Azapeptides with unique covalent warheads as SARS-CoV-2 main protease inhibitors.

The main protease (MPro) of SARS-CoV-2, the causative agent of COVID-19, is a pivotal nonstructural protein critical for viral replication and pathogenesis. Its protease function relies on three active site pockets for substrate recognition and a catalytic cysteine for enzymatic activity. To develop potential SARS-CoV-2 antivirals, we successfully synthesized a diverse range of azapeptide inhibitors with various covalent warheads to target MPro's catalytic cysteine. Our characterization identified potent MPro inhibitors, including MPI89 that features an aza-2,2-dichloroacetyl warhead with a remarkable EC50 value of 10 nM against SARS-CoV-2 infection in ACE2+ A549 cells and a selective index of 875. MPI89 is also remarkably selective and shows no potency against SARS-CoV-2 papain-like protease and several human proteases. Crystallography analyses demonstrated that these inhibitors covalently engaged the catalytic cysteine and used the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 stands as one of the most potent MPro inhibitors, suggesting the potential for further exploration of azapeptides and the aza-2,2-dichloroacetyl warhead for developing effective therapeutics against COVID-19.

A Systematic Survey of Reversibly Covalent Dipeptidyl Inhibitors of the SARS-CoV-2 Main Protease.

SARS-CoV-2, the COVID-19 pathogen, relies on its main protease (MPro) for replication and pathogenesis. MPro is a demonstrated target for the development of antivirals for SARS-CoV-2. Past studies have systematically explored tripeptidyl inhibitors such as nirmatrelvir as MPro inhibitors. However, dipeptidyl inhibitors especially those with a spiro residue at their P2 position have not been systematically investigated. In this work, we synthesized about 30 dipeptidyl MPro inhibitors and characterized them on enzymatic inhibition potency, structures of their complexes with MPro, cellular MPro inhibition potency, antiviral potency, cytotoxicity, and in vitro metabolic stability. Our results indicated that MPro has a flexible S2 pocket to accommodate inhibitors with a large P2 residue and revealed that dipeptidyl inhibitors with a large P2 spiro residue such as (S)-2-azaspiro [4,4]nonane-3-carboxylate and (S)-2-azaspiro[4,5]decane-3-carboxylate have favorable characteristics. One compound, MPI60, containing a P2 (S)-2-azaspiro[4,4]nonane-3-carboxylate displayed high antiviral potency, low cellular cytotoxicity, and high in vitro metabolic stability.

An amber-encoding helper phage for more efficient phage display of noncanonical amino acids.

Using an amber suppression-based noncanonical amino acid (ncAA) mutagenesis approach, the chemical space in phage display can be significantly expanded for drug discovery. In this work, we demonstrate the development of a novel helper phage, CMa13ile40, for continuous enrichment of amber obligate phage clones and efficient production of ncAA-containing phages. CMa13ile40 was constructed by insertion of a Candidatus Methanomethylophilus alvus pyrrolysyl-tRNA synthetase/PylT gene cassette into a helper phage genome. The novel helper phage allowed for a continuous amber codon enrichment strategy for two different libraries and demonstrated a 100-fold increase in packaging selectivity. CMa13ile40 was then used to create two peptide libraries containing separate ncAAs, Nϵ-tert-butoxycarbonyl-lysine and Nϵ-allyloxycarbonyl-lysine, respectively. These libraries were used to identify peptide ligands that bind to the extracellular domain of ZNRF3. Each selection showed differential enrichment of unique sequences dependent upon the ncAA used. Peptides from both selections were confirmed to have low micromolar affinity for ZNRF3 that was dependent upon the presence of the ncAA used for selection. Our results demonstrate that ncAAs in phages provide unique interactions for identification of unique peptides. As an effective tool for phage display, we believe that CMa13ile40 can be broadly applied to a wide variety of applications.

An Azapeptide Platform in Conjunction with Covalent Warheads to Uncover High-Potency Inhibitors for SARS-CoV-2 Main Protease.

Main protease (M Pro ) of SARS-CoV-2, the viral pathogen of COVID-19, is a crucial nonstructural protein that plays a vital role in the replication and pathogenesis of the virus. Its protease function relies on three active site pockets to recognize P1, P2, and P4 amino acid residues in a substrate and a catalytic cysteine residue for catalysis. By converting the P1 Cα atom in an M Pro substrate to nitrogen, we showed that a large variety of azapeptide inhibitors with covalent warheads targeting the M Pro catalytic cysteine could be easily synthesized. Through the characterization of these inhibitors, we identified several highly potent M Pro inhibitors. Specifically, one inhibitor, MPI89 that contained an aza-2,2-dichloroacetyl warhead, displayed a 10 nM EC 50 value in inhibiting SARS-CoV-2 from infecting ACE2 + A549 cells and a selectivity index of 875. The crystallography analyses of M Pro bound with 6 inhibitors, including MPI89, revealed that inhibitors used their covalent warheads to covalently engage the catalytic cysteine and the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 represents one of the most potent M Pro inhibitors developed so far, suggesting that further exploration of the azapeptide platform and the aza-2,2-dichloroacetyl warhead is needed for the development of potent inhibitors for the SARS-CoV-2 M Pro as therapeutics for COVID-19.

