While population declines among Adélie penguins and population increases among gentoo penguins on the Western Antarctic Peninsula are well established, the logistical challenges of operating in the sea ice-heavy northern tip of the Antarctic Peninsula have prohibited reliable monitoring of seabirds in this region. Here we describe the findings of an expedition to the northern and eastern sides of the Antarctic Peninsula—a region at the nexus of two proposed Marine Protected Areas—to investigate the distribution and abundance of penguins in this region. We discovered several previously undocumented penguin colonies, completed direct surveys of three colonies initially discovered in satellite imagery, and re-surveyed several colonies last surveyed more than a decade ago. Whereas our expectation had been that the Peninsula itself would divide the areas undergoing ecological transition and the apparently more stable Weddell Sea region, our findings suggest that the actual transition zone lies in the so-called "Adélie gap," a 400-km stretch of coastline in which Adélies are notably absent. Our findings suggest that the region north and east of this gap represents a distinct ecoregion whose dynamics stand in sharp contrast to surrounding areas and is likely to be impacted by future conservation measures. One of the notable features of penguin range distributions in this region is the complete absence of breeding Adélie penguins over a 400-km stretch extending between Anvers Island in the south and King George Island and the Duroch Islands on the west coast of Trinity Peninsula—a feature known as the "Adélie gap"5 Sea Fig. . Biologi14. In contrast, in the Weddell Sea, overall sea-ice extent showed moderate increases between 1979 and 2015, before more recent and sudden decreases beginning in 201617. Kumar et al.16 found that a reduced sea ice extent year in 2015/16 closely followed a strong El Nino Southern Oscillation (ENSO) event, an irregular periodic variation in winds and sea surface temperatures over the tropical eastern Pacific Ocean that affects the climate of much of the tropics and subtropics. Coincident with the ENSO, a positive phase of the Southern Annual Mode co-occurred that same year, causing major ice loss and potentially shifting the Weddell Sea into an alternative state of sea-ice decline16. Moreover, the Weddell Sea gyre may be perpetuating a stabilizing negative feedback loop, where a decrease in sea ice leads to a strengthening of the gyre, resulting in surface temperatures that support higher sea-ice concentration18. Under the assumption of relative stability in annual sea-ice extent, the Weddell Sea has been projected to have increasingly suitable habitat for Antarctic krill, while the WAP is expected to have an increase in habitat in the spring, but a decline in the summer and fall19.Waters off the WAP have experienced notable declines in annual sea-ice extent over the past several decades20, trends remain less well-understood among the eastern populations in the Weddell Sea region, the northeastern-most area of their breeding range along the Peninsula. Several years ago, satellite-based observations of penguin guano in the Danger Islands archipelago, near the eastern tip of Joinville Island, motivated a major expedition to the region21. In addition to finding several large Adélie colonies not previously documented, the expedition discovered that 55% of all Adélie penguins in the Antarctic Peninsula region nest in the Danger Islands, making it an unexpected hotspot of Adélie abundance in a region of Antarctica projected to remain favorable for Adélie reproduction even under forecasted climate change scenarios22. Importantly, Borowicz et al.21 compared recent high-resolution satellite imagery with older Landsat imagery and historic aerial photography, finding evidence that Adélie populations in the Danger Islands appeared broadly stable over the last 60 years, in marked contrast to the long-reported Adélie population declines on the WAP23, and in areas of the South Shetland24 and South Orkney Islands25. This apparent dichotomy between the trends of Adèlie penguins on the east and west sides of the Antarctic Peninsula, alongside the effort to establish two Marine Protected Areas in the region28, motivated an expedition in the 2021/22 austral summer to the lesser-surveyed areas of the northern Peninsula and Weddell Sea.An ongoing question is the extent to which differences in habitat and climate patterns along the Eastern Antarctic Peninsula differ relative to the WAP. While it is well established that Adélie penguin populations along the WAP and sub-Antarctic islands, have declined sharply over the last several decadesPygoscelis papua), a historically sub-antarctic species of Pygoscelis penguin, have shown consistent southward range expansions along the Peninsula and concurrent increases in population size30. While previous work has shown that Adélie penguins rely on areas that experience extensive annual sea ice, an important over-wintering habitat for their primary dietary staple (krill), gentoo penguins are known for greater flexibility in diet composition and foraging behaviour32, and occupy regions characterized by reduced ice-cover33. As a resident species that remains in the vicinity of the breeding colony year round, gentoos are particularly sensitive to winter sea ice conditions, as extensive winter sea ice coverage near their breeding colony could preclude easy access to the ocean required for winter foraging8. So, while declining sea-ice extent around the WAP may be beneficial for gentoo penguin expansion, the relatively icy environment of the Weddell Sea, particularly during the overwintering months, may be less hospitable.In contrast to patterns of Adélie penguin population decline throughout much of the Antarctic Peninsula, gentoo penguins  prior to our arrival. Penguin chick fraction was estimated using portions of the RPAS captured scenes at each site where chicks could be unambiguously identified . This expedition was permitted under Antarctic Conservation Act Permit ACA 2022-017 and ACA 2022-018. An Initial Environmental Evaluation was approved by the US Environmental Protection Agency on 14 October 2021, and Advance Notification provided to the US Department of State.24. Wherever possible, observers maintained a minimum of 5 m distance from the penguin colony to minimize disturbance of wildlife. Colonies were surveyed by direct counting of penguin chicks using handheld tally counters. To ensure count accuracy, penguin chicks were counted individually three times, with the requirement of counts being within 5% of their mean. Using this method, the mean number of chicks was assumed to be accurate to ± 5%, the accuracy threshold corresponding to the ’N1’ level of precision described by Croxall and Kirkwood35 and used regularly to report penguin abundance in the Antarctic38.Manual ground counts were conducted by experienced observers following previously-established methods and proceduresRPAS surveys were performed using a DJI Phantom 4 Pro V2  quadcopter using the stock CMOS FC6310S camera. Depending on conditions, the RPAS was flown either manually or automatically using DJI Ground Station Pro. At each survey site, weather permitting, a RPAS team consisting of a Federal Avaiation Administration licensed remote pilot and an observer conducted RPAS survey operations. While the pilot operated the RPAS, the observer maintained visual contact with the RPAS at all times, in accordance with US FAA regulations. At one location, challenging terrain precluded landing and executing a ground-based RPAS survey. In this case, the RPAS was deployed from a rigid-hulled inflatable boat. In all other locations, surveys were conducted on land within the vicinity of the colony.https://www.dji.com/ground-station-pro). Images were captured every 2 s , with a constant velocity of 3 m/s (low speed), to minimize motion blur and to ensure sharp and correctly-exposed images. Flight heights were determined by the geography, take off locations, and ruggedness of the terrain but were no less than 30 m above the colony. No disturbance to nesting penguins was observed during flight operations. Image collection parameters were set to obtain image overlap of 70% in both front and side directions to ensure reliable alignment of images and to reduce the distortions in the resulting orthomosaic data products39. After completing each RPAS survey, RPAS-captured images were processed on Agisoft PhotoScan Professional  commercial photogrammetry software using structure-from-motion processing40.RPAS-captured images were collected along flight transects using the autonomous flight planning software DJI Ground station Pro . At two sites, Cockburn Island and Eden Rocks, image clarity was insufficient to separate adults from chicks in some areas. To circumvent this issue, analysts counted every penguin at each colony regardless of age class, then selected a sample of high quality image sections from each colony and differentiated between penguin adults and chicks to obtain the ratios of chicks to adults. The ratios were then applied across each colony to estimate the total number of chicks at each site. This process was also employed for Devil Island, a colony where accurate hand counts were obtained the same day as the RPAS survey. This allowed us to compare our extrapolation method to the more traditional method of hand counting chicks from the ground.41 and acquired via the US National Snow and Ice Data Center42. Sea-ice concentration products used in this study were derived from two data sets: The Near-Real-Time Defense Meteorological Satellite Program (DMSP) Special Sensor Microwave Imagery Sounder (SSMIS) Daily Polar Gridded sea-ice concentrations and the sea-ice concentrations from Nimbus-7 Scanning Multichannel Microwave Radiometer Passive Microwave data. A 25 km × 25 km land mask was applied to all sea-ice concentration rasters to eliminate pixels with readings from both sea ice and land ice cover. We grouped adjacent penguin colonies into three sub-regions .To investigate patterns of change in sea-ice concentration across regions of the Antarctic Peninsula through time 1979–2021) data, we used 25 km × 25 km resolution gridded monthly sea-ice concentration (the fraction of ocean surface covered by ice) product obtained from satellite-based passive-microwave sensors. The data were processed by the NASA Team algorithm979–2021 i) throughout the study period (1979–2018); we assumed region-specific intercepts (βj0 ) and slopes (βj1 ) as followsLinear models were used to fit annual and quarterly sea-ice concentration (SIC) in each Year . We used three Markov Chain Monte Carlo (MCMC) sequences from the posterior distribution of each model parameter. Posterior distributions were derived using three chains with 5000 samples (after applying a thinning rate of 4 from each respective chain) following a "burn-in" period of 5500 draws . Model convergence was assessed using a visual analysis of the posterior chains, in addition to the use of the Gelman-Rubin convergence diagnostic46. Posterior analysis indicated that models unambiguously converged. Parameter estimate plots were generated using the "MCMCvis" package47, while other plots were generated using the "ggplot2" package48 in the R statistical environment. Posterior predictive checks of estimated parameters were calculated using the ppcheck function in the "jagsUI" package49.Models were fit in a Bayesian framework using the R package "rjags"12. Five sites  had been previously surveyed at least once by a ground-based science team.We surveyed nine Adélie penguin sites around the northern tip of the Antarctic Peninsula and into the Weddell Sea in January and February of 2022 Fig. . At the At Cockburn Island and Eden Rocks, surveyed solely by RPAS in this study, the total abundance was estimated by multiplying the total number of individuals (chicks and adults) visible at the site by an estimate of the fraction of all individuals that were chicks. At Cockburn Island, the fraction of chicks was estimated at 