Illuminating protein space with a programmable generative model.

Three billion years of evolution has produced a tremendous diversity of protein molecules1, but the full potential of proteins is likely to be much greater. Accessing this potential has been challenging for both computation and experiments because the space of possible protein molecules is much larger than the space of those likely to have functions. Here we introduce Chroma, a generative model for proteins and protein complexes that can directly sample novel protein structures and sequences, and that can be conditioned to steer the generative process towards desired properties and functions. To enable this, we introduce a diffusion process that respects the conformational statistics of polymer ensembles, an efficient neural architecture for molecular systems that enables long-range reasoning with sub-quadratic scaling, layers for efficiently synthesizing three-dimensional structures of proteins from predicted inter-residue geometries and a general low-temperature sampling algorithm for diffusion models. Chroma achieves protein design as Bayesian inference under external constraints, which can involve symmetries, substructure, shape, semantics and even natural-language prompts. The experimental characterization of 310 proteins shows that sampling from Chroma results in proteins that are highly expressed, fold and have favourable biophysical properties. The crystal structures of two designed proteins exhibit atomistic agreement with Chroma samples (a backbone root-mean-square deviation of around 1.0 Å). With this unified approach to protein design, we hope to accelerate the programming of protein matter to benefit human health, materials science and synthetic biology.

Tumor-localized catalases can fail to alter tumor growth and transcriptional profiles in subcutaneous syngeneic mouse tumor models.

Catalase is an antioxidant enzyme that catalyzes the rapid conversion of hydrogen peroxide to water and oxygen. Use of catalase as a cancer therapeutic has been proposed to reduce oxidative stress and hypoxia in the tumor microenvironment, both activities which are hypothesized to reduce tumor growth. Furthermore, exposing murine tumors to exogenous catalase was previously reported to have therapeutic benefit. We studied the therapeutic effect of tumor-localized catalases with the aim to further elucidate the mechanism of action. To do this, we engineered two approaches to maximize intratumoral catalase exposure: 1) an injected extracellular catalase with enhanced tumor retention, and 2) tumor cell lines that over-express intracellular catalase. Both approaches were characterized for functionality and tested for therapeutic efficacy and mechanism in 4T1 and CT26 murine syngeneic tumor models. The injected catalase was confirmed to have enzyme activity >30,000 U/mg and was retained at the injection site for more than one week in vivo. The engineered cell lines exhibited increased catalase activity and antioxidant capacity, with catalase over-expression that was maintained for at least one week after gene expression was induced in vivo. We did not observe a significant difference in tumor growth or survival between catalase-treated and untreated mice when either approach was used. Finally, bulk RNA sequencing of tumors was performed, comparing the gene expression of catalase-treated and untreated tumors. Gene expression analysis revealed very few differentially expressed genes as a result of exposure to catalase and notably, we did not observe changes consistent with an altered state of hypoxia or oxidative stress. In conclusion, we observe that sustained intratumoral catalase neither has therapeutic benefit nor triggers significant differential expression of genes associated with the anticipated therapeutic mechanism in the subcutaneous syngeneic tumor models used. Given the lack of effect observed, we propose that further development of catalase as a cancer therapeutic should take these findings into consideration.

Alum-anchored intratumoral retention improves the tolerability and antitumor efficacy of type I interferon therapies.

Effective antitumor immunity in mice requires activation of the type I interferon (IFN) response pathway. IFNα and IFNß therapies have proven promising in humans, but suffer from limited efficacy and high toxicity. Intratumoral IFN retention ameliorates systemic toxicity, but given the complexity of IFN signaling, it was unclear whether long-term intratumoral retention of type I IFNs would promote or inhibit antitumor responses. To this end, we compared the efficacy of IFNα and IFNß that exhibit either brief or sustained retention after intratumoral injection in syngeneic mouse tumor models. Significant enhancement in tumor retention, mediated by anchoring these IFNs to coinjected aluminum-hydroxide (alum) particles, greatly improved both their tolerability and efficacy. The improved efficacy of alum-anchored IFNs could be attributed to sustained pleiotropic effects on tumor cells, immune cells, and nonhematopoietic cells. Alum-anchored IFNs achieved high cure rates of B16F10 tumors upon combination with either anti-PD-1 antibody or interleukin-2. Interestingly however, these alternative combination immunotherapies yielded disparate T cell phenotypes and differential resistance to tumor rechallenge, highlighting important distinctions in adaptive memory formation for combinations of type I IFNs with other immunotherapies.

An affinity threshold for maximum efficacy in anti-PD-1 immunotherapy.

Monoclonal antibodies targeting the programmed cell death protein 1 (PD-1) remain the most prevalent cancer immunotherapy both as a monotherapy and in combination with additional therapies. Despite the extensive success of anti-PD-1 monoclonal antibodies in the clinic, the experimental relationship between binding affinity and functional potency for anti-PD-1 antibodies in vivo has not been reported. Anti-PD-1 antibodies with higher and lower affinity than nivolumab or pembrolizumab are entering the clinic and show varied preclinical efficacy. Here, we explore the role of broad-ranging affinity variation within a single lineage in a syngeneic immunocompetent mouse model. By developing a panel of murine anti-PD-1 antibodies with varying affinity (ranging from KD = 20 pM - 15 nM), we find that there is a threshold affinity required for maximum efficacy at a given dose in the treatment of the MC38 adenocarcinoma model with anti-PD-1 immunotherapy. Physiologically based pharmacokinetic modeling complements interpretation of the experimental results and highlights the direct relationship between dose, affinity, and PD-1 target saturation in the tumor.

Early stages of speciation with gene flow in the Amazilia Hummingbird (Amazilis amazilia) subspecies complex of Western South America.

