The concept of Halogen-Enriched Fragment Libraries (HEF) has gained significant traction in medicinal chemistry, supported by multiple successful screening campaigns.^[1,2] These libraries are designed to explore halogen-mediated interactions, which can play a critical role in molecular recognition and binding affinity. Halogen bonding, the key interaction exploited by HEF, has been extensively studied since its initial discovery via X-ray crystallography.^[3] Its electrostatic nature is now well understood, and it has been shown to significantly influence both selectivity and efficacy in drug-target interactions.^[4,5,6]

Halogen bonding through σ-hole interactions

A major factor in halogen bonding is the σ-hole, whose characteristics can be “tuned” by varying (hetero)aromatic ring systems and their substitution patterns. This tunability allows for precise control over interaction strength and directionality.

Enamine, a global leader in heterocyclic chemistry, has synthesized thousands of novel halogenated scaffolds, creating the most diverse collection of halogenated core heterocycles available. Our carefully designed Halogen-Enriched Fragment Library comprises the most relevant fragments for exploring affinity towards binding via these non-covalent halogen-mediated interactions.

Key features

• Diverse (hetero)aromatic fragments set for probing halogen bonds
• All compounds include at least one non-fluorine halogen atom
• No PAINS, only MedChem friendly compounds
• Express follow-up from stock and from Enamine REAL

Examples of compounds from HEF Library

Library design

The fragment library was constructed from a curated database of over 250,000 compounds, all strictly compliant with the Rule of Three (Ro3) and filtered using medicinal chemistry criteria. Each compound includes at least one heavy halogen atom (Cl, Br, or I), with 95% containing only one. The distribution is as follows: 183 iodides (9.5%), 963 bromides (50.1%), and 684 chlorides (35.6%). The remaining 4.8% are dichloro derivatives. Additionally, the library includes 194 fluorinated compounds (10.1%). Fragments were selected with heavy atom counts (HA) ranging from 6 to 19, with a majority (1,206 fragments) falling within the 12–16 HA range. Each fragment contains between one and three rings, with 97% featuring one or two. We also included 154 fragments from 200 originally described as HEFLib by Frank M. Boeckler. Scaffold analysis revealed the most frequently occurring (hetero)aromatic ring systems.

Most Frequently Used Heteroaromatic Ring Systems in the HEF Library

Nuisance compounds are commonly known for their interference with bioassay readouts in high-throughput screening (HTS), causing false positives that are inapplicable to the hit-finding process. Recently, Jonathan Baell provided a description of nuisance compound classes, including colloidal aggregates, PAINS (Pan-Assay Interference Compounds), electrophiles and redox cyclers, chelators, as well as optical and phenotypic assay interference compounds.

We provide a curated list of more than 80 nuisance chemicals known to obstruct assay readouts in target-based and phenotypic screenings. Conveniently available in an assay-ready screening plate, this nuisance compound set is expected to be of great interest to the research community, helping to develop high-quality HTS assays that yield promising, optimizable hits. Hits arising from this small set of nuisance compounds would be highly informative in screening campaigns, assisting target-owners in identifying the types of interference compounds that their assays might be susceptible to.

Key features

1. Enhances HTS assay reliability by addressing potential interference
2. Over 40 different modes of action
3. Readily accessible in an assay-ready screening plate
4. References for compounds are available

Library design

The global literature overview and data analysis served as the foundation for the design. Over 80 compounds are categorized based on their specific targets, structural elements, and types of interactions. The collection includes compounds widely recognized and validated as promiscuous across a range of independent and distinct screening methodologies.

Example of structures

Tuberculosis (TB) is among the most threatening diseases that can cause the next pandemic across the world. It is one of the most rampant infectious diseases owing largely to the methods of transmission and high death ratio. The absence of efficient treatment and the rapid appearance of drug resistance make TB one of the most emerging tasks to resolve in public health. Continuously mutated, Mtb remains one of the most dangerous and difficult-to-treat bacterial diseases. Lack of early diagnosis and the prevalence of resistant strains make TB extremely difficult for timely detection and treatment. Antituberculosis drug development is a high priority to prevent future pandemics. It needs further investment in scientific research and rational drug design to combat the high death rate caused by this disease.

