High-power screening (HPS) empowered by DNA-encoded libraries
Authors: Yashoda Krishna Sunkari, Vijay Kumar Siripuram, Thu-Lan Nguyen, Marc Flajolet
Journal: Trends in Pharmacological Sciences (2021)

We propose to group under the umbrella name HPS (high-power screening) technologies that screen an extremely large numbers [thousands to million times larger than HTS] (high-throuput screening) of chemical/biological compounds very effectively. The synergistic forces that differentiate HPS from HTS are the extra-large size of HPS libraries (combinatorial nature) and refinements for extremely efficient drug screening.HTS tests 1 million compounds in 1 million wells (50 000 compounds/week); HPS tests 1 billion compounds in a one tube (1 billion compounds/week).HPS offers novel screening options such as multiplex screening (parallel screening), thus allowing several screens to be performed at the same time (repeats, wild type vs mutants, different isoforms, competition with known compounds).Ex vivo screening is easily accessible with HPS.DNA-encoded libraries (DEL) technology offers new paths to druggable alternatives such as proteolysis targeting chimeras (PROTACs). The world is totally dependent on medications. As science progresses, new, better, and cheaper drugs are needed more than ever. The pharmaceutical industry has been predominantly dependent on high-throughput screening (HTS) for the past three decades. Considering that the discovery rate has been relatively constant, can one hope for a much-needed sudden trend uptick? DNA-encoded libraries (DELs) and similar technologies, that have several orders of magnitude more screening power than HTS, and that we propose to group together under the umbrella term of high-power screening (HPS), are very well positioned to do exactly that. HPS also offers novel screening options such as parallel screening, ex vivo and in vivo screening, as well as a new path to druggable alternatives such as proteolysis targeting chimeras (PROTACs). Altogether, HPS unlocks novel powerful drug discovery avenues. The world is totally dependent on medications. As science progresses, new, better, and cheaper drugs are needed more than ever. The pharmaceutical industry has been predominantly dependent on high-throughput screening (HTS) for the past three decades. Considering that the discovery rate has been relatively constant, can one hope for a much-needed sudden trend uptick? DNA-encoded libraries (DELs) and similar technologies, that have several orders of magnitude more screening power than HTS, and that we propose to group together under the umbrella term of high-power screening (HPS), are very well positioned to do exactly that. HPS also offers novel screening options such as parallel screening, ex vivo and in vivo screening, as well as a new path to druggable alternatives such as proteolysis targeting chimeras (PROTACs). Altogether, HPS unlocks novel powerful drug discovery avenues. Scientific progress and novel technologies are helping to uncover disease causes faster than ever before. Novel emergencies such as the advent of new pathogens (e.g., SARS-CoV2) are further pressuring the pharmaceutical world. To be even faster, cheaper, and more accessible to academia, a new type of power is urgently needed. High-throughput screening (HTS; see Glossary) has been extremely useful over the past three decades (Box 1) and remains at the center of most drug discovery campaigns. However, multiple unsolvable HTS hurdles persist and several technologies that have higher screening power than regular HTS are well positioned to tackle these limitations. These technologies, that have several orders of magnitude increased screening power than HTS, could be grouped together under the umbrella term of high-power screening (HPS). Those include, for example, all types of DNA-encoded library (DEL) technologies and combinatorial protein libraries (e.g., phage display). For reasons of space, we focus only on DEL technologies.Box 1Historical perspective of HTS and limitationsThe 1980s saw the birth of large-scale screening campaigns grouped under the code name of 'high-throughput screening' (HTS). The early evolution of HTS started in around 1984 in large pharmaceutical companies, and Pfizer was the uncontested leader. The catalyzer of the exponential growth of HTS was the birth of modern biochemistry and molecular biology that uncovered novel therapeutic targets (e.g., the h-Ras oncogene). Today, although the universal usefulness of HTS is obvious, its recognition did not take place without effort. It was with some skepticism, and under-preparedness, that the world welcomed HTS in the mid-1980s. In addition to the drug discovery process per se, the driving forces underlying HTS embraced, and revolutionized, diverse disciplines of biology (e.g., DNA sequencing, digital microscopy, protein crystallization). On