65% and our final estimate of the number of chicks at this site during the survey was 4274. At Eden Rocks, which was surveyed 2 days later, the fraction of chicks was 49%. Devil Island was surveyed by both RPAS and manual field counting, the latter of which provided a means of comparing the RPAS-derived estimate to the count completed in the field. At Devil Island, the chick fraction was 48%, yielding a chick estimate of 14,521. This RPAS-based estimate was 21% higher than the ground-based count of 11,978. We designated the RPAS counts as N3, meaning they are estimated to be within 10–25% of the true population size.r = 0.08  over the period from 1996 to 2021. Vortex Island, Eden Rocks, and Tay Head, and Penguin Point did not show significant trends in the number of breeding pairs over their survey histories . See Table The number of breeding pairs on Devil Island grew at an annual rate of  0.03, 0.2) over t9, were documented breeders. Unfortunately, we were not able to conduct a detailed survey to establish how many gentoo penguin pairs are currently breeding at this location. While gentoo penguins were the only penguin species breeding on Cape Scrymgeour, the southernmost Weddell Sea gentoo penguin colony surveyed during this expedition (63.566 °S), they co-occurred with Adélie penguins on Northeast of D’Urville, Medley Rocks, and North of Medley Rocks, and with Adélie and chinstrap penguins on Wideopen Islands. See Table We counted 3694 gentoo penguin chicks across four breeding colonies. At the time of our survey, penguin chicks had already begun créching so individual chick counts were performed rather than nest counts. Two of the sites, Cape Scrymgeour and Northeast of D’Urville, had not previously been documented, and had 75 and 1917 chicks respectively. We identified the penguins previously noted (but not identified to species) at Medley Rocks  and North of Medley Rocks  as gentoo penguins with abundances of 1658 and 44 gentoo penguin chicks, respectively. We also confirmed the presence of breeding gentoo penguins (with chicks) at Wideopen Islands, a site where Adélie and chinstrap, but not gentoo, penguins35 or were identified in Landsat imagery as having guano-like stains that might reflect penguin colonies . Our expedition was able to confirm the absence of penguin colonies at the following locations: Correy Island, Beak Island, and Red Island. We were unable to locate a previously reported penguin colony on Dundee Island nearby Petrel Station, and though the exact location of the historical account is unclear, we do not believe the colony remains. We also confirmed that Rosamel Island and Cape Gordon on Vega Island, the latter of which was thought might contain breeding seabirds based on Landsat satellite imagery, have no breeding seabirds.In addition to the surveys reported in Table We analyzed annual and quarterly patterns  of sea-ice concentration across the three regions: (1) South Shetland Islands, (2) ’northeast-of-gap’, and (3) ’southwest-of-gap’) from 1979 to 2021 , our expedition found a small gentoo colony at Cape Scrymgeour that appears to be a new colonization and, with the exception of a single nest reported from nearby Jade Point, the most southerly colony of this species on the Weddell Sea side of the Peninsula. Based on patterns of growth of new gentoo colonies50, we estimate that the Cape Scrymgeour population has been in existence at least 5 years and possibly longer absent continued immigration from other established populations. While gentoo penguin population expansions along their southern range margin on the western side have been well reported29, our findings provide further evidence of gentoo range expansion along the southern margins of their range east of the Antarctic Peninsula in the northern Weddell Sea.This survey expands the knowledge of Adélie and gentoo penguin abundance throughout the northern tip of the Antarctic Peninsula region and includes previously unsurveyed colony locations. Based on our survey estimates, we find that the Adélie penguin populations northeast and northwest of the so-called “Adélie gap” have remained stable or even increased over the last 30–40 years despite dramatic declines across colonies farther south along the western side of the Antarctic Peninsula. While Adélie and gentoo penguin habitat preferences are usually considered a zero-sum game in which the former climate change "loser" declines in step with increases in the latter—considered a climate change "winner"—we find that both species appear to be flourishing northeast and northwest of the Adélie gap. While previous studies have documented growing or stable gentoo populations at established colonies in this region  populations, important prey species for Adélie penguins58, as sea ice provides spawning habitat, food resources and nursery regions for eggs and early life stages61. Chapman et al.62 showed that silverfish in Adélie penguin diet plays an important role in the survival of fledged chicks and La Mesa et al.64 found that decreased silverfish abundance in the southern WAP was correlated with decreases in sea ice. In this context, it is important to highlight the striking asymmetry in annual and quarterly sea ice trends. The magnitude of decline in sea-ice concentration observed across penguin colonies south of the Adélie gap was considerably larger in all but winter (July–September) relative to trends observed northeast-of-gap and in the South Shetland Islands. Along the WAP, substantial declines in Adélie penguin population abundance has been associated with multi-year and decadal scale trends of reduced lower sea-ice concentrations. While the logistical difficulties of tracking Adélie penguins during the overwinter period unavoidably limits our understanding of winter foraging behavior and its relationship to environmental conditions, it’s clear that sea ice can have both direct and indirect influences on winter foraging habitat with concomitant changes in the size of breeding colonies through time. While much of the discussion has focused on Adélie losses, our findings provide hope that where sea ice is being maintained, Adélie populations remain robust.The findings of this survey provide further support for existence of two separate and geographically distinct populations of Adélie penguins. The apparent asymmetry of Adélie penguin abundance observed north and east of the “Adélie gap” was initially hypothesized by Fraser et al.Despite the seemingly intuitive connection between sea ice and population trends, we recognize the challenge of suggesting any direct mechanistic relationship that such an analysis may provide in the absence of higher resolution datasets. The 25 km × 25 km spatial resolution of the passive microwave data used is likely inadequate to identify the precise mechanisms at play as ice-dependent wildlife interact with sea ice on an extremely localized basis. The characteristics of sea ice that might influence individual and group decisions about movement, foraging, or reproduction occur at scales far smaller than the resolution of most publicly available sea-ice data products. With this consideration in mind, we believe future investigations may benefit by incorporating newer satellite-based platforms such as the European Space Agency’s Sentinel-1 or NASA’s ICESat-2.66. With consideration to the large and apparently stable Adélie penguin populations in the northeast-of-gap region, as well as potential for further colonization by expanding gentoo penguins, the northeast-of-gap region deserves particular consideration in the design and implementation of conservation measures. Of particular relevance is the Domain 1 MPA planning process, which has already indicated the northeast-of-gap area as being of high conservation value28. Moreover, evidence for more stable sea ice dynamics Northeast-of-gap relative to the region south of the Adélie gap is consistent with the idea that this region, especially further around the tip of the Peninsula into the Weddell Sea, may act as a habitat refuge under future climate change that deserves particular protection from other, non climate-related, disturbances.Previous work already identified the northern Weddell Sea as a penguin hotspot of significant conservation value21. In particular, the phenology of breeding at the time of the survey plays an important role in the ease with which survey images can be used to estimate abundance. Ideally, surveys should be completed during nest incubation, when each nest is attended by a single stationary adult. By contrast, our surveys occurred later in the season when the site was comprised of both adults and large chicks. Adults and chicks could be distinguished in some portions of the imagery, and while we were able to use this to estimate the percentage of all individuals that were chicks, a more accurate survey would have been achieved if we had been able to visit the study locations nearer to late December or early January.The use of RPAS for penguin surveys is rapidly becoming a standard tool in Antarctic wildlife surveys, and while some of our surveys simply could not have been completed using manual counts alone , the unavoidable delay in survey work due to the Covid-19 pandemic created challenges in the use of RPAS-based imagery not shared by earlier expeditions we had undertaken in the same region in previous yearsSupplementary Information.


Motor vehicle crashes are a leading cause of injury and death for older adults in the United States. Braking reaction time (BRT), how quickly a driver responds to the situational demands of driving, is a known predictor of driver fitness and hypothesized as sensitive to difficulties with executive functioning. Unclear is how BRT may vary across different levels of executive functioning, including objective test performance and perceived executive dysfunction, and if awareness of executive dysfunction in the presence of objective difficulties jointly predicts BRT. Using data from a simulated driving study, 50 adults aged 65-94 years old  completed computerized EF tests , the Behavior Rating Inventory of Executive Function, the Big Five Inventory, and a simulated drive. Multilevel modeling analyses controlling for covariates of empirical and design relevance  were performed. Our results show a significant interaction between perceived executive dysfunction and objective test performance on BRT. Specifically, associations with BRT were stronger for individuals who perceived more executive dysfunction in the presence of worse inhibition  and worse working memory . Findings provide further justification for the role of executive functioning in monitoring driver fitness in older age. Future directions and implications are discussed.