The library is available in pre-plated formats for quick access and is supported with multiple benefits on hit follow-up studies:

• Analogs and hit samples resupply from dry stock of over 4.4 M compounds with immediate QC check.
• MedChem support in hit follow-up and optimization to lead series.
• Same-day ADME tests for design and synthesis prioritization.

Library Design

To design the library and select the most promising molecules, we pay attention to the complexity of Mtb cell-wall and the difficulties of membrane penetration. Despite the absence of certain ruleless and defined filters, we tried to remove all compounds that could have issues with penetration based on the reported data and structure analysis of cell active vs inactive compounds.

Having studied some special features related to the biochemical pathways and morphological properties inherent to Mtb, a group of protein targets has been selected and prioritized to perform in silico screening (vHTS) and select compounds with the alleged activity. Generally, the protein targets in this library can be divided into two subsets: Cell Wall Synthesis-associated proteins and Mtb-specific Essential protein targets distinct from those present in eukaryotic organisms.

Alanine racemase (Alr), which catalyzes the interconversion of L-alanine and D-alanine. Which in turn plays a key role in peptidoglycan cross-linking.

dTDP-4-dehydrorhamnose 3,5-epimerase (RmlC) is involved in the biosynthesis of the dTDP-L-rhamnose which is required for connection of the galactan region of arabinogalactan to peptidoglycan molecule.

Enoyl-[acyl-carrier-protein] reductase [NADH] (InhA) is involved in the biosynthesis of mycolic acids playing a key role in the fatty acid elongation process.

Decaprenylphosphoryl-beta-D-ribose oxidase (Dpre1) is a component of the complex that catalyzes the formation of decaprenyl-phospho-arabinose (DPA), a key precursor required for the synthesis of cell-wall arabinans.

N-Acetylglucosamine-1-phosphate uridyltransferase (GLMU) is an enzyme that catalyzes the final two steps in the biosynthesis of UDP-GlcNAc and is essential for the synthesis of the lipid A of lipopolysaccharide (LPS).

Shikimate kinase (AroK) is necessary for the synthesis of the common precursor of aromatic amino acids and secondary metabolites by phosphorylation of the 3-hydroxyl group of shikimic acid using ATP. The amino acids biosynthesis is also a very attractive direction for drug design, particularly as it is represented with a broad family of enzymes.

Beta-lactamase responsible for resistance to beta-lactam antibiotics.

Isocitrate lyase is in charge of the first step of the glyoxylate shunt, namely the synthesis of C4 dicarboxylic acids from C2 compounds.

ATPases (ClpC, ClpX) – chaperon proteins that induce degradation of proteins by bacterial proteasome. Play an essential role in bacterial proteostasis.

Mtb regulatory Serine Proteases (ClpP1P2) – serine proteases that form bacterial proteasomes and are responsible for protein homeostasis and degradation in bacteria.

Peptidyl tRNA hydrolase (Pth) is an essential Mtb-specific RNA hydrolase that participte in bacteria homeostasis and protein synthesis control.

The vHTS procedure assumes the application of grid-based representation of the binding site and designation of constraints based on literature data (mutation experiments) or ligand-protein interaction map with subsequent partial or full matching. Besides the rating of compounds by empirical docking score, the result conformations were also visually checked for the reliability of the binding mode. At the same time, the number of reference compounds against selected targets is relatively small and possesses a low degree of diversity inside each target-based reference set.

The example of the binding pose of Z1603549506 in the Shikimate kinase binding site.

Predicted binding conformation in ribbon and shape protein representation of Z1313265676 to RmlC reductase.

Examples of predicted hits Z111780734 (left) and Z189105794 binding conformation in the active site of InhA enzyme.