A Systematic Survey of Reversibly Covalent Dipeptidyl Inhibitors of the SARS-CoV-2 Main Protease.

SARS-CoV-2 is the coronavirus pathogen of the currently prevailing COVID-19 pandemic. It relies on its main protease (M Pro ) for replication and pathogenesis. M Pro is a demonstrated target for the development of antivirals for SARS-CoV-2. Past studies have systematically explored tripeptidyl inhibitors such as nirmatrelvir as M Pro inhibitors. However, dipeptidyl inhibitors especially those with a spiro residue at their P2 position have not been systematically investigated. In this work, we synthesized about 30 reversibly covalent dipeptidyl M Pro inhibitors and characterized them on in vitro enzymatic inhibition potency, structures of their complexes with M Pro , cellular M Pro inhibition potency, antiviral potency, cytotoxicity, and in vitro metabolic stability. Our results indicated that M Pro has a flexible S2 pocket that accommodates dipeptidyl inhibitors with a large P2 residue and revealed that dipeptidyl inhibitors with a large P2 spiro residue such as ( S )-2-azaspiro[4,4]nonane-3-carboxylate and ( S )-2-azaspiro[4,5]decane-3-carboxylate have optimal characteristics. One compound MPI60 containing a P2 ( S )-2-azaspiro[4,4]nonane-3-carboxylate displayed high antiviral potency, low cellular cytotoxicity, and high in vitro metabolic stability and can be potentially advanced to further preclinical tests.

A multi-pronged evaluation of aldehyde-based tripeptidyl main protease inhibitors as SARS-CoV-2 antivirals.

As an essential enzyme of SARS-CoV-2, the COVID-19 pathogen, main protease (MPro) is a viable target to develop antivirals for the treatment of COVID-19. By varying chemical compositions at both P2 and P3 positions and the N-terminal protection group, we synthesized 18 tripeptidyl MPro inhibitors that contained also an aldehyde warhead and ß-(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 position. Systematic characterizations of these inhibitors were conducted, including their in vitro enzymatic inhibition potency, X-ray crystal structures of their complexes with MPro, their inhibition of MPro transiently expressed in 293T cells, and cellular toxicity and SARS-CoV-2 antiviral potency of selected inhibitors. These inhibitors have a large variation of determined in vitro enzymatic inhibition IC50 values that range from 4.8 to 650 nM. The determined in vitro enzymatic inhibition IC50 values reveal that relatively small side chains at both P2 and P3 positions are favorable for achieving high in vitro MPro inhibition potency, the P3 position is tolerable toward unnatural amino acids with two alkyl substituents on the α-carbon, and the inhibition potency is sensitive toward the N-terminal protection group. X-ray crystal structures of MPro bound with 16 inhibitors were determined. In all structures, the MPro active site cysteine interacts covalently with the aldehyde warhead of the bound inhibitor to form a hemithioacetal that takes an S configuration. For all inhibitors, election density around the N-terminal protection group is weak indicating possible flexible binding of this group to MPro. In MPro, large structural variations were observed on residues N142 and Q189. Unlike their high in vitro enzymatic inhibition potency, most inhibitors showed low potency to inhibit MPro that was transiently expressed in 293T cells. Inhibitors that showed high potency to inhibit MPro transiently expressed in 293T cells all contain O-tert-butyl-threonine at the P3 position. These inhibitors also exhibited relatively low cytotoxicity and high antiviral potency. Overall, our current and previous studies indicate that O-tert-butyl-threonine at the P3 site is a key component to achieve high cellular and antiviral potency for tripeptidyl aldehyde inhibitors of MPro.

A systematic exploration of boceprevir-based main protease inhibitors as SARS-CoV-2 antivirals.

Boceprevir is an HCV NSP3 inhibitor that was explored as a repurposed drug for COVID-19. It inhibits the SARS-CoV-2 main protease (MPro) and contains an α-ketoamide warhead, a P1 ß-cyclobutylalanyl moiety, a P2 dimethylcyclopropylproline, a P3 