G12C shows increased antitumoractivity in a preclinical model of KRASG12C mutant NSCLC that exhibits resistance to KRASG12C inhibitor monotherapy.Hyperactivation of mTOR kinase by mutations in the PI3K/mTORpathwayor by crosstalk with other mutant cancer drivers, such as RAS, isa feature of many tumors. Multiple allosteric inhibitors of mTORC1and orthosteric dual inhibitors of mTORC1 and mTORC2 have been developedas anticancer drugs, but their clinical utility has been limited.To address these limitations, we have developed a novel class of “bi-stericinhibitors” that interact with both the orthosteric and theallosteric binding sites in order to deepen the inhibition of mTORC1while also preserving selectivity for mTORC1 over mTORC2. In thisreport, we describe the discovery and preclinical profile of the developmentcandidate RMC-5552 and the in vivo preclinical tool compound RMC-6272.We also present evidence that selective inhibition of mTORC1 in combinationwith covalent inhibition of KRAS However, it was over a decade before a detailedmechanistic understanding of the biological activity for rapamycinbegan to be revealed. Rapamycin and a structurally related immuno-suppressivenatural product FK-506 (tacrolimus) 2 into thenucleus.22 In contrast, rapamycin 1, although related in its chemical structure to FK-506 2, was recognized as having a differing mechanism of action.23 Studies in yeast identified a gene encoding for a homologue of FKBPtogether with two additional genes that participate in rapamycin biology.24 The genes were named Target of Rapamycin 1 and2 (TOR1 and TOR2) with a suggestion that subunits of rapamycin-FKBP12and TOR interacted as a protein complex.24 Thereafter, independent studies identified a mammalian homologueof TOR.27 In time, this homologue became known as mammalianTarget of Rapamycin (mTOR) or, more recently, mechanistic Target ofRapamycin.28Rapamycin  olimus) 2,17 bind 29 sitting downstream of receptor tyrosine kinase(RTK) and phosphatidylinositol 3-kinase (PI3K) signaling. mTOR containskinase31 and FKBP12-rapamycin-binding (FRB) domains,32 along with multiple N-terminalHuntington elongation factor 1A-protein phosphatase 2A-A subunit-TOR(HEAT) repeats, FAT , and C-terminal FAT (FATC)domains, among other important structural elements.33 mTOR is recognized to form two distinct complexes, knownas (rapamycin-sensitive) mTOR complex 1 (mTORC1) and (rapamycin-insensitive)mTOR complex 2 (mTORC2).34 mTORC1 coordinateswith auxiliary protein Regulatory-associated protein of mTOR (Raptor)35 to phosphorylate multiple substrates, includingribosomal protein S6 kinase (S6K) and eukaryotic initiation factor4E-binding protein 1 (4EBP1).39 Phosphorylation of 4EBP1 releases eIF4E, relievinginhibition of cap-dependent translation and driving growth and proliferationof normal and cancer cells.40 mTORC2 isdefined by the presence of rapamycin-insensitive companion of mTOR(Rictor) and mammalian stress-activated protein kinase interactingprotein 1 (Sin1)43 and phosphorylates and activates AKT in responseto cellular stimuli, including activation of PI3K.44 mTORC2 activation leads to effects on glucose metabolism,proliferation, cell survival, and growth.45mTOR is a 289 kDa serine/threonine protein kinasebelonging tothe phosphatidylinositol 3-kinase-related kinases (PIKK) family1 being approved as an immunosuppressantin 1999.46 More recently, mTOR inhibitionas a cancer therapeutic has been of particular interest, especiallyin the context of effects upon the 4EBP1-eIF4E axis.40 There are three broad classes of mTOR inhibitors.47 Rapamycin and its analogs are first-generation mTOR inhibitors and inhibit mTORC1-mediated phosphorylationof S6K but only weakly inhibit phosphorylation of 4EBP1 and have negligibleeffects on mTORC2.48 Second-generationmTOR inhibitors, such as sapanisertib , are ATP-competitiveinhibitors of the mTOR kinase and thus inhibit phosphorylation ofsubstrates of mTORC1 and mTORC2, including S6K, 4EBP1, and AKT. Theclinical activity of second-generation mTOR inhibitors remains marginal,potentially due to dose-limiting toxicities that prevent optimal inhibitionof 4EBP1 phosphorylation in the clinical setting.49 In 2016, Shokat and colleagues introduced a third generationof mTOR inhibitor, exemplified by RapaLink-1 3,51 which links an FKBP12-FRB allosteric mTOR inhibitor based on rapamycintogether with an active-site (orthosteric) inhibitor, based on sapanisertib.53 RapaLink-1 3 inhibits mTOR activity more potently thanother mTOR inhibitors, overcomes some mechanisms of resistance, andalso shows an approximate 3–4-fold selectivity for inhibitionof mTORC1 over mTORC2, as illustrated by inhibition of phosphorylationof 4EBP1 (IC50 = 1.7 nM) over inhibition of phosphorylationof AKT (IC50 = 6.7 nM) in MDA-MB-468 cells. The therapeuticpotential of a bi-steric inhibitor such as RapaLink-1 3 inspired our own interest in mTOR inhibition.66 Our aim was to design a compound with further enhanced selectivityfor mTORC1 over mTORC2. Such a compound would potently inhibit bothS6K and 4EBP1 phosphorylation while limiting unwanted effects on glucosemetabolism and relief of AKT-dependent feedback inhibition of receptortyrosine kinase (RTK) expression that result from inhibition of mTORC2.70 In this paper, we build upon the findings with RapaLink-1 3 to describe how each component of the bi-steric molecule can be modified to obtain a compound with enhancedselectivity for mTORC1 over mTORC2. Such mTORC1-selective bi-stericinhibitors also demonstrate selectivity over related off-target lipidkinases and inhibition of mTORC1-mediated substrate phosphorylationin tumors, which translated to antitumor activity in xenograft modelsas a single agent and in combination with other targeted inhibitors.Our work culminated in the discovery of a development candidate RMC-5552 38, which is currently undergoing evaluation in clinical studies.71Inhibition of mTOR has been of historical interest to thepharmaceuticalindustry, with rapamycin 1 is a 31-membered lactam–lactone macrolidecontaining a rich density of carbon–oxygen functional groups,numerous stereodefined olefins, including an acid- and air-sensitiveconjugated triene, and a delicate beta-keto lactoneat the C32 carbonyl that is particularly prone to base-induced eliminationof pipecolinate (vide infra).73 Functionalization ofrapamycin to introduce linking groups en route to bi-steric finalcompounds optimally occurs at precise locations without affectingthe stereochemical integrity of the 15-chiral centers nor initiatingbase- or acid-catalyzed degradation pathways.75 To complicate matters further,rapamycin and close analogs exist as two interconverting hemiketalstructural isomers.76The synthesis of bi-steric compounds containinga modified rapamycinunit as a key constituent presents numerous challenges. For example,rapamycin 77 and alkylationof this position has been used to prepare the approved compound everolimus 4.78 In addition, functionalizationof the C40 oxygen in rapamycin was also used to prepare RapaLink-1 3.51 Building upon these results,we prepared numerous bi-steric inhibitors using the chemistry outlinedin 10 component was prepared from rapamycin 1 (30%yield) by alkylation with freshly purified hex-5-yn-1-yl trifluoromethanesulfonateand 2,6-di-tert-butyl-4-methylpyridine  carbonate.80 For rapamycin itself, the C40-PNP carbonate 13 was synthesized in 59% yield from rapamycin 1 and p-nitrophenyl chloroformate using pyridineas base  domain of Rictor sits on top ofthe mTOR FRB domain in mTORC2, blocking the binding of FKBP-rapamycinto mTORC2 and explaining mTORC2 insensitivity to rapamycin.88 These studies supported our rationale that thestructural differences between mTORC1 and mTORC2 could be exploitedfurther to increase the selectivity of a bi-steric hybrid. Therefore,we applied a rational drug design strategy to improve on the selectivityby systematically tuning the affinities of the rapamycin core andactive-site binding moieties in the bi-steric molecule and also examinedthe effect of linker length and the chemical handle of attachmentto rapamycin.Aspreviously outlined, mTORC1 associates with auxiliary protein Raptor(149 kDa) while mTORC2 associates with the auxiliary proteins Rictor(192 kDa) and Sin1. We reasoned that the reduced affinity of rapamycinfor mTORC2, in comparison to mTORC1, may be due to a partial occlusionof the FKBP12-rapamycin binding (FRB) motif in mTORC2 by the Rictor-Sin1complex 2.Recent3 resulted inan early observationthat an oxygen atom between the C40 position of rapamycin and a triazolegroup could be replaced with carbon to give compound 16 , as measured bycomparing the potencies for inhibition of 4EBP1 T37/T46 phosphorylation(mTORC1) and AKT S473 phosphorylation (mTORC2) in MDA-MB-468 breastcancer cells. Improved mTORC1/mTORC2 selectivity was noted when theactive-site inhibitor was changed to a version of PP242, a close relativeof sapanisertib, in both oxygen51 and carbonC40-triazole spacers (entries 5 and 6). The resulting bi-steric inhibitors 17(18 (RMC-4627)58 were approximately equipotent with RapaLink-1 3 for mTORC1 inhibition  but showed reduced inhibition of mTORC2 signaling, cf.RapaLink-1 3 , thereby improving the mTORC1/mTORC2 ratio from ca. 4-foldfor RapaLink-1 (entry 1) up to approximately 13-fold for bi-stericcompound 18 (entry 6). This observation necessitatedthe investigation of a larger set of active-site ATP inhibitors inour bi-steric ensembles with a predominant focus on carbon-linkedanalogs. To enable an appropriate reference, where possible, biologicalactivity for the active-site inhibitor is also included in 19 (entry 8) exhibited highpotency for inhibition of mTORC1 signaling (p4EBP1 IC50 = 0.06 nM) while also demonstrating good selectivity over inhibitionof mTORC2 signaling . Even greater selectivity for mTORC1/mTORC2 inhibitionwas realized when a bi-steric compound contained an active-site inhibitorderived from PP121, leading to compound 20 (entry 10)with mTORC1 p4EBP1 IC50 = 1.0 nM and mTORC2 pAKT IC50 = 17 nM (mTORC1/mTORC2 ratio of 17.0). The mTORC1/mTORC2ratio could be increased further again if a tetrahydroisoquinoline-linkedand rigidified version of MLN (“rigid-MLN”) was usedin the bi-steric construct to give compound 21 (entry12), exhibiting mTORC1 p4EBP1 IC50 = 0.44 nM and mTORC2pAKT IC50 = 8.8 nM (mTORC1/mTORC2 ratio of 20). We believethe higher level of mTORC1/mTORC2 selectivity of bi-steric constructscontaining an active-site inhibitor based around “rigid-MLN”(3 (22 (entry 13), containing a piperidine-linked “modified-MLN”active-site inhibitor, exhibited an mTORC1/mTORC2 ratio of 2.7, whichis approximately the same level of selectivity to matched-pair RapaLink-1 3 (entry 1). Also of note was the effect of the N,N-dimethylation of MLN-derived active-site inhibitorswhen applied to a bi-steric compound. Compound 23 wasinactive up to a maximal test concentration of 1 μM againstp4EBP1 and pAKT inhibition (entry 14), thus demonstrating the importanceof the primary amino-pyrimidinyl group in MLN-derived active-siteinhibitors.Our SAR studies with 50 Inhibition of AKTphosphorylation by mTORC2 was also reduced with a shorter PEG chainlength. However, mTORC2 inhibition was shifted less than mTORC1 inhibition,generally resulting in diminished mTORC1/mTORC2 selectivity ratiosfor bi-steric inhibitors containing a linker with fewer than eight(8) PEG units. This sensitivity to the PEG chain length was most pronouncedfor the more selective bi-steric inhibitors containing active-siteinhibitors with moderate intrinsic potency toward mTORC2 inhibition.For example, in a bi-steric compound containing PP121 as the mTORactive-site inhibitor, varying the PEG chain length from 8 PEG unitsto 6 PEG units resulted in a >30-fold reduction in mTORC1 p4EBP1activity.Although mTORC2 activity was also reduced with a shorter 6 PEG unitlinker , the relative shift was lower than themTORC1 p4EBP1 activity. Thus, the net effect was both to reduce on-targetmTORC1 activity and to diminish the mTORC1/mTORC2 selectivity ratio(from 17 to 3.5) upon reducing the PEG linker from 8 to 6 PEG units(entries 1 and 2). A similar observation was apparent when a bi-stericcompound utilized PP242 as the active-site inhibitor (entries 3 and4). In this case, on-target mTORC1 activity dropped >250-fold (p4EBP1IC50 = 1.4 nM to 370 nM) and the mTORC1/mTORC2 selectivityratio dropped from approximately 13 to 0.3 upon changing the PEG chainlength from 8 to 6 PEG units . Interestingly, a bi-steric compoundcontaining BGT226 as the active-site inhibitor and with 6 insteadof 8 PEG units in the linker resulted in a slight reduction of on-targetmTORC1 activity  and yet hadlittle effect on the mTORC1/mTORC2 selectivity ratio . Finally, for a bi-stericcompound using MLN as the active-site inhibitor, a reduction of PEG-linkerlength from 8 to 6 PEG units did not change on-target mTORC1 activitysignificantly . Surprisingly, the mTORC1/mTORC2 selectivity ratio improvedslightly for the 6 PEG linker, compound 27, over the8 PEG linker, compound 16 (entries 7 and 8). However,when the linker length was reduced further to 5 PEG units to givecompound 28, a significant diminution of on-target mTORC1activity (p4EBP1 IC50 = 25 nM) and a lowering of the mTORC1/mTORC2selectivity ratio was again observed (entry 9), demonstrating a generaltrend toward reduced mTORC1 inhibitory potency and mTORC1/mTORC2 selectivitywith shorter linker lengths. Of all of the linker lengths