An anti-HER2 biparatopic antibody that induces unique HER2 clustering and complement-dependent cytotoxicity.

Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that plays an oncogenic role in breast, gastric and other solid tumors. However, anti-HER2 therapies are only currently approved for the treatment of breast and gastric/gastric esophageal junction cancers and treatment resistance remains a problem. Here, we engineer an anti-HER2 IgG1 bispecific, biparatopic antibody (Ab), zanidatamab, with unique and enhanced functionalities compared to both trastuzumab and the combination of trastuzumab plus pertuzumab (tras + pert). Zanidatamab binds adjacent HER2 molecules in trans and initiates distinct HER2 reorganization, as shown by polarized cell surface HER2 caps and large HER2 clusters, not observed with trastuzumab or tras + pert. Moreover, zanidatamab, but not trastuzumab nor tras + pert, elicit potent complement-dependent cytotoxicity (CDC) against high HER2-expressing tumor cells in vitro. Zanidatamab also mediates HER2 internalization and downregulation, inhibition of both cell signaling and tumor growth, antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), and also shows superior in vivo antitumor activity compared to tras + pert in a HER2-expressing xenograft model. Collectively, we show that zanidatamab has multiple and distinct mechanisms of action derived from the structural effects of biparatopic HER2 engagement.

Multidimensional Global Optimization and Robustness Analysis in the Context of Protein-Ligand Binding.

Accuracy of protein-ligand binding free energy calculations utilizing implicit solvent models is critically affected by parameters of the underlying dielectric boundary, specifically, the atomic and water probe radii. Here, a global multidimensional optimization pipeline is developed to find optimal atomic radii specifically for protein-ligand binding calculations in implicit solvent. The computational pipeline has these three key components: (1) a massively parallel implementation of a deterministic global optimization algorithm (VTDIRECT95), (2) an accurate yet reasonably fast generalized Born implicit solvent model (GBNSR6), and (3) a novel robustness metric that helps distinguish between nearly degenerate local minima via a postprocessing step of the optimization. A graph-based "kT-connectivity" approach to explore and visualize the multidimensional energy landscape is proposed: local minima that can be reached from the global minimum without exceeding a given energy threshold (kT) are considered to be connected. As an illustration of the capabilities of the optimization pipeline, we apply it to find a global optimum in the space of just five radii: four atomic (O, H, N, and C) radii and water probe radius. The optimized radii, ρW = 1.37 Å, ρC = 1.40 Å, ρH = 1.55 Å, ρN = 2.35 Å, and ρO = 1.28 Å, lead to a closer agreement of electrostatic binding free energies with the explicit solvent reference than two commonly used sets of radii previously optimized for small molecules. At the same time, the ability of the optimizer to find the global optimum reveals fundamental limits of the common two-dielectric implicit solvation model: the computed electrostatic binding free energies are still almost 4 kcal/mol away from the explicit solvent reference. The proposed computational approach opens the possibility to further improve the accuracy of practical computational protocols for binding free energy calculations.

Strongly Bent Double-Stranded DNA: Reconciling Theory and Experiment.

The strong bending of polymers is poorly understood. We propose a general quantitative framework of polymer bending that includes both the weak and strong bending regimes on the same footing, based on a single general physical principle. As the bending deformation increases beyond a certain (polymer-specific) point, the change in the convexity properties of the effective bending energy of the polymer makes the harmonic deformation energetically unfavorable: in this strong bending regime the energy of the polymer varies linearly with the average bending angle as the system follows the convex hull of the deformation energy function. For double-stranded DNA, the effective bending deformation energy becomes non-convex for bends greater than ~ 2° per base-pair, equivalent to the curvature of a closed circular loop of ~ 160 base pairs. A simple equation is derived for the polymer loop energy that covers both the weak and strong bending regimes. The theory shows quantitative agreement with recent DNA cyclization experiments on short DNA fragments, while maintaining the expected agreement with experiment in the weak bending regime. Counter-intuitively, cyclization probability (j-factor) of very short DNA loops is predicted to increase with decreasing loop length; the j-factor reaches its minimum for loops of ≃ 45 base pairs. Atomistic simulations reveal that the attractive component of the short-range Lennard-Jones interaction between the backbone atoms can explain the underlying non-convexity of the DNA effective bending energy, leading to the linear bending regime. Applicability of the theory to protein-DNA complexes, including the nucleosome, is discussed.

Accurate evaluation of charge asymmetry in aqueous solvation.

