. Consequently, the exposure estimates derived from central-site ambient measurements are uncertain and tend to underestimate actual exposures. The Exposure Classification Project (ECP) was conducted to measure pollutant concentrations for common urban microenvironments (MEs) for use in evaluating the results of regulatory human exposure models. Nearly 500 sets of measurements were made in three Los Angeles County communities during fall 2008, winter 2009, and summer 2009. MEs included in-vehicle, near-road, outdoor and indoor locations accessible to the general public. Contemporaneous 1- to 15-min average personal breathing zone concentrations of carbon monoxide (CO), carbon dioxide (CO2), volatile organic compounds (VOCs), nitric oxide (NO), nitrogen oxides (NO(x)), particulate matter (< 2.5 microm diameter; PM2.5) mass, ultrafine particle (UFP; < 100 nm diameter) number black carbon (BC), speciated HAPs (e.g, benzene, toluene, ethylbenzene, xylenes [BTEX], 1,3-butadiene), and ozone (O3) were measured continuously. In-vehicle and inside/outside measurements were made in various passenger vehicle types and in public buildings to estimate penetration or infiltration factors. A large fraction of the observed pollutant concentrations for on-road MEs, especially near diesel trucks, was unrelated to ambient measurements at nearby monitors. Comparisons of ME concentrations estimated using the median ME/ambient ratio versus regression slopes and intercepts indicate that the regression approach may be more accurate for on-road MEs. Ranges in the ME/ambient ratios among ME categories were generally greater than differences among the three communities for the same ME category, suggesting that the ME proximity factors may be more broadly applicable to urban MEs. Implications: Estimates of population exposure to air pollutants extrapolated from ambient measurements at ambient fixed site monitors or exposure surrogates are prone to uncertainty. This study measured concentrations of mobile source air toxics (MSAT) and related criteria pollutants within in-vehicle, outdoor near-road, and indoor urban MEs to provide multipollutant ME measurements that can be used to calibrate regulatory exposure models.

Design and Synthesis of Functionally Active 5-Amino-6-Aryl Pyrrolopyrimidine Inhibitors of Hematopoietic Progenitor Kinase 1.

Immune activating agents represent a valuable class of therapeutics for the treatment of cancer. An area of active research is expanding the types of these therapeutics that are available to patients via targeting new biological mechanisms. Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of immune signaling and a target of high interest for the treatment of cancer. Herein, we present the discovery and optimization of novel amino-6-aryl pyrrolopyrimidine inhibitors of HPK1 starting from hits identified via virtual screening. Key components of this discovery effort were structure-based drug design aided by analyses of normalized B-factors and optimization of lipophilic efficiency.

The Choice of Osteosynthesis for Distal Radius Fractures: A Matter of Taste, Fracture Instability, or Patient-Related Factors? A Retrospective Study of Functional Outcome in 346 Distal Radius Fractures Operated With Percutaneous Wires or Volar Plate Fixation.

BACKGROUND: Surgery with volar locking plate (VLP) for distal radius fractures (DRFs) has become dominant over percutaneous Kirschner wire (K-wire) (PKW) fixation. Not many studies have proved advantages of the VLP and the increasing dominance of the VLP is thus not derived from evidence of superiority but influenced by other factors. METHODS: By retrospectively classifying 346 DRFs treated with either PKW or VLP fixation, according to the Buttazzoni classification system, we aimed to investigate the determining factors for choice of surgical method, and by review of the patients' medical records, the functional outcome, duration, and frequentness of the rehabilitation period were correlated to Buttazzoni type and surgical method. RESULTS: The odds ratio of having volar plate fixation was negatively correlated to age and positively correlated to a higher Buttazzoni type. We found no clinically significant differences in the functional outcome for different Buttazzoni type of fractures within the VLP and PKW groups, respectively, nor between the 2 methods of surgery for any Buttazzoni type of fracture. CONCLUSION: Younger patients and fractures with higher grade of instability were more likely to be treated with VLP than PKW; however, neither fracture instability nor surgical method had any impact on functional outcome.

LTK fusions: A new target emerges in non-small cell lung cancer.

Identification of targetable fusions as oncogenic drivers in non-small cell lung cancer has transformed its diagnostic and therapeutic paradigm. In a recent article in Nature, Izumi et al. report the discovery of CLIP1-LTK fusion as a novel oncogenic driver in lung cancer, targetable using the ALK tyrosine kinase inhibitor lorlatinib.

Analysis of lorlatinib analogs reveals a roadmap for targeting diverse compound resistance mutations in ALK-positive lung cancer.

Lorlatinib is currently the most advanced, potent and selective anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor for the treatment of ALK-positive non-small cell lung cancer in the clinic; however, diverse compound ALK mutations driving therapy resistance emerge. Here, we determine the spectrum of lorlatinib-resistant compound ALK mutations in patients, following treatment with lorlatinib, the majority of which involve ALK G1202R or I1171N/S/T. We further identify structurally diverse lorlatinib analogs that harbor differential selective profiles against G1202R versus I1171N/S/T compound ALK mutations. Structural analysis revealed increased potency against compound mutations through improved inhibition of either G1202R or I1171N/S/T mutant kinases. Overall, we propose a classification of heterogenous ALK compound mutations enabling the development of distinct therapeutic strategies for precision targeting following sequential tyrosine kinase inhibitors.

Wastewater sequencing uncovers early, cryptic SARS-CoV-2 variant transmission.