investigated,our studies showed a preference for 8 PEG units, particularly formore selective bi-steric inhibitors.Next, we examined the effect of the PEG linker chainlength onthe biological activity and mTORC1/mTORC2 selectivity of bi-stericinhibitors 2. As a gp-nitrophenoxy chloroformate chemistry or “rigid-MLN” (entry 2) as the active-site inhibitor,respectively, exhibited potent mTORC1 inhibition . The mTORC1 activity was comparable to triazole-linkedcounterparts 16 , also displayed potent on-target mTORC1 inhibition(p4EBP1 IC50 = 0.70 nM) with a moderate degree of mTORC1/mTORC2selectivity (ratio = 4.1).We also examined the effect of removing a triazolein the linker.The inspiration behind this investigation was to identify moietiesthat may be attached to the C40 oxygen of rapamycin more efficientlythan an ether, which suffered from poor yields of attachment and difficultywith purification of the product. In addition, formation of the triazolering requires an azide-containing precursor that introduces potentialsafety concerns when larger scale preparations are required. An attractivesolution was found by linking the C40 oxygen of rapamycin via a carbamategroup, which could be attached reproducibly and in high yield withwell-known hemistry2.80 We wectivity 3. For in16 was administered to arat (iv) or mouse (ip), a major species arising from ring openingof the rapamycin macrocycle was observed, which resulted from theelimination of the β-keto lactone at the C32 carbonyl. In thecase of a rat, this seco-species could account for approximately 35%of the parent on an AUClast basis. Other bi-steric compounds,such as 30, were also susceptible to a similar ring-openingprocess to yield significant amounts of a seco-species after dosingto rats (data not shown). Comparable seco-species are a well-knowndegradant from rapamycin itself (where it is known as secorapamycin 34), which forms in vivo,92 under enzymaticprocesses (CYP 3A4)90 and under basic conditions.75 Testing of the seco-products 32 and 33, formed from bi-steric inhibitors 16 and 30, respectively, indicated that theywere much less potent as mTORC1 or mTORC2 inhibitors  relative to rapamyicin 1 (Kd = 0.44 nM). The incorporation of this largergroup necessitates slight rearrangement of residue F47. However, byreplacing the methoxy with a C32 hydroxyl group with compound 11, the steric bulk was removed and an intramolecular hydrogenbond with the adjacent C34 ester oxygen was formed, which resultsin a 30-fold decrease in affinity for FKBP12 (Kd = 13.3 nM) relative to rapamycin 1 (Kd = 0.44 nM). Despite these differences in FKBP12 Kd binding, modified rapamycin cores 11 and 12 were potent in a FKBP12-FRB TR-FRET assay (EC50 < 10 nM), all being measured at the assay detectionlimit (11 maintained comparableactivity with rapamycin 1 (pS6K IC50 = 0.04nM for 11 and 0.06 nM for 1), whereas theC32 methyl ether resulted in a 6-fold decrease in potency (pS6K IC50 = 0.37 nM for 12).Due to the prevalence of a seco-product when bi-stericinhibitorscontaining a C32 carbonyl were dosed to rodents or when subjectedto mildly basic conditions 3, we sou11−83 and a11. This comparesfavorably with its rapamycin counterpart (C32 carbonyl group), bi-stericcompound 29 . Similarly,C32-methoxy rapamycin bi-steric inhibitors 37 and 39 , thus affording an impressive level of mTORC1/mTORC2 selectivityof ca. 21.9 for bi-steric inhibitor 37 and ca. 36.1 forbi-steric inhibitor 39. We also explored the incorporationof the C32-hydroxy rapamycin core in bi-steric inhibitors, as C32-hydroxyrapamycin had improved binding to FKBP12 compared with the C32-methoxymodification of rapamycin . RMC-5552 38 showed very potent p4EBP1 inhibition(IC50 = 0.48 nM) with much lower pAKT inhibition (IC50 = 19 nM), resulting in mTORC1/mTORC2 selectivity approaching40-fold. Finally, combination of the C32-hydroxy rapamycin core witha modified XL388 active-site inhibitor afforded another impressivebi-steric inhibitor compound RMC-6272 40 (entry 6), whichhad a p4EBP1 inhibition IC50 of 0.44 nM and significantlylower pAKT inhibition IC50 of 12 nM, thus displaying anapproximate 27-fold selectivity for mTORC1 over mTORC2.The FKBP12-FRB TR-FRET assay results in apamycin 5. Combin87 The mTORC1-RMC-5552-FKBP12 particles displayed2-fold symmetry; therefore, each particle was split into its two monomersgiving ∼1 600 000 particles for further 3D refinement.After postprocessing, a final monomeric map of 2.9 Å was usedfor modeling to determinethe structure of the mTORC1-RMC-5552-FKBP12 complex. Three-dimensional(3D) reconstruction of about 800 000 particles yielded a mapof 3.1 Å with clear secondary, tertiary, and quaternary structureelements and fit to previously obtained cryo-EM models.modeling 5A. The dling  injectionare shown in 38 showed a high Cmax (3.19 ± 0.62 μM) and high exposure (AUClast = 25.9 ± 3.0 μM × h), providing plasma concentrationsseveral multiples above the cellular IC50 for p4EBP1 inhibition(IC50 = 0.48 nM) for an extended period. Compound RMC-6272 40 also demonstrated an attractive pharmacokinetic profilewith extended plasma concentrations above the IC50 forp4EBP1 inhibition (IC50 = 0.44 nM), although Cmax (0.97 μM ± 0.10 μM) and overall exposure(AUClast = 8.7 ± 1.2 μM × h) were somewhatlower than those for RMC-5552 38.ADME/PK parameters for compounds RMC-5552 38 was studied in a CDX model using the Michigan Cancer Foundation-7(MCF-7) breast cancer cell line, which bears an activating mutationin the p110α catalytic subunit of Phosphoinositide 3-kinase(PIK3CAE545K). Administration of a single intraperitoneal(ip) dose of RMC-5552 38 resulted in a dose-dependent,prolonged, and profound inhibition of tumor p4EBP1 levels up to 48h . The dual mTORC1/mTORC2 active-site inhibitor sapanisertib 5, which is moderately potent for inhibition of 4EBP1 phosphorylationin vitro (p4EBP1 IC50 = 19 nM), caused significant (>80%inhibition) inhibition of tumor p4EBP1 at 4 h after intraperitonealdosing at 1 mg/kg and reduced inhibition after 24 h, consistent withthe elimination kinetics of this compound  studies using human cancer cell line-derived xenograft(CDX) tumor models in mice. The leading bi-steric inhibitor RMC-5552 38 and RMC-6272 40 translated into antitumoractivityupon repeat dosing when each compound was examined in a 28-day studyin the MCF-7 (PIK3CAE545K) cell line-derived xenograft(CDX) model  model 7. RMC-5593 One combination that is particularly appealing is withinhibitors of mutated oncogenic forms of Kirsten rat sarcoma (KRAS),such as KRASG12C.96 We investigated the combinationof RMC-6272 40 and sotorasib (AMG-510), the first approvedKRASG12C inhibitor for KRASG12C-mutated locally advanced or metastatic nonsmallcell lung cancer (NSCLC),98 in the NCI-H2122 NSCLCCDX model. Despite harboring a KRASG12C mutation, this model is relatively insensitive to KRASG12C inhibition.95 Significantly, NCI-H2122also harbors a STK11LOF mutation. The STK11 gene product negatively regulates mTORC1 andfunctions as a tumor suppressor. Up to 25% of KRASG12C NSCLC patients have co-occurring STK11mut with an estimate ofover 5400 new patients per annum in the United States.62 This patient population appears refractory toanti-PD-1 treatment and is a significant unmet medical need.99 Neither RMC-6272 40 (10 mg/kg ip once weekly) nor sotorasib induced tumor regressions when dosed as singleagents in this model  ion channel waslow when the compounds were screened at 10 μM. RMC-5552 38, our most selective inhibitor with broad tolerability,emerged as a preferred clinical candidate. In addition, when RMC-5552 38 (10 μM) was profiled in the Eurofins Safety Screen44, a well-known “cross pharma”-recommended panel againstundesirable off-targets,101 no significantinhibition was observed.RMC-5552 38 and compound RMC-6272 40 is detailed in The profile of compound RMC-5552 38 and RMC-6272 40 that potentlyand selectively inhibit mTORC1-mediated phosphorylation of 4EBP1 andother substrates. These inhibitors resulted from a systematic examinationof the active-site inhibitor, linker, and rapamycin core in orderto refine on-target mTORC1 activity, selectivity over mTORC2, pharmacokineticproperties, chemical stability, and synthetic tractability. Selectiveinhibition of mTORC1 could be beneficial for treating cancers withaberrant activation of mTORC1 via mutations in the PI3K/mTOR pathway,and indeed, RMC-5552 38 and RMC-6272 40 eachexhibit single-agent antitumor activity in a human xenograft modelof PIK3CA mutant breast cancer in mice in vivo. Aberrant activationof mTORC1 can also co-occur with mutations in other cancer drivers,such as RAS, limiting the antitumor activity of targeted RAS inhibitors.An mTORC1 inhibitor may have utility as a companion to RAS inhibitorsin this context. Consistent with this hypothesis, RMC-6272 40 induces widespread apoptosis and deep tumor regressions in a xenograftmodel of KRASG12C mutantNSCLC bearing a loss of function mutation in STK11, a negative regulator of mTORC1. This model exhibits only modestinhibition of tumor growth in response to single-agent treatment withthe KRASG12C inhibitor sotorasib, suggesting that mTORC1activation can contribute to resistance to KRASG12C inhibitors,which can be overcome by combination with an mTORC1 inhibitor. RMC-5552 38 has now advanced to clinical studies for further evaluation.71Herein, we have highlighted the developmentof two bi-steric inhibitorsRMC-5552 2O with 0.1% formic acid. All key compounds were >95% purityby HPLC. The purity for compounds and low-resolution mass spectrawere determined using liquid chromatography mass spectrometry (LCMS)on a Shimadzu LC-20 instrument using electrospray ionization. LCMSconditions were as follows: Uptisphere Strategy C18-HQ 5 μm150 × 4.6 mm, 55% → 100% MeCN (0.1% TFA) in H2O (0.1% TFA), 20 min run, oven temperature 60 °C, flow rate0.5 mL/min, UV detection (λ = 280 nm). HRMS were performed ona Thermo Fisher LTQ Orbitrap using high-performance liquid chromatographywith electrospray ionization Orbitrap mass spectrometry (HPLC ESIOrbitrap/MS). Liquid-state 1H NMR experiments for intermediateswere recorded on 400, 500, or 600 MHz Bruker Avance III NMR spectrometers.Liquid-state 1H, COSY, 13C, and HMBC spectrawere recorded on a 600 MHz Bruker Avance III NMR spectrometer  using a triple-resonance 1H,15N,13C CP-TCI 5 mm cryoprobe . All of the experiments used for the resonance assignmentprocedure and the elucidation of the final products structures  were recordedat 300 K. 1H chemical shifts are reported in δ (ppm)as s (singlet), d (doublet), t (triplet), q (quartet), dd (doubledoublet), m (multiplet), br s (broad singlet), and o (overlay) andare referenced to TMS as an internal standard.All solventsand commercially available reagents were used as received. All reactionswere followed by TLC analysis or LCMS. Column chromatography was performedon prepacked silica gel columns (Biotage SNAP KP-Sil) using a BiotageIsolera One system. Reverse-phase preparative chromatography was performedon a Uptisphere Strategy C18-HQ 5 μm 150 mm × 7 mm columnusing an Interchim PuriFlash system. The column was eluted with MeCN/H10  and SI-7  in DMSO (5.2 mL) was added Cu(MeCN)4PF6  followed by TBTA . The reaction mixture was then stirred at roomtemperature for 5 h. Purification of the reaction mixture by reverse-phasechromatography (40% → 90% MeCN/H2O) afforded thedesired product  as a colorless amorphous solid.LCMS (ESI) m/z: [M+H] calcd forC92H140N12O23 1782.02;found 1782.2.To a solution of 10  and SI-13  in DMSO (9.1 mL) was added Cu(MeCN)4PF6  followed by TBTA . The reaction mixture was then stirred at room temperaturefor 6 h. Purification of the reaction mixture by reverse-phase chromatography(40% → 90% MeCN/H2O) afforded the desired product as a colorless amorphous solid. LCMS (ESI) m/z: [M+H] calcd for C93H141N11O23 1781.02; found 1781.2To a solution of 10  and SI-15 in DMSO (8 mL) was added Cu(MeCN)4PF6  followed by TBTA .The reaction mixture was then stirred at room temperature for 6 h.Purification of the reaction mixture by reverse-phase chromatography(40% → 90% MeCN/H2O) afforded the desired product as