Disentangling the factors underlying the diversification of geographically variable species with a wide geographical range is essential to understanding the initial stages and drivers of the speciation process. The Amazilia Hummingbird, Amazilis amazilia, is found along the Pacific coast from northern Ecuador down to the Nazca Valley of Peru, and is currently classified as six phenotypically differentiated subspecies. We aimed to resolve the evolutionary relationships of the six subspecies, to assess the geographical pattern and extent of evolutionary divergence, and to test for introgression using both a mtDNA marker and a genome-by-sequencing dataset from 86 individuals from across the species range. The consensus phylogenetic tree separated the six subspecies into three distinct clades, corresponding with the Ecuador lowlands (A. amazilia dumerilii), the Ecuador highlands (A. amazilia alticola and A. amazilia azuay), and the Peruvian coast (A. amazilia leucophoea, A. amazilia amazilia, and A. amazilia caeruleigularis). However, an unresolved mtDNA network suggests that the diversification of the subspecies was recent and rapid. We found evidence of gene flow among the subspecies A. amazilia dumerilii, A. amazilia alticola, and A. amazilia leucophoea, with strong genetic isolation of the subspecies A. amazilia azuay in the isolated Yunguilla Valley of Ecuador. Finally, environmental data from each subspecies' capture locations were concordant with the three distinct clades. Overall, our results suggest that both expansions into new habitats and geographic isolation shaped the present-day phylogeny and range of the A. amazilia subspecies, and that A. amazilia azuay may be genetically divergent enough to be considered a separate species.

Comparison of adult census size and effective population size support the need for continued protection of two Solomon Island endemics.

Because a population's ability to respond to rapid change is dictated by standing genetic variation, we can better predict a population's long-term viability by estimating and then comparing adult census size (N) and effective population size (N e ). However, most studies only measure N or N e , which can be misleading. Using a combination of field and genomic sequence data, we here estimate and compare N and N e in two range-restricted endemics of the Solomon Islands. Two Zosterops White-eye species inhabit the small island of Kolombangara, with a high elevation species endemic to the island (Z. murphyi) and a low elevation species endemic to the Solomon Islands (Z. kulambangrae). Field observations reveal large values of N for both species with Z. kulambangrae numbering at 114,781 ± 32,233 adults, and Z. murphyi numbering at 64,412 ± 15,324 adults. In contrast, genomic analyses reveal that N e was much lower than N, with Z. kulambangrae estimated at 694.5 and Z. murphyi at 796.1 individuals. Further, positive Tajima's D values for both species suggest that they have experienced a demographic contraction, providing a mechanism for low values of N e . Comparison of N and N e suggests that Z. kulambangrae and Z. murphyi are not at immediate threat of extinction but may be at genetic risk. Our results provide important baseline data for long-term monitoring of these island endemics, and argue for measuring both population size estimates to better gauge long-term population viability.

Rapid, complete reproductive isolation in two closely related Zosterops White-eye bird species despite broadly overlapping ranges.

Examining what happens when two closely related species come into secondary contact provides insight into the later stages of the speciation process. The Zosteropidae family of birds is one of the most rapidly speciating vertebrate lineages. Members of this family are highly vagile and geographically widespread, raising the question of how divergence can occur if populations can easily come into secondary contact. On the small island of Kolombangara, two closely related nonsister species of white-eyes, Zosterops kulambangrae and Zosterops murphyi, are distributed along an elevational gradient and come into secondary contact at mid-elevations. We captured 134 individuals of both species along two elevational transects. Using genotyping-by-sequencing data and a mitochondrial marker, we found no evidence of past hybridization events and strong persistence of species boundaries, even though the species have only been diverging for approximately 2 million years. We explore potential reproductive barriers that allow the two species to coexist in sympatry, including premating isolation based on divergence in plumage and song. We also conducted a literature review to determine the time it takes to evolve complete reproductive isolation in congeneric avian species/subspecies in secondary contact (restricted to cases where congeneric taxa are parapatric or have a hybrid zone), finding our study is one of the youngest examples of complete reproductive isolation studied in a genomic context reported in birds.

Repurposing niclosamide as a versatile antimicrobial surface coating against device-associated, hospital-acquired bacterial infections.

Device-associated and hospital-acquired infections remain amongst the greatest challenges in regenerative medicine. Furthermore, the rapid emergence of antibiotic resistance and lack of new classes of antibiotics has made the treatment of these bacterial infections increasingly difficult. The repurposing of Food and Drug Administration approved drugs for antimicrobial therapies is a powerful means of reducing the time and cost associated with drug discovery and development. In this work, niclosamide, a commercially available anthelmintic drug with recently identified antimicrobial properties, was found to prevent the formation of, and combat existing biofilms of, several relevant Gram-positive bacteria, namely strains of Staphylococcus aureus, including methicillin resistant S. aureus (MRSA), and Staphylococcus epidermidis, all common causes of hospital-acquired and device-associated infections. This anti-biofilm activity was demonstrated at niclosamide concentrations as low as 0.01 µg ml-1. We then assessed niclosamide activity as an antibacterial coating, which could potentially be applied to medical device surfaces. We developed solvent cast niclosamide coatings on a variety of surfaces common amongst medical devices including glass, titanium, stainless steel, and aluminum. Niclosamide-coated surfaces exhibited potent in vitro activity against S. aureus, MRSA, and S. epidermidis. At niclosamide surface concentrations as low as 1.6 × 10-2 µg mm-2, the coatings prevented attachment of these bacteria. The coatings also cleared bacteria inoculated suspensions at niclosamide surface concentrations of 3.1 × 10-2 µg mm-2. Hemolysis was not observed at any of the antimicrobial coating concentrations tested. We report a facile, effective means of coating devices with niclosamide to both clear and prevent biofilm formation of common bacteria encountered in hospital-acquired and device-associated infections.