Charge hydration asymmetry (CHA)-a characteristic dependence of hydration free energy on the sign of the solute charge-quantifies the asymmetric response of water to electric field at microscopic level. Accurate estimates of CHA are critical for understanding hydration effects ubiquitous in chemistry and biology. However, measuring hydration energies of charged species is fraught with significant difficulties, which lead to unacceptably large (up to 300%) variation in the available estimates of the CHA effect. We circumvent these difficulties by developing a framework which allows us to extract and accurately estimate the intrinsic propensity of water to exhibit CHA from accurate experimental hydration free energies of neutral polar molecules. Specifically, from a set of 504 small molecules we identify two pairs that are analogous, with respect to CHA, to the K(+) /F(-) pair-a classical probe for the effect. We use these "CHA-conjugate" molecule pairs to quantify the intrinsic charge-asymmetric response of water to the microscopic charge perturbations: the asymmetry of the response is strong, ∼50% of the average hydration free energy of these molecules. The ability of widely used classical water models to predict hydration energies of small molecules correlates with their ability to predict CHA.

Systematic reverse engineering of network topologies: a case study of resettable bistable cellular responses.

A focused theme in systems biology is to uncover design principles of biological networks, that is, how specific network structures yield specific systems properties. For this purpose, we have previously developed a reverse engineering procedure to identify network topologies with high likelihood in generating desired systems properties. Our method searches the continuous parameter space of an assembly of network topologies, without enumerating individual network topologies separately as traditionally done in other reverse engineering procedures. Here we tested this CPSS (continuous parameter space search) method on a previously studied problem: the resettable bistability of an Rb-E2F gene network in regulating the quiescence-to-proliferation transition of mammalian cells. From a simplified Rb-E2F gene network, we identified network topologies responsible for generating resettable bistability. The CPSS-identified topologies are consistent with those reported in the previous study based on individual topology search (ITS), demonstrating the effectiveness of the CPSS approach. Since the CPSS and ITS searches are based on different mathematical formulations and different algorithms, the consistency of the results also helps cross-validate both approaches. A unique advantage of the CPSS approach lies in its applicability to biological networks with large numbers of nodes. To aid the application of the CPSS approach to the study of other biological systems, we have developed a computer package that is available in Information S1.

Introducing Charge Hydration Asymmetry into the Generalized Born Model.

The effect of charge hydration asymmetry (CHA)-non-invariance of solvation free energy upon solute charge inversion-is missing from the standard linear response continuum electrostatics. The proposed charge hydration asymmetric-generalized Born (CHA-GB) approximation introduces this effect into the popular generalized Born (GB) model. The CHA is added to the GB equation via an analytical correction that quantifies the specific propensity of CHA of a given water model; the latter is determined by the charge distribution within the water model. Significant variations in CHA seen in explicit water (TIP3P, TIP4P-Ew, and TIP5P-E) free energy calculations on charge-inverted "molecular bracelets" are closely reproduced by CHA-GB, with the accuracy similar to models such as SEA and 3D-RISM that go beyond the linear response. Compared against reference explicit (TIP3P) electrostatic solvation free energies, CHA-GB shows about a 40% improvement in accuracy over the canonical GB, tested on a diverse set of 248 rigid small neutral molecules (root mean square error, rmse = 0.88 kcal/mol for CHA-GB vs 1.24 kcal/mol for GB) and 48 conformations of amino acid analogs (rmse = 0.81 kcal/mol vs 1.26 kcal/mol). CHA-GB employs a novel definition of the dielectric boundary that does not subsume the CHA effects into the intrinsic atomic radii. The strategy leads to finding a new set of intrinsic atomic radii optimized for CHA-GB; these radii show physically meaningful variation with the atom type, in contrast to the radii set optimized for GB. Compared to several popular radii sets used with the original GB model, the new radii set shows better transferability between different classes of molecules.

Statistical mechanics model for the dynamics of collective epigenetic histone modification.

Epigenetic histone modifications play an important role in the maintenance of different cell phenotypes. The exact molecular mechanism for inheritance of the modification patterns over cell generations remains elusive. We construct a Potts-type model based on experimentally observed nearest-neighbor enzyme lateral interactions and nucleosome covalent modification state biased enzyme recruitment. The model can lead to effective nonlocal interactions among nucleosomes suggested in previous theoretical studies, and epigenetic memory is robustly inheritable against stochastic cellular processes.

BMP receptor 1A regulates development of hypothalamic circuits critical for feeding behavior.