a colorless amorphous solid. LCMS (ESI) m/z: [M+H] calcd for C104H147F3N10O24 1978.06; found1977.9.To a solution of 10  and SI-19  in DMSO (8 mL) was added Cu(MeCN)4PF6  followed by TBTA . The reaction mixture was then stirred at room temperaturefor 6 h. Purification of the reaction mixture by reverse-phase chromatography(40% → 90% MeCN/H2O) afforded the desired product as a colorless amorphous solid. LCMS (ESI) m/z: [M+H] calcd for C92H140N12O22 1766.03; found 1765.9.To a solution of 10  and SI-24  in DMSO (8 mL) was added Cu(MeCN)4PF6  followed by TBTA . The reaction mixture was then stirred at roomtemperature for 6 h. Purification of the reaction mixture by reverse-phasechromatography (40% → 90% MeCN/H2O) afforded thedesired product  as a colorless amorphous solid.LCMS (ESI) m/z: [M+H] calcd forC98H142N12O23 1856.04;found 1856.0.To a solution of 10  and SI-28  in DMSO (2 mL) was added Cu(MeCN)4PF6  followed by TBTA . The reaction mixture was then stirredat room temperature for 3 h. Purification of the reaction mixtureby reverse-phase chromatography (40% → 90% MeCN/H2O) afforded the desired product  as a colorlessamorphous solid. LCMS (ESI) m/z:[M+H] calcd for C94H142N12O23 1808.04; found 1808.1.To a solution of 10  and SI-32  in DMSO (3 mL) was added Cu(MeCN)4PF6  followed by TBTA . The reaction mixture was then stirred at roomtemperature for 4 h. Purification of the reaction mixture by reverse-phasechromatography (40% → 90% MeCN/H2O) afforded thedesired product  as a colorless amorphous solid.LCMS (ESI) m/z: [M+H] calcd forC94H144N12O23 1810.05;found 1810.0.To a solution of 10  and SI-20  in MeOH (10 mL) was added a 1 M solution ofCuSO4 in H2O  followed by a 1 M solution of sodium ascorbate in H2O . After stirring overnight,a 1 M solution of CuSO4 in H2O  followed by a 1 M solution of sodium ascorbate in H2O  was added. After stirring overnight,the reaction mixture was concentrated under reduced pressure, dissolvedin DMSO, and filtered, and formic acid (300 μL) was added. Purificationof the reaction mixture by reverse-phase chromatography (10% →60% MeCN/H2O) afforded the desired product  as a colorless amorphous solid. LCMS (ESI) m/z: [M+H] calcd for C88H132N12O20 1677.98; found 1677.9.To a solution of 10  and SI-14  in MeOH (10 mL) was added a 1 M solution of CuSO4 in H2O  followedby a 1 M solution of sodium ascorbate in H2O . After stirring overnight, the reaction mixturewas concentrated under reduced pressure, dissolved in DMSO, and filtered,and formic acid (300 μL) was added. Purification of the reactionmixture by reverse-phase chromatography (10% → 60% MeCN/H2O) afforded the desired product  as acolorless amorphous solid. LCMS (ESI) m/z: [M+H] calcd for C89H133N11O21 1692.98; found 1692.9.To a solution of 10  and SI-16  in MeOH (8 mL) was added a 1 M solution of CuSO4 in H2O  followedby a 1 M solution of sodium ascorbate in H2O . After stirring overnight, the reaction mixturewas concentrated under reduced pressure, dissolved in DMSO, and filtered,and formic acid (300 μL) was added. Purification of the reactionmixture by reverse-phase chromatography (10% → 60% MeCN/H2O) afforded the desired product  as acolorless amorphous solid. LCMS (ESI) m/z: [M+H] calcd for C100H139F3N10O22 1890.1; found 1890.0.To a solution of 10  and SI-8  in MeOH (10.5 mL) was added a 1 M solution of CuSO4 in H2O  followed by a1 M solution of sodium ascorbate in H2O . After 2 h, the reaction mixture was concentrated under reducedpressure, dissolved in DMSO, and filtered, and formic acid (300 μL)was added. Purification of the reaction mixture by reverse-phase chromatography(10% → 65% MeCN/H2O) afforded the desired product as a colorless amorphous solid. LCMS (ESI) m/z: [M+H] calcd for C88H132N12O21 1693.97; found 1694.0.To a solution of 10  and SI-9  in MeOH (10 mL) was added a 1 M solution of CuSO4 in H2O  followed by a 1 Msolution of sodium ascorbate in H2O . After stirring overnight, a 1 M solution of CuSO4 in H2O  followed by a 1 Msolution of sodium ascorbate in H2O  was added. After stirring overnight, the reaction mixture wasconcentrated under reduced pressure, dissolved in DMSO, and filtered,and formic acid (300 μL) was added. Purification of the reactionmixture by reverse-phase chromatography (10% → 60% MeCN/H2O) afforded the desired product  as acolorless amorphous solid. LCMS (ESI) m/z: [M+H] calcd for C86H128N12O20 1649.94; found 1650.0.To a solution of 13  and SI-34  in DMA (0.2 mL) was added DIPEA . The reaction was stirred for 7 h, at whichpoint the reaction mixture was purified by reverse-phase chromatography(40% → 100% MeCN/H2O) to afford the desired product as a colorless amorphous solid. LCMS (ESI) m/z: [M+H] calcd for C87H132N10O24 1701.95; found 1701.8.To a solution of SI-36  in DMA (2.3 mL) at 0 °C was addedDIPEA  followed by 13 . The reaction mixture was warmedto room temperature and stirred for 5 h. The reaction mixture wasacidified with formic acid and purified by reverse-phase chromatography(40% → 100% MeCN/H2O) to afford the desired product as a colorless amorphous solid. LCMS (ESI) m/z: [M+H] calcd for C93H134N10O24 1775.97; found 1775.9.To a solution of SI-44  in DMA (8 mL) was added DIPEA  followed by 13 . The reaction mixture was stirred overnightat room temperature. The reaction mixture was acidified with formicacid and purified by reverse-phase chromatography (40% → 100%MeCN/H2O) to afford the desired product as a colorless amorphous solid. LCMS (ESI) m/z: [M+H] calcd for C95H139FN6O27S 1847.95; found 1848.0.To a solution of 16  in DMA (2 mL) was added NH4OAc . The reaction mixture washeated to 40 °C, and after 6 h it was cooled to room temperature.Purification of the reaction mixture by reverse-phase chromatography(40% → 100% MeCN/H2O) afforded the desired product as a colorless amorphous solid.LCMS (ESI) m/z: [M+Na] calcd forC92H140N12O23 1804.01;found 1804.0.To a solution of 30  in DMA (1.4 mL) was added NH4OAc . The reaction mixture washeated to 40 °C, and after 5 h it was cooled to room temperature.Purification of the reaction mixture by reverse-phase chromatography(10% → 100% MeCN/H2O) afforded the desired product as a colorless amorphous solid.LCMS (ESI) m/z: [M+Na] calcd forC93H134N10O24 1775.97;found 1775.8.To a solution of 15  and SI-34  in DMA (1.15 mL) was added DIPEA . The reaction was stirred overnight, andthen the reaction mixture was purified by reverse-phase chromatography(40% → 100% MeCN/H2O) to afford the desired product as a colorless amorphous solid. LCMS (ESI) m/z: [M+H] calcd for C88H136N10O24 1717.98; found 1718.0.To a solution of 14  and SI-34  in DMA (1.15 mL) was added DIPEA . The reaction was stirred overnight, and then the reactionmixture was purified by reverse-phase chromatography (40% →100% MeCN/H2O) to afford the desired product  as a colorless amorphous solid. LCMS (ESI) m/z: [M+H] calcd for C87H134N10O24 1703.97; found 1703.7.To a solution of SI-36  in DMA (6.8 mL) was added DIPEA  followed by 15 . The reaction mixture was stirred overnightat room temperature. The reaction mixture was acidified with formicacid and purified by reverse-phase chromatography (40% → 100%MeCN/H2O) to afford the desired product as a colorless amorphous solid. LCMS (ESI) m/z: [M+H] calcd for C93H136N10O24 1777.98; found 1777.7.To a solution of SI-36  in DMA (1.4 mL) at 0 °C was addedDIPEA  followed by 14 . The reaction mixture was warmedto room temperature and stirred for 5 h. The reaction mixture wasacidified with formic acid and purified by reverse-phase chromatography(40% → 100% MeCN/H2O) to afford the desired product as a colorless amorphous solid. HRMS (ESI) m/z: [M+H] calcd for C93H136N10O24 1777.9807; found 1777.9813.To a solution of SI-44  in DMA (1.4 mL) at 0 °C was addedDIPEA  followed by 15 . The reaction mixture was stirredovernight at room temperature. The reaction mixture was acidifiedwith formic acid and purified by reverse-phase chromatography (10%→ 100% MeCN/H2O) to afford the desired product  as a colorless amorphous solid. LCMS (ESI) m/z: [M+H] calcd for C96H143FN6O27S 1863.98 found 1864.0.To a solution of SI-44  in DMA (6.7 mL) at 0 °C was addedDIPEA  followed by 14 . The reaction mixture was stirredovernight at room temperature. The reaction mixture was acidifiedwith formic acid and purified by reverse-phase chromatography (10%→ 90% MeCN/H2O) to afford the desired product  as a colorless amorphous solid. LCMS (ESI) m/z: [M+H] calcd for C95H141FN6O27S 1849.96 found 1850.0.To a solution of


Structural colors have a demonstrated value in constructing various functional materials. Efforts in this area are devoted to developing stratagem for generating heterogeneous structurally colored materials with new architectures and functions. Here, inspired by icing process in nature and ice-templating technologies, we present freeze-derived heterogeneous structural color hydrogels with multiscale structural and functional features. We find that the space-occupying effect of ice crystals is helpful for tuning the distance of non-close-packed colloidal crystal nanoparticles, resulting in corresponding reflection wavelength shifts in the icing area. Thus, by effectively controlling the growth of ice crystals and photo-polymerizing them, structural color hydrogels with the desired structures and morphologies can be customized. Other than traditional monochromatic structure color hydrogels, the resultant hydrogels can be imparted with heterogeneous structured multi-compartment body and multi-color with designed patterns through varying the freezing area design. Based on these features, we have also explored the potential value of these heterotypic structural color hydrogels for information encryptions and decryptions by creating spatiotemporally controlled icing areas. We believe that these inverse ice-template structural color hydrogels will offer new routes for the construction and modulation of next generation smart materials with desired complex architectures. While most of the structural colored materials only present isotropic colors, limiting their functions and many practical applications, while realizing anisotropic structural color materials remains challenging. Here, the authors develop a freeze-derived heterogeneous structural color hydrogels for information encryption and decryption. Inspired by natural creatures, various strategies have emerged for preparing artificial structurally colored materials with specific nanoarchitectures13. Among them, the self-assembled colloidal crystals (CCs) have offered unprecedented opportunities, because of their cheap cost, facile preparation procedures, the comparative simplicity of scaling up, and flexibility of building complicated nanometer-precision architectures15. In particular, the non-close-packed CCs formed by the charge repulsion of highly charged nanoparticles are attracting great interest for their relatively simple fabrication and flexible color adjustment16. Benefitting from these advantages, a variety of functional structurally colored materials have been fabricated for displaying17, anti-counterfeiting18, coding20, etc21. However, most of the structurally colored materials obtained in these traditional ways were with single nanostructures and could only present isotropic colors, which limits their functions and many practical applications24. Therefore, stratagem for generating anisotropic structurally colored materials with new architectures are still anticipated in various fields.Structural color, resulting from the interaction of light with periodic nanoscale structures exhibiting varied refractive indices, has sparked widespread concerns by its prevalence in nature28, we proposed freeze-derived heterogeneous structural color hydrogel films with a series of distinctive features, as schemed in Fig. 29, such as ceramics31, carbon nanotubes32, graphene34, polymers35, and boron nitride36, have been integrated into mass complex composites possessing nacre-like morphology via ice-templating41. In addition, benefitting from the porous nature of ice-template materials, the freeze-casting method is creatively used to prepare aerogel42, smart sponges44, polymeric