As SARS-CoV-2 continues to spread and evolve, detecting emerging variants early is critical for public health interventions. Inferring lineage prevalence by clinical testing is infeasible at scale, especially in areas with limited resources, participation, or testing/sequencing capacity, which can also introduce biases. SARS-CoV-2 RNA concentration in wastewater successfully tracks regional infection dynamics and provides less biased abundance estimates than clinical testing. Tracking virus genomic sequences in wastewater would improve community prevalence estimates and detect emerging variants. However, two factors limit wastewater-based genomic surveillance: low-quality sequence data and inability to estimate relative lineage abundance in mixed samples. Here, we resolve these critical issues to perform a high-resolution, 295-day wastewater and clinical sequencing effort, in the controlled environment of a large university campus and the broader context of the surrounding county. We develop and deploy improved virus concentration protocols and deconvolution software that fully resolve multiple virus strains from wastewater. We detect emerging variants of concern up to 14 days earlier in wastewater samples, and identify multiple instances of virus spread not captured by clinical genomic surveillance. Our study provides a scalable solution for wastewater genomic surveillance that allows early detection of SARS-CoV-2 variants and identification of cryptic transmission.

Atypical Presentations among Older Adults with COVID-19 Disease: A Need for Broadening the Differential Diagnosis.

Typical presenting symptoms of COVID-19 have been reported to be common in older adults. Current guidelines by the World Health Organization (WHO) and Centers for Disease Control (CDC) for testing and diagnosis are based on the presence of these typical symptoms. Several older adults seen at our hospital have presented atypically with symptoms such as delirium, falls, increasing the need for attention to diagnostic protocols since this has significant implications for early detection and patient outcomes, infection control and promotion of safety among healthcare providers. With the increased risk of fatality among older adults with COVID-19, appropriate diagnostic protocols are needed to ensure early diagnosis and management. Recognizing these atypical presentations in nursing homes would also facilitate early screening and cohorting in these congregate living facilities where older adults have had disproportionately high morbidity and mortality rates. We present two patients who presented with delirium and falls, found to have COVID-19 infection.

Spectrum of Mechanisms of Resistance to Crizotinib and Lorlatinib in ROS1 Fusion-Positive Lung Cancer.

PURPOSE: Current standard initial therapy for advanced, ROS proto-oncogene 1, receptor tyrosine kinase fusion (ROS1)-positive (ROS1+) non-small cell lung cancer (NSCLC) is crizotinib or entrectinib. Lorlatinib, a next-generation anaplastic lymphoma kinase/ROS1 inhibitor, recently demonstrated efficacy in ROS1+ NSCLC, including in crizotinib-pretreated patients. However, mechanisms of lorlatinib resistance in ROS1+ disease remain poorly understood. Here, we assessed mechanisms of resistance to crizotinib and lorlatinib. EXPERIMENTAL DESIGN: Biopsies from patients with ROS1 + NSCLC progressing on crizotinib or lorlatinib were profiled by genetic sequencing. RESULTS: From 55 patients, 47 post-crizotinib and 32 post-lorlatinib biopsies were assessed. Among 42 post-crizotinib and 28 post-lorlatinib biopsies analyzed at distinct timepoints, ROS1 mutations were identified in 38% and 46%, respectively. ROS1 G2032R was the most commonly occurring mutation in approximately one third of cases. Additional ROS1 mutations included D2033N (2.4%) and S1986F (2.4%) post-crizotinib and L2086F (3.6%), G2032R/L2086F (3.6%), G2032R/S1986F/L2086F (3.6%), and S1986F/L2000V (3.6%) post-lorlatinib. Structural modeling predicted ROS1L2086F causes steric interference to lorlatinib, crizotinib, and entrectinib, while it may accommodate cabozantinib. In Ba/F3 models, ROS1L2086F, ROS1G2032R/L2086F, and ROS1S1986F/G2032R/L2086F were refractory to lorlatinib but sensitive to cabozantinib. A patient with disease progression on crizotinib and lorlatinib and ROS1 L2086F received cabozantinib for nearly 11 months with disease control. Among lorlatinib-resistant biopsies, we also identified MET amplification (4%), KRAS G12C (4%), KRAS amplification (4%), NRAS mutation (4%), and MAP2K1 mutation (4%). CONCLUSIONS: ROS1 mutations mediate resistance to crizotinib and lorlatinib in more than one third of cases, underscoring the importance of developing next-generation ROS1 inhibitors with potency against these mutations, including G2032R and L2086F. Continued efforts are needed to elucidate ROS1-independent resistance mechanisms.

The temporal patterning microRNA let-7 regulates several transcription factors at the larval to adult transition in C. elegans.

The let-7 microRNA is phylogenetically conserved and temporally expressed in many animals. C. elegans let-7 controls terminal differentiation in a stem cell-like lineage in the hypodermis, while human let-7 has been implicated in lung cancer. To elucidate let-7's role in temporal control of nematode development, we used sequence analysis and reverse genetics to identify candidate let-7 target genes. We show that the nuclear hormone receptor daf-12 is a let-7 target in seam cells, while the forkhead transcription factor pha-4 is a target in the intestine. Additional likely targets are the zinc finger protein die-1 and the putative chromatin remodeling factor lss-4. Together with the previous identification of the hunchback ortholog hbl-1 as a let-7 target in the ventral nerve cord, our findings show that let-7 acts in at least three tissues to regulate different transcription factors, raising the possibility of let-7 as a master temporal regulator.

4-methylpteridinones as orally active and selective PI3K/mTOR dual inhibitors.

Pteridinones were designed based on a non-selective kinase template. Because of the uniqueness of the PI3K and mTOR binding pockets, a methyl group was introduced to C-4 position of the peteridinone core to give compounds with excellent selectivity for PI3K and mTOR. This series of compounds were further optimized to improve their potency against PI3Kα and mTOR. Finally, orally active compounds with improved solubility and robust in vivo efficacy in tumor growth inhibition were identified as well.