Hypothalamic neural circuits are known to regulate energy homeostasis and feeding behavior, but how these circuits are established during development is not well understood. Here we report that embryonic neural progenitors that express the transcription factor OLIG1 contribute neurons to the ventral hypothalamus including the arcuate nucleus (ARH), a center that regulates feeding behavior. Ablation of bone morphogenetic protein receptor 1a (BMPR1A) in the OLIG1 lineage resulted in hypophagia, hypoglycemia, and weight loss after the second postnatal week with death by week 4. Differentiation and specification of inhibitory hypothalamic neurons contributing to melanocortin and dopaminergic systems were abnormal in the BMPR1A-deficient ARH. Although the hypophagia promoted expression of the orexigenic neuropeptide agouti related protein (AgRP) in the BMPR1A-deficient ARH, there was a profound decrease of AgRP(+) axonal terminals in the mutant ARH targets including dorsomedial and paraventricular hypothalamic nuclei. Projection of AgRP(+) neurons to these nuclei is known to be regulated by leptin. Leptin injection in neonatal mice increased bone morphogenic protein (BMP) signaling in the ventral hypothalamus, and blocking BMP signaling prevented leptin-induced neurite outgrowth in ARH explant cultures. These findings suggest that BMPR1A signaling is critical for postnatal establishment of leptin-responsive orexigenic fibers from ARH to multiple hypothalamic nuclei. More generally these observations indicate that BMPR1A signaling regulates postnatal establishment of OLIG1 lineage-derived ARH neuronal circuits that are critical for leptin-mediated feeding behavior.

Charge hydration asymmetry: the basic principle and how to use it to test and improve water models.

Charge hydration asymmetry (CHA) manifests itself in the experimentally observed strong dependence of free energy of ion hydration on the sign of the ion charge. This asymmetry is not consistently accounted for by popular models of solvation; its magnitude varies greatly between the models. While it is clear that CHA is somehow related to charge distribution within a water molecule, the exact nature of this relationship is unknown. We propose a simple, yet general and rigorous criterion that relates rotational and charge inversion properties of a water molecule's charge distribution with its ability to cause CHA. We show which electric multipole components of a water molecule are key to explain its ability for asymmetric charge hydration. We then test several popular water models and explain why specific models show none, little, or strong CHA in simulations. We use the gained insight to derive an analogue of the Born equation that includes the missing physics necessary to account for CHA and does not rely on redefining the continuum dielectric boundary. The proposed formula is as simple as the original, does not contain any fitting parameters, and predicts hydration free energies and entropies of spherical cations and anions within experimental uncertainty. Our findings suggest that the gap between the practical continuum electrostatics framework and the more fundamental explicit solvent treatment may be reduced considerably by explicitly introducing CHA into the existing continuum framework.

HeyL promotes neuronal differentiation of neural progenitor cells.

Members of the Hes and Hey families of basic helix-loop-helix transcription factors are regarded as Notch target genes that generally inhibit neuronal differentiation of neural progenitor cells. We found that HeyL, contrary to the classic function of Hes and Hey factors, promotes neuronal differentiation of neural progenitor cells both in culture and in the embryonic brain in vivo. Furthermore, null mutation of HeyL decreased the rate of neuronal differentiation of cultured neural progenitor cells. HeyL binds to and activates the promoter of the proneural gene neurogenin2, which is inhibited by other Hes and Hey family members, and HeyL is a weak inhibitor of the Hes1 promoter. HeyL is able to bind other Hes and Hey family members, but it cannot bind the Groucho/Tle1 transcriptional corepressor, which mediates the inhibitory effects of the Hes family of factors. Furthermore, although HeyL expression is only weakly augmented by Notch signaling, we found that bone morphogenic protein signaling increases HeyL expression by neural progenitor cells. These observations suggest that HeyL promotes neuronal differentiation of neural progenitor cells by activating proneural genes and by inhibiting the actions of other Hes and Hey family members.

BMPR1a and BMPR1b signaling exert opposing effects on gliosis after spinal cord injury.

Astrogliosis following spinal cord injury (SCI) involves an early hypertrophic response that is beneficial and a subsequent formation of a dense scar. We investigated the role of bone morphogenetic protein (BMP) signaling in gliosis after SCI and find that BMPR1a and BMPR1b signaling exerts opposing effects on hypertrophy. Conditional ablation of BMPR1a from glial fibrillary acidic protein (GFAP)-expressing cells leads to defective astrocytic hypertrophy, increased infiltration by inflammatory cells, and reduced axon density. BMPR1b-null mice conversely develop "hyperactive" reactive astrocytes and consequently have smaller lesion volumes. The effects of ablation of either receptor are reversed in the double knock-out animals. These findings indicate that BMPR1a and BMPR1b exert directly opposing effects on the initial reactive astrocytic hypertrophy. Also, BMPR1b knock-out mice have an attenuated glial scar in the chronic stages following injury, suggesting that it has a greater role in glial scar progression. To elucidate the differing roles of the two receptors in astrocytes, we examined the effects of ablation of either receptor in serum-derived astrocytes in vitro. We find that the two receptors exert opposing effects on the posttranscriptional regulation of astrocytic microRNA-21. Further, overexpression of microRNA-21 in wild-type serum-derived astrocytes causes a dramatic reduction in cell size accompanied by reduction in GFAP levels. Hence, regulation of microRNA-21 by BMP signaling provides a novel mechanism for regulation of astrocytic size. Targeting specific BMPR subunits for therapeutic purposes may thus provide an approach for manipulating gliosis and enhancing functional outcomes after SCI.