woods45, and multifunctional cellular plastics46. Remarkably, strong tough hydrogels48 and fatigue-resistant hydrogels49, which are difficult to prepare by other methods, are successfully fabricated through the ice-template strategy50. However, the preparation of structural color hydrogel materials using freeze-casting methods has never been investigated.In this paper, inspired by the icing process in nature and freeze-casting technologiesHere, we carried out the desired structurally colored materials generation stratagem through a freeze-photopolymerization method. We found that the space-occupying effect of ice crystals was helpful for modulating the distance of non-close-packed nanoparticles, thus resulting in a corresponding blue shift of reflection wavelengths in the icing area. After being photo-polymerizing, the ice-template hydrogel with the same vivid structural color could be easily obtained. It was worth mentioning that compared with uniform ice growth, directional ice growth could further promote the assembly of colloidal nanoparticles through long-range orderly ice crystals arrangement and improve structural color performance on their surfaces. Based on these characteristics, heterogeneous structural color films with dual-color compartments were successfully fabricated, whose color could be flexibly adjusted by varying parameters such as nanoparticles solution concentration, subcooling, and water content in colloidal suspension. More attractively, the heterogeneous inverse ice-templated structural color films could be imparted with more complicated patterns via selectively controlling the freezing areas. Benefitting from the macroscopically visible color differences and spectral shift caused by icing, we demonstrated that the heterogeneous structural color films were valuable for information decryption. These features impart the inverse ice-template structural color hydrogels with great potentials in sensors, optical devices, anticounterfeiting, and so on.Freeze-casting (or ice-templating) can be employed as a method to construct sophisticated composites with desired structures. As shown in Supplementary Fig. In a typical experiment, we employed the ice-template to assemble non-close-packed colloidal crystal nanoparticles and adapted photopolymerization to fix the nanoparticle-assembled structure and replicate the ice-template structures, named the freeze-photopolymerization method. As illuminated by Fig. 2 nanoparticles did not collapse was that the position of the nanoparticles was fixed by the gelatin of PEGDA. Additionally, due to the electrostatic repulsion effect, the highly charged nanoparticles presented a face-centered cubic non-close-packed arrangement  diffractive layers’ spacing, nave denotes the average refraction index. In addition, when freezing–melting cycles were performed at a constant freezing temperature for the unpolymerized pregel suspension, the dynamic reflective peak also cycled between ~535 nm and ~610 nm  images, which presented a micro-nano hierarchical structure Fig. . For theent Fig. , which went Fig. . We founent Fig. . The corent Fig. . The pho nm Fig. , indicatThe micrometer-level architectures of II-SCHFs are templated by the ice crystal, endowing the II-SCHFs with designable micrometer-sized structure features. Especially, the porous lamellar structures are successfully constructed in structural colors materials. The growing ice displays strong anisotropic growth kinetics, enabling the possibility of shaping ice flakes with a very high aspect ratio under steady-state temperature gradient conditions. Directional structure in II-SCHFs can be achieved by oriented aligned ice platelets. Taking advantage of traveling temperature gradients, the unidirectional ice platelets growth was achieved after ice nucleation  was prepared. Subsequently, by mixing a constant volume PEGDA solution with divergent amounts of surface-charged nanoparticles, the colloidal suspension with various nanoparticles solution concentration was obtained. After freezing and UV light solidifying, we obtained the II-TSCHFs of various colors, and the reflection spectra were measured correspondingly, as shown in Fig. The structural color of II-TSCHFs was tunable via non-close-packed nanoparticles solution concentrations. According to Eq., as the Besides, the presented freeze-photopolymerization method was generalizable to a wide range of hydrogels. The hydrogels that satisfy the following two conditions were applicable, which were that surface-charged nanoparticles were able to disperse into the solution to produce a colloidal suspension with vivid structural color and the hydrogels were photopolymerizable, respectively. To verify this hypothesis, besides PEGDA hydrogels Fig. , we alsoIn order to clarify the spectrum difference between the frozen and non-frozen structure color film, we proposed a mechanism interpretation, named icing blue shift. There was a liquid water phase that could be frozen and a non-aqueous phase (nanoparticles and PEGDA) that could not be frozen in the colloidal suspension. On the low-temperature cold substrate, when the nucleation energy barrier was reached, the liquid water in the suspension firstly nucleated to form an ice embryo. Next, ice grew from the embryo, free liquid water molecules near the ice embryo were captured and fixed at the ice-water interface, and a liquid-solid phase transition occurred. Simultaneously, ice crystals squeezed out the non-aqueous phase. Because of the space-occupying effect of ice crystals, the distance between nanoparticles was reduced. Corresponding reflection spectra of obtained II-TSCHFs would be blue-shifted according to Bragg Eq., as scheIn order to verify the icing blue shift principle proposed above and reveal the influence of icing behavior on the reflection spectrum of the structured color film, we designed a heterogeneous structural color film, in which the half area was frozen and the rest kept non-frozen , respectively. The same weight of nanoparticles was dispersed into the same volume of pre-solutions with different H2O/PEGDA volume ratios to obtain orange colloidal crystals suspensions L1~L6. The dual color heterogeneous structural color films F1~F6 were prepared by applying freeze-photopolymerization technology to the six groups of orange suspensions L1~L6, as shown in Fig. 2O content increasing. This result showed that as the H2O content in the pre-solution increased, the value of icing blue shifts gradually increased  diacrylate , 2-hydroxy-2-methyl-1-phenyl-1-propanone (HMPP), acrylamide (AM), N-isopropylacrylamide (NIPAM), and N, N’-methylenebisacrylamide (BIS) were acquired from Sigma Aldrich . Unless otherwise specified, PEGDA (M.W. 700) was employed.Highly-charged SiO2 nanoparticles. Firstly, PEGDA and H2O were mixed at the v/v ratio of 1:1, followed by adding the HMPP (1 v/v %) to the solution. After adequately mixing them, the PEGDA aqueous solution was obtained. Next, the highly-charged SiO2 nanoparticles were dispersed into the prepared PEGDA solution until the final nanoparticle concentration was 20 wt %. After ultrasound treatment for 30 min and vortex oscillation for 10 min, the colloidal suspension with vivid structural color was acquired. Remarkably, when the silica nanoparticles are completely dispersed in the PEGDA hydrogel precursor solution, an appropriate amount of ion exchange resin should be added to adsorb the cations in the system and ensure the stability of the self-assembled structure of the silica nanoparticles. Besides, by varying the amounts of nanoparticles added to the PEGDA solution, the pregel suspensions of various nanoparticles solution concentration (15–33%) were prepared. Unless otherwise specified, nanoparticles with a diameter of 145 nm were employed.The colloidal suspension was composed of the PEGDA aqueous solution and surface-charged SiO2 to replicate the ice-crystals structure. Finally, the II-TSCHF with randomly oriented pore structures was obtained after removing ice-template at room temperature. Unless otherwise specified, UV light was irradiated for 10 s at an intensity of 500 mW/cm2 for all experiments.The II-TSCHF was fabricated based on freeze-photopolymerization methods. Two clean, hydrophilic slides were separated by spacers with a certain thickness to obtain a Hele–Shaw cell. The pregel was injected uniformly and slowly into the gap between the two slides. Then the Hele-Shaw cell was moved to a low-temperature Peltier cooler. After the water in the suspension is completely frozen, the Hele–Shaw cell was polymerized under UV light exposure for approximately 10 s with an intensity of 500 mW/cmThe Hele-Shaw cell containing nanoparticles pregel suspension was placed on the Peltier cooler with a thin ethanol liquid film separating them. The syringe pump (Longer Pump LSP01) was used to push the Hele–Shaw cell structure at a constant speed, so as to realize a fixed freezing speed of the colloidal suspension along the velocity direction. After the suspension was completely frozen, UV light was used to solidify it. Thus, the directional II-TSCHF was fabricated.We fabricated heterogeneous structural color films with half-frozen area and half non-frozen area to validate the icing blue-shift mechanism. The Hele-Shaw cell with nanoparticle suspension was prepared based on the above method. A tiny part of the Hele-Shaw cell (denoted as A) was directly in contact with the square Peltier cooler platform, the temperature of which was set relatively low, while the rest (denoted as B) was suspended in the air without contact with the cold platform. After ice nucleation occurred in part A, a half (denoted as C) of the Hele-Shaw cell was pushed onto the Peltier cooler, simultaneously the temperature of Peltier cooler was set to the specified value. Subsequently, ice crystals grew from Part A to the rest of Part C. When Part C was completely frozen, the suspension was immediately polymerized under UV light exposure, the dual-color heterogeneous structural color film with half-frozen area and half non-frozen area was finally formed.2. Subsequently, the temperature was raised to remove the ice templates, followed by photopolymerizing and the rest, thus patterned heterogeneous hydrogel films were obtained. Similarly, the suspension film was firstly spatially irradiated (UV light was irradiated for 2 s with an intensity of 500 mW/cm2) at ambient temperature via masks and then directly frozen at low temperature. Subsequently, the entire film was polymerized in a frozen state (UV light was irradiated for 10 s at an intensity of 500 mW/cm2), and the patterned hydrogel films were also fabricated.Spatially defined patterns are achieved via masked UV irradiations at frozen state and molten state. With the help of embossing molds, different patterned masks were prepared. The Hele-Shaw cell containing the suspension was put on the Peltier cooler to freeze at a specific temperature, and then we used masks to selectively photopolymerize the pregels, UV light was irradiated for 2 s with an intensity of 500 mW/cm2. SEM images were obtained by scanning electron microscopy . Optical images were taken using an optical microscope (Olympus BX51) equipped with a CCD camera (Media Cybernetics Evolution MP5.0) and a digital camera .Reflection spectra were acquired at a constant glancing angle employing an optical microscope equipped with a fiber-optic spectrometer . The spectra of the structural color hydrogels were measured before water volatilization. The light beam size for the reflectance measurement is ~0.636 mmSupplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6