the other hand, some of its limitations remained large obstacles. The technology has remained relatively cumbersome and rigid in some ways, limiting the number of compounds that can be tested (1 million compounds require 1 million wells) and the size/number of screening campaigns that can be accomplished. HTS libraries are typically smaller than 1 million compounds and are often over-used by the drug industry and academia. The target source, often E. coli, results in products that are often not optimal (e.g., folding, PTMs). Large amounts of protein targets and/or cells compatible with 105 wells are needed, necessitating costly steps, expensive equipment, and logistical requirements, as well as extensive infrastructure. The growing number of therapeutic targets combined with HTS limitations are driving the need for more screening power. This is how combinatorial chemistry erupted a few years after HTS; however, because of the lack of addressability, the application of combinatorial chemistry did not meet its expectations. As a result, the DNA-encoded library (DEL) technology was born, burgeoning from the combinatorial chemistry field. DEL technology, a concept proposed by Drs Richard Lerner and Sidney Brenner, was based on their proof of concept published in 1992 that aimed to solve 'addressability' by adding a unique DNA tag sequence to each drug-like molecule, thus allowing every molecule to be identified at the end of a screening campaign. DNA synthesis capabilities were very limited in the early 1990s, and large-scale sequencing had yet to be invented. The combination of a constantly decreasing cost of DNA synthesis and the emergence of next-generation DNA sequencing ('massively parallel sequencing'; this emerged in the 1990s and was first commercialized in 2005) led to the birth of DEL technology in the mid-2000s. If Pfizer has been associated with the birth of HTS, Drs Lerner and Brenner will certainly be associated with the birth of HPS, begetting it with their initial proof of principle in 1992, and being instrumental in its development since then; Dr Lerner remains at the forefront of the field. The 1980s saw the birth of large-scale screening campaigns grouped under the code name of 'high-throughput screening' (HTS). The early evolution of HTS started in around 1984 in large pharmaceutical companies, and Pfizer was the uncontested leader. The catalyzer of the exponential growth of HTS was the birth of modern biochemistry and molecular biology that uncovered novel therapeutic targets (e.g., the h-Ras oncogene). Today, although the universal usefulness of HTS is obvious, its recognition did not take place without effort. It was with some skepticism, and under-preparedness, that the world welcomed HTS in the mid-1980s. In addition to the drug discovery process per se, the driving forces underlying HTS embraced, and revolutionized, diverse disciplines of biology (e.g., DNA sequencing, digital microscopy, protein crystallization). On the other hand, some of its limitations remained large obstacles. The technology has remained relatively cumbersome and rigid in some ways, limiting the number of compounds that can be tested (1 million compounds require 1 million wells) and the size/number of screening campaigns that can be accomplished. HTS libraries are typically smaller than 1 million compounds and are often over-used by the drug industry and academia. The target source, often E. coli, results in products that are often not optimal (e.g., folding, PTMs). Large amounts of protein targets and/or cells compatible with 105 wells are needed, necessitating costly steps, expensive equipment, and logistical requirements, as well as extensive infrastructure. The growing number of therapeutic targets combined with HTS limitations are driving the need for more screening power. This is how combinatorial chemistry erupted a few years after HTS; however, because of the lack of addressability, the application of combinatorial chemistry did not meet its expectations. As a result, the DNA-encoded library (DEL) technology was born, burgeoning from the combinatorial chemistry field. DEL technology, a concept proposed by Drs Richard Lerner and Sidney Brenner, was based on their proof of concept published in 1992 that aimed to solve 'addressability' by adding a unique DNA tag sequence to each drug-like molecule, thus allowing every molecule to be identified at the end of a screening campaign. DNA synthesis capabilities were very limited in the early 1990s, and large-scale sequencing had yet to be invented. The combination of a constantly decreasing cost of DNA synthesis and the emergence of next-generation DNA sequencing ('massively parallel sequencing'; this emerged in the 1990s and was first commercialized in 2005) led to the birth of DEL technology in the mid-2000s. If Pfizer has been associated with the