Opinion formation on adaptive networks with intensive average degree.

We study the evolution of binary opinions on a simple adaptive network of N nodes. At each time step, a randomly selected node updates its state ("opinion") according to the majority opinion of the nodes that it is linked to; subsequently, all links are reassigned with probability p̃ (q̃) if they connect nodes with equal (opposite) opinions. In contrast to earlier work, we ensure that the average connectivity ("degree") of each node is independent of the system size ("intensive"), by choosing p̃ and q̃ to be of O(1/N). Using simulations and analytic arguments, we determine the final steady states and the relaxation into these states for different system sizes. We find two absorbing states, characterized by perfect consensus, and one metastable state, characterized by a population split evenly between the two opinions. The relaxation time of this state grows exponentially with the number of nodes, N. A second metastable state, found in the earlier studies, is no longer observed.

HeyL regulates the number of TrkC neurons in dorsal root ganglia.

The basic-helix-loop-helix transcription factor HeyL is expressed at high levels by neural crest progenitor cells (NCPs) that give rise to neurons and glia in dorsal root ganglia (DRG). Since HeyL expression was observed in these NCPs during the period of neurogenesis, we generated HeyL null mutants to help examine the factor's role in ganglion neuronal specification. Homozygous null mutation of HeyL reduced the number of TrkC(+) neurons in DRG at birth including the subpopulation that expresses the ETS transcription factor ER81. Conversely, null mutation of the Hey paralog, Hey1, increased the number of TrkC(+) neurons. Null mutation of HeyL increased expression of the Hey paralogs Hey1 and Hey2, suggesting that HeyL normally inhibits their expression. Double null mutation of both Hey1 and HeyL rescued TrkC(+) neuron numbers to control levels. Thus, the balance between HeyL and Hey1 expression regulates the differentiation of a subpopulation of TrkC(+) neurons in the DRG.

Differential effects of BMP signaling on parvalbumin and somatostatin interneuron differentiation.

Several different populations of interneurons in the murine cortex, including somatostatin (SST)- or parvalbumin (PV)-expressing cells, are born in the ventral ganglionic eminences during mid-gestation and then migrate tangentially to the cortex. SST is expressed by some interneuron progenitors in the cerebral cortex and in migrating populations in the ventrolateral cortex at birth. However, PV (also known as PVALB) is not expressed by interneurons until the second postnatal week after reaching the cortex, suggesting that molecular cues in the cerebral cortex might be involved in the differentiation process. BMP4 is expressed at high levels in the somatosensory cortex at the time when the PV(+) interneurons differentiate. Treatment of cortical cultures containing interneuron precursors is sufficient to generate PV(+) interneurons prematurely and inhibit SST differentiation. Furthermore, overexpression of BMP4 in vivo increases the number of interneurons expressing PV, with a reduction in the number of SST(+) interneurons. PV(+) interneurons in the cortex express BMP type I receptors and a subpopulation displays activated BMP signaling, assessed by downstream molecules including phosphorylated SMAD1/5/8. Conditional mutation of BMP type I receptors in interneuron precursors significantly reduces the number of cortical PV(+) interneurons in the adult brain. Thus, BMP4 signaling through type I receptors regulates the differentiation of two major medial ganglionic eminence-derived interneuron populations and defines their relative numbers in the cortex.

BMPR1a signaling determines numbers of oligodendrocytes and calbindin-expressing interneurons in the cortex.

Progenitor cells that express the transcription factor olig1 generate several neural cell types including oligodendrocytes and GABAergic interneurons in the dorsal cortex. The fate of these progenitor cells is regulated by a number of signals including bone morphogenetic proteins (BMPs) secreted in the dorsal forebrain. BMPs signal by binding to heteromeric serine-threonine kinase receptors formed by type I (BMPR1a, BMPR1b, Alk2) and type II (BMPRII) subunits. To determine the specific role of the BMPR1a subunit in lineage commitment by olig1-expressing cells, we used a cre/loxP genetic approach to ablate BMPR1a in these cells while leaving signaling from other subunits intact. There was a reduction in numbers of immature oligodendrocytes in the BMPR1a-null mutant brains at birth. However, by postnatal day 20, the BMPR1a-null mice had a significant increase in the number of mature and immature oligodendrocytes compared with wild-type littermates. There was also an increase in the proportion of calbindin-positive interneurons in the dorsomedial cortex of BMPR1a-null mice at birth without any change in the number of parvalbumin- or calretinin-positive cells. These effects were attributable, at least in part, to a decrease in the length of the cell cycle in subventricular zone progenitor cells. Thus, our findings indicate that BMPR1a mediates the suppressive effects of BMP signaling on oligodendrocyte lineage commitment and on the specification of calbindin-positive interneurons in the dorsomedial cortex.