Using a combination of biochemical, pharmacological, neurophysiological, and behavioral approaches, we find that glutamate-dependent reduction in cyclin-dependent kinase 5 (Cdk5)-dependent RIIβ phosphorylation alters the PKA holoenzyme autoinhibitory state to increase PKA signaling in response to dopamine. Furthermore, we show that disruption of RIIβ phosphorylation by Cdk5 enhances cortico-ventral striatal synaptic plasticity. In addition, we demonstrate that acute and chronic stress in rats inversely modulate RIIβ phosphorylation and ventral striatal infusion of a small interfering peptide that selectively targets RIIβ regulation by Cdk5 improves behavioral response to stress. We propose this new signaling mechanism integrating ventral striatal glutamate and dopamine neurotransmission is important to brain function, may contribute to neuropsychiatric conditions, and serves as a possible target for the development of novel therapeutics for stress-related disorders.Cortical glutamate and midbrain dopamine neurotransmission converge to mediate striatum-dependent behaviors, while maladaptations in striatal circuitry contribute to mental disorders. However, the crosstalk between glutamate and dopamine signaling has not been entirely elucidated. Here we uncover a molecular mechanism by which glutamatergic and dopaminergic signaling integrate to regulate cAMP-dependent protein kinase (PKA)  Understanding of the molecular mechanisms by which this is achieved is incomplete.A fundamental function of brain circuitry is to impart emotional salience upon sensory input so that environmental experiences may be acted upon with appropriate behavioral responses. This requires the integration of fast ligand-gated ionotropic synaptic signals with those mediated by G-protein–coupled neurotransmitter receptors. Subserving these synaptic receptors, CaThe nucleus accumbens  is a central input structure within the basal ganglia and mediates reinforced learning and behavioral response processing. The medium spiny neurons (MSNs) in the NAc receive glutamatergic input from the medial prefrontal cortex, hippocampus, and other regions as well as dopaminergic input from the ventral tegmental area , 2. Coincat: Cα, Cβ, or Cγ isoforms) bound to a homodimer of regulatory subunits . When RI subunits are incorporated, it is classified as Type I PKA, when bound to RII subunits as Type II PKA. R subunits possess an N-terminal dimerization domain (D/D), responsible for both dimerization and subcellular localization via binding to members of the A-kinase anchoring protein (AKAP) family of scaffolding molecules  indicates that the RIIβ subunit is most selectively expressed in brain compared to the other regulatory isoforms. Moreover, its brain region tissue distribution mimics the expression pattern of Cdk5. For both RIIβ and Cdk5, highest levels occur in limbic regions including prefrontal cortex, dorsal and ventral striatum, and hippocampus.Messenger RNA transcripts encoding all four isoforms of the PKA regulatory subunit are present in brain, with expression of the RIIβ isoform enriched in the central nervous system , 18, 19.Fig. 1B). RIIβ colocalized with Cdk5 in dissociated rat striatal neurons grown in culture (C) and within cells and neuropil of rat striatal tissue. Furthermore, analysis of a rat NAc single nucleus RNA (snRNA-seq) library  and Cdk5 genes are co-expressed within subclusters of rat NAc cell populations including D1 and D2 dopamine expressing MSNs  and GABAergic and glutamatergic neurons (A–E). Interestingly, RIIβ expression was absent from populations of astrocytic and oligodendritic cells (B and D). Similarly, Cdk5 expression occurred in populations of D1-MSN, D2-MSN, GABAergic, and glutamatergic neurons and was also absent in astrocytic and oligodendritic cell clusters (C and E). Thus, Cdk5 and RIIβ are co-expressed within subcellular populations of the rat ventral striatum.Immunostains of ventral striatum showed RIIβ in the soma and neuropil of MSNs where it colocalized with its scaffolding protein, AKAP150 , 21 FigB. RIIβ c culture C and wit library  revealed neurons , A–E. Inic cells , B and Dclusters , C and Ein vitro, achieving a maximal stoichiometry of 0.6 mol PO4/mol of substrate under saturating conditions (E and F). Mass spectrometry positively identified Thr69 as the site of phosphorylation, and the T69A site-directed mutant form of RIIβ was not phosphorylated by Cdk5 at all. To determine if this phosphorylation occurred in brain, a phosphorylation state-specific antibody for phospho-Thr69 RIIβ was derived (F–H). Phospho-Thr69 RIIβ was detected within MSNs throughout the striatum by immunostaining (G). Together these data demonstrate that the RIIβ regulatory subunit of PKA is phosphorylated at Thr69 by Cdk5 and that this phosphorylation event occurs in vivo within striatal neurons.The amino acid sequence of RIIβ includes a proline-directed Cdk5 consensus motif (S/TPXK/H/R) within the N-terminal linker region predicting Thr69, a site unique to this particular R subunit, as a potential Cdk5 phosphorylation site. Indeed, Cdk5 phosphorylated pure recombinant RIIβ nditions , E and F derived , F–H. Phstaining G. Togethcat activity. However, in vitro analysis indicated that Thr69 phosphorylation did not alter the inhibition of PKA catalytic activity by RIIβ (I). Furthermore, phospho-memetic T69D mutation had no effect on cAMP-dependent activation of PKA in the RIIβ/PKA holoenzyme complex (J). As an alternative, we considered that Cdk5-dependent phosphorylation of Thr69 RIIβ could alter structure/function aspects of the holoenzyme inhibitory state. As mentioned above, the RIIβ D/D forms a four-helix bundle that docks with AKAPs through hydrophobic interactions (A). The Cdk5 phosphorylation site on RIIβ is encompassed within the flexible linker region that occurs between the D/D and the cAMP binding domains. This site is proximal to the IS in Type II R subunits, which contains the PKA phosphorylation site Ser114 (B). Therefore, we assessed the effect of phospho-memetic T69D mutation on PKA-dependent phosphorylation of Ser114 RIIβ (C). Interestingly, T69D phospho-mimetic mutation attenuated the efficiency of Ser114 phosphorylation by PKA in comparison to WT RIIβ, significantly reducing the maximum velocity of the reaction under linear conditions. Thus, the phosphorylation state of RIIβ at Thr69 may govern PKA “autophosphorylation” at Ser114.Both genetic deletion and pharmacologic inhibition of Cdk5 increases phosphorylation of PKA substrates in striatum . Thus, w by RIIβ I. Furthe complex J. As an ractions , 24 . Cdk5 inhibition attenuated the phosphorylation of phospho-Thr69, with corresponding increase in PKA-dependent phosphorylation of Ser114. Together, these data reveal a novel intramolecular mechanism by which RIIβ phosphorylation is reciprocally regulated at two proximal sites in vitro and ex vivo.To determine if this functional relationship between Thr69 and Ser114 RIIβ phosphorylation occurs CP681301 D. Cdk5 ivia RII subunit autophosphorylation has not previously been examined as a neuronal signaling mechanism. This may be because phosphorylation of RII by PKA characterizes the inhibited state of type II PKA holoenzymes  (A). Thus, the level of PKA-dependent phosphorylation of Ser114, which occurs with formation of the PKA/RIIβ holoenzyme inhibitory state, may be controlled by the phosphorylation state of Thr69 RIIβ and may determine PKA inhibition potency by RIIβ. Therefore, neuronal activity which regulates Thr69 levels could modulate the efficacy of activators of G protein–coupled receptors that invoke the cAMP/PKA signaling cascade.PKA regulation oenzymes . Howeverociation . Consist, S114D) A. Thus, B). Concomitantly, PKA-dependent phosphorylation at Ser114 was increased, consistent with the reciprocal modulation of these sites detected in vitro and in response to Cdk5 inhibition in intact brain tissue. The effect of NMDA on phospho-Thr69 appeared to be mediated by the activation of the Ca2+-dependent serine/threonine protein phosphatase, PP2B , as the NMDA-induced reduction was blocked by the PP2B inhibitor cyclosporin A (A). Inhibition of protein phosphatases PP1 and PP2A by 1 μM okadaic acid also attenuated the effect of NMDA. Moreover, the basal phosphorylation state of phospho-Thr69 was increased by PP2B inhibition with cyclosporin A, PP1 inhibition by 1 μM okadaic acid, or selective PP2A inhibition with 200 nM okadaic acid (B). Together these data indicate that phospho-Thr69, and thereby phospho-Ser114, are regulated by activation of ionotropic NMDA class glutamate receptors. This is mediated, at least in part, through Ca2+-dependent protein phosphatases including PP2B and 2A. These phosphatases together with PP1 also determine the basal phosphorylation state of phospho-Thr69 RIIβ. The diversity in protein phosphatases dephosphorylating RIIβ may not be surprising as both RII subunits interact with the serine/threonine phosphatase calcineurin (PP2B), Cdk5 complexes with PP2A  treatment  strongly reduced the phosphorylation state of Thr69 B. Concomsporin A A. Inhibiaic acid B. Togethith PP2A , and AKAith PP2A .C). Specifically, dopamine (10 μM) was bath-applied for 15 min with NMDA (25 μM) added for the last 5 min in reduced Mg2+ buffer with a 30 min delay following treatments. Effects on phospho-Thr69 RIIβ compared with PKA activity, as indicated by changes in PKA-dependent phosphorylation states of two key effectors, Ser845 GluA1 and Thr34 DARPP-32 . Under these conditions, NMDA again induced a decrease in phospho-Thr69 RIIβ, without significantly altering PKA activity. Dopamine alone also did not sustain PKA activity for 30 min. However, phospho-Thr69 reduction by combined dopamine and NMDA treatment corresponded to increased PKA activity 30 min after the treatments were removed . These data suggest a compound mechanism in which relatively high levels of phospho-Thr69 occur under basal conditions. Robust excitatory glutamatergic neurotransmission reduces phospho-Thr69 via protein phosphatase activation, resulting in increased PKA-dependent phosphorylation of Ser114 RIIβ. In the context of dopamine receptor activation, this leads to sustained elevated PKA activity involving downstream effector signaling pathways known to mediate synaptic plasticity and striatal function.Phosphorylation at the Thr69 site modulates phosphorylation at Ser114, which in turn affects PKA inhibition by dissociated RIIβ. Therefore, NMDA-dependent modulation of Thr69 has the potential to impart effects upon PKA activation. To test this, ventral striatal slices were treated with NMDA to activate fast neurotransmission, dopamine to activate slow neurotransmission, or both. As both NMDA and dopamine receptor activation are essential to the induction of striatal long-term potentiation (LTP) , these tFig. 3A, top). This Thr69 RIIβ siP (RIIβ siP) potently inhibited Cdk5-dependent phosphorylation of RIIβ (IC50 = 7.14 ± 2.28 μM) (A). Treatment of ventral striatal slices with this peptide caused reduction in phospho-Thr69 RIIβ but had no effect on Cdk5-dependent phosphorylation of Thr75 DARPP-32 . RIIβ siP/dopamine cotreatment of ventral striatal slices resulted in a sustained increase in PKA activity, similar to the previously observed effects of NMDA/dopamine treatment (B). Consequently, we established the means to selectively modulate phospho-Thr69 RIIβ levels in intact brain tissue and facilitate prolonged PKA activation.To directly assess the contribution of the RIIβ regulatory phosphorylation mechanism to striatal function, a small interfering peptide (siP) was developed which corresponded to 18 amino acid residues from the RIIβ linker region encompassing Thr69 and included a membrane-permeabilizing penetratin tag A, top. T2.28 μM) A. TreatmDARPP-32 A, bottomreatment B. ConseqA). LTP was induced by high-frequency stimulation (HFS) and could be enhanced by 15 min bath application of the D1-type dopamine receptor agonist SKF81297 (B), as previously observed for dorsolateral striatum (B and C). The RIIβ siP had no effect on synaptic excitation  or paired pulse response ratios. RIIβ siP caused a transient reduction in basal fEPSP amplitude but significantly enhanced LTP compared to scrambled control siP, causing elevations in fEPSP amplitudes that were maintained through 50 min following HFS (0.31 ± 0.03 mV scrambled siP versus 0.45 ± 0.06 mV for RIIβ siP). Interestingly, the enhancement of LTP by the RIIβ siP was partly occluded in slices pretreated with SKF-81297 (2 μM) prior to HFS (C and D).With this reagent in hand, we