birth of HTS, Drs Lerner and Brenner will certainly be associated with the birth of HPS, begetting it with their initial proof of principle in 1992, and being instrumental in its development since then; Dr Lerner remains at the forefront of the field. HTS has some strong advantages and will continue to be highly valuable for the drug discovery industry. HTS libraries of compounds can be highly curated libraries (size, functionalities, chemical space). HTS includes functional screening, often in live cells, and library noise is often well established. However, HTS also has several limitations that HPS can address: (i) because of heavy and highly specialized automation, robot maintenance, and a large real-estate footprint, the high price of HTS reflects the cost of building a library [HTS, 1 billion dollars for 1 million compounds ($1000 per compound); compared to HPS, <1 million dollars for 1 billion compounds for DEL (<1 cent per DEL compound)] and for the cost of using it (HTS, 0.05 to 1 dollars per well; HPS, negligible on a per-compound basis); (ii) the relatively limited number of molecules that can be reasonably tested in a single screening campaign (typically a hundred thousand to a million), the inconveniency of introducing new compounds and pharmacophores into an existing library (need for relatively pure compounds), and the limited number of HTS compounds (a total of 16 million in all HTS libraries) indicate that the same compound libraries are used over and over; (iii) an overwhelmingly large fraction of HTS compounds are constrained by the Lipinski rule of five and are therefore not adapted to targeting larger areas such as protein–protein interactions; (iv) the format in multi-well plates necessitates large amounts of the target, thus increasing the cost and the constraints on target purification/origin [HTS requires Escherichia coli recombinants and a few micrograms per compound (500 mg per million compounds), whereas HPS requires only 25 micrograms comprising 1 billion compounds]; (v) local contamination of stock plates from reusing the same library is another significant drawback and requires compound/library maintenance (occasional liquid chromatography/mass spectrometry verification and well replenishment); (vi) a significant number of colored compounds and dyes interfere with various types of assays leading to higher false positive rates; and (vii) cellular assays represent a major improvement for HTS, but limitations persist and the constrained access and scalability of primary cultures and stem cells impedes their use for HTS. These limitations alone or in combination lead to overall low data quality. The two synergistic advantages of HPS that are new compared to HTS, and that differentiate between the two by several orders of magnitude, are the extra-large sizes of the libraries, reflecting their combinatorial nature and modifications that allow extremely efficient drug screening. For DEL technology, the DNA-tagging system allows very large numbers of compounds to coexist in a single tube and brings an entirely new set of screening prospects not seen before. The father of HPS, Dr Richard Lerner, was of primary and tremendous importance regarding phage display screening and perhaps even more for DEL technologies. He coinvented the DEL concept together with the late Dr Sidney Brenner about 30 years ago, long before DNA synthesis was mainstream and about 15 years before next-generation sequencing (NGS) was invented. The libraries built/screens performed and the therapeutic targets used are summarized in Table 1 and Figure 1A , respectively. Table 1 shows the great diversity in terms of library size, with the disappearance over the past few years of excessively large libraries (hundreds of billions and trillions), demonstrating some level of adaptability and evolution of the field. HPS is well positioned to significantly and positively shake up the drug discovery process and could do so in several ways that we review in the following sections.Table 1Studies published describing the construction of DNA-encoded libraries and DEL screening campaignsaAbbreviations: B, billion; BMS, Bristol-Myers Squibb; ETH, Eidgenössische Technische Hochschule; GSK, GlaxoSmithKline; M, million; N.S., not specified; T, trillion., bHPS library sizes: categories 2XS through to 4XL are defined in Figure 1; sizes are color-coded from extra-small to medium (black), large (green), to extra-large (orange and red). 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Chem. 2021; 64: 4196-4205Crossref PubMed Scopus (8) Google Scholar]a Abbreviations: B, billion; BMS, Bristol-Myers Squibb; ETH, Eidgenössische Technische Hochschule; GSK, GlaxoSmithKline; M, million; N.S., not specified; T, trillion.b HPS library sizes: categories 2XS through to 4XL are defined in Figure 1; sizes are color-coded from extra-small to mediu