chose to assess the role of RIIβ phosphorylation in cortico-ventral striatal plasticity as one measure of physiological function. Cortical afferents within corpus callosum of sagittal brain slices were stimulated, and field excitatory postsynaptic potentials (fEPSPs) were recorded in ventral striatum core A. LTP waSKF81297 B, as prestriatum . To assestriatum , B and Cr to HFS , C and DD). This effect was attributable to increased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated function, as AMPA/NMDA ratios were increased by the RIIβ siP , consistent with elevated phospho-Ser845 GluR1, which is linked to increased synaptic surface AMPAR trafficking . In contrast, chronic exposure to stress caused elevation in the phosphorylation state of this site. Thus, environmental exposure to stressful conditions can alter the basal signaling state of RIIβ, raising the intriguing possibility that the regulation of RIIβ phosphorylation may contribute to behavioral responses induced by stress. Additionally, this mechanism may be targeted to modify behavioral responses to stress.Synaptic plasticity and cognition are affected by stress , 31, 32.Fig. 5via cannula were conducted. Delivery of FITC-tagged RIIβ siP to ventral striatal core MSNs was confirmed by immunostains (B). RIIβ siP infusion induced increased PKA-dependent phosphorylation of Ser114 RIIβ, Ser845 GluA1, and Thr34 DARPP-32 in vivo (C), similar to its effects in slices. Incorporating this approach with behavioral studies, animals were subjected to the 2-day forced swim test (FST. D). On day 1, animals that had undergone intra-accumbal cannulation were made to swim for 8 min. The following day, subjects were infused with either the RIIβ siP or a scrambled control peptide, then re-exposed to the stress of forced swimming. RIIβ siP-infused subjects spent significantly less time immobile . The RIIβ siP effect was completely blocked by co-administration of the PKA inhibitor, PKI. No changes in immobility time were detected when PKI alone was infused. Moreover, none of these infusions had a significant effect on latency to immobility in the FST or locomotor activity (A and B). Immobility was significantly less for animals that received the RIIβ siP during the 4 to 6 and 6 to 8 min periods of forced swimming compared to controls receiving scramble peptide (C). Also, the RIIβ siP’s effects closely mimicked those of the rapid-acting antidepressant, ketamine, administered at subanesthetic levels via bilateral intra-accumbal infusion. These findings demonstrate that RIIβ phosphorylation in the NAc mediates behavioral reactions to a stressful environment and that these responses may be altered by pharmacological targeting of this integrative mechanism.To examine this possibility, bilateral intra-accumbens infusions of the RIIβ siP nostains B. RIIβ s in vivo C, similaest FST. D. On dayimmobile E, left. activity , A and B peptide C. Also, F) where the PKA/RIIβ holoenzyme can be shifted to a form where Thr69 RIIβ is less phosphorylated in response to excitatory glutamatergic neurotransmission and more phosphorylated at Ser114. In this state, upon activation of Gs-coupled D1 dopamine receptor signaling, PKA activation may be prolonged, thereby contributing to synaptic plasticity and behavioral responses. Thus, the equilibrium between different regulatory states of PKA may be an important determinant of behavioral responses to stress and overall mood.Together these findings support a signaling scheme F where t2+, respectively. These signaling modes can act antagonistically by activating protein phosphorylation or dephosphorylation of the same substrate site. However, they may function most critically when working synergistically to invoke downstream effectors that alter neuronal excitability . For single unit patch clamp recordings, Drd2-EGFP mice in an FVB background were used. All manipulations were approved by UT Southwestern Medical Center and University of Alabama at Birmingham Institutional Animal Care and Use Committees and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.Ten- to 14 week-old male Sprague-Dawley rats  were used for most animal experiments and maintained on a 12 h light/dark cycle, single- or double-housed (chronic stress paradigm only) in standard cages, with food and water available All chemicals were purchased from Sigma-Aldrich unless otherwise specified. The sequence of the penetratin-tagged RII β-siP and penetratin-tagged scramble control peptides were RQIKIWFQNRRMKWKK-AGAAAGGGTPSKGVNFAEE and RQIKIWFQNRRMKWKK-AAGAGSGAVAFKGANGEA or RQIKIWFQNRRMKWKK-AGAAAGGGAPRKGVNFAEE, respectively. The FITC-tagged siP was synthesized with an N-terminal FITC label.in vitro by Cdk5 or PKA using standard conditions (18 resin (Millipore) for purification. Analysis was conducted using nanoelectrospray-QSTAR Pulsar I quadrupole time-of-flight tandem mass spectrometry (MDS Proteomics). Structural model of AKAP/RIIβ dimer interaction was based on published data of AKAP (PDB: 2HWN) (1CX4) . RIIβ was phosphorylated nditions . Cdk5/p2nditions  or DARPPnditions . For phonditions . DigestsB: 2HWN)  and RIIβ) (1CX4) .AB_2225905; 1:200) and RIIβ . For rat striatal immunofluorescent colocalization of Cdk5 and RIIβ, anti-Cdk5 (Rockland Cat.# 200-301-163) with goat anti-mouse Alexa488 and goat anti-rabbit Cy3 (Jackson Immuno) were used with DAPI  blue-fluorescent DNA counterstain (Millipore Sigma). For phospho-T69 RIIβ staining, paraffin-embedded rat brain cryosections (5 μ) were made. The 1° antibody was used at 1:200 dilution. A phospho-T69 RIIβ antibody was generated as previously described  and counterstained with NeuN .Immunostains were performed as previously described . Brieflyescribed  using thin vitro on 12-mm coverslips, fixed, immunostained for Cdk5 and RIIβ using the above indicated antibodies and imaged as previously described  from Long Evans rats (Charles River Labs) were cultured 14 to 21 days escribed .Using an open-source software of single-nucleus RNA sequencing data from the rat ventral striatum (Ratlus)  Cdk5 and2+ (0.5 mM) and a 30 min tissue recovery in drug-free buffer prior to harvest. Tissue was snap-frozen on dry ice. Immunoblotting was as previously described , phospho-S114 RIIβ (BD Biosciences Cat# 612550 RRID:AB_399845), and NMDAR1 (BD Biosciences Cat# 556308 RRID:AB_396353), phospho-S845 GluA1 (PhosphoSolutions Cat# p1160-845 RRID:AB_2492128) and phospho-T34 DARPP-32 (PhosphoSolutions Cat# p1025-34 RRID:AB_2492068), and total GluA1 (Millipore Cat# 05-855R RRID:AB_1587070). Total DARPP-32 antibody was from H. Hemmings .Slice pharmacology was conducted as previously described . Brieflyescribed  and anal2PO4, 7 MgCl2, 0.5 CaCl2, 25 NaHCO3, and 10 glucose . Parasagittal vibratome slices (350 μ thick) containing prefrontal cortex and NAc core were transferred into saline solution containing 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1.3 MgCl2, 2 CaCl2, 25 NaHCO3, and 25 glucose  and maintained for 20 min at 30 °C, then transferred to room temperature (22–25 °C) and allowed 1 h recovery. Slices were transferred to perfusion chambers placed within an upright microscope stage and visualized by infrared differential interference microscopy and a CCD Super Low Luminance camera . Slices were perfused continuously with oxygenated saline solution (2–3 ml/min).Adult rats were anaesthetized by isoflurane , and brains were placed in ice-cold sucrose saline solution (4 °C) containing (in mM): 87 NaCl, 75 Sucrose, 2.5 KCl, 1.25 NaHA antagonist  and were evoked by square current pulses (0.2 ms) at 0.033 Hz with a bipolar stimulation electrode (FHC) placed at the border of corpus callosum separated by ∼300 to 500 μ from the recording electrode. The perfusion saline solution was partially modified (in mM): 0.5 MgCl2, 1 CaCl2, 3.5 KCl. Results were obtained using a stimulus intensity to induce 60% of the maximal fEPSP amplitude taken from the I/O curve of each slice. The paired pulse ratio (PPR) paradigm was applied using different inter-stimulus intervals and PPR changes were measured as PPR = second fEPSP amplitude/first fEPSP amplitude. After recording stable baseline for at least 10 min, LTP was induced by HFS . fEPSP amplitude was monitored for at least 50 min after HFS to evaluate LTP. All recordings were performed using a Multiclamp 700B amplifier and filtered at 4 kHz and digitized with a Digidata 1440 with pClamp 10 software for data acquisition . Recording pipette was filled with the same extracellular solution as the perfusion bath (2–4 MΩ resistance).Extracellular recordings: fEPSPs were obtained at 25 °C in the presence of the GABA2, 7 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, 25 glucose, and 75 sucrose, bubbled with 95% O2/5% CO2. Parasagittal slices were cut transversely at 300 μm using a vibrating blade microtome . Slices were transferred to normal saline solution containing (in mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgCl2, 1.3 NaH2PO4, 26 NaHCO3, and 20 glucose at 32 °C for 30 min and then allowed to recover for 1 h at room temperature before recordings. Individual slices were transferred to a submerged chamber mounted on a fixed-stage upright microscope (Zeiss Axioskop FS) and continuously perfused at 30 °C with normal saline solution containing (in μM): 0 MgCl2, 50 picrotoxin, and 10 glycine. GFP-negative D1 cells were visualized by infrared differential interference contrast microscopy with a water-immersion 63X objective . Current clamp recordings were performed with unpolished pipettes containing (in mM): 140 K-gluconate, 5 KCl, 2 MgCl2, 10 Hepes, 4 Mg-ATP, 0.3 Na-GTP, 10 Na2-creatine phosphate, 0.2 Na-EGTA, 290 to 300 mOsm, pH 7.3 . LTP of EPSPs was induced by HFS in control and RIIβ siP-treated D1 cells (at −70 mV). Slices were incubated in the presence of 10 μM control or RIIβ siP for 1 h and washed out prior to recordings. Following LTP, slices were perfused with the NMDAR antagonist D-AP5 (20 μM) and the AMPAR antagonist NBQX (10 μM). All recordings were acquired with Axopatch-200B amplifiers (Molecular Devices), filtered at 2 kHz, and digitized at 10 kHz with ITC-18 A/D-D/A interfaces (InstruTech) controlled by custom-written software in G5 PowerMac computers .For whole-cell recordings, Drd2-EGFP FVB mice were deeply anesthetized and transcardially perfused with ice-cold cutting artificial cerebrospinal fluid containing (in mM): 87 NaCl, 2.5 KCl, 0.5 CaClRats were anesthetized with isoflurane and placed in a stereotaxic frame (Kopf Instruments). Bilateral cannulas  were placed in NAc core . Peptides were bilaterally infused 1 week later in awake and mobile animals. Injection volume was 1 μl of peptide . Animals were allowed to recover for 1 h prior to behavioral testing. Accuracy was confirmed by infusion of methylene blue dye or direct visualization of cannula tracts postbehavioral testing.The Porsolt FST was conducted as described previously . BrieflyThe chronic unpredictable stress paradigm was performed as adapted from Willner . Animalst test. In addition, for some comparisons the Wilcoxon signed-rank test was used where indicated. In some cases, the means for more than one group were compared by ANOVA with the appropriate post hoc comparison as specified in the figure legends using GraphPad Prism 6.0 unless stated otherwise. Where specified, two categorical variables were compared using a two-way ANOVA. For all experiments, ∗p <0 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 were considered significant. No statistical methods were used to predetermine sample sizes, but sample sizes are similar to those generally employed in comparable studies. Statistical analysis was conducted with n ≥ 4 except for in vitro biochemistry (n = 3).Data are reported as mean or normalized mean ± standard deviation, unless specified otherwise. Statistical analysis comparing two means was performed using the Student’s jbibb@uab.edu).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr James A. Bibb (This paper is dedicated to the memory of Paul Greengard.The authors declare there are no conflicts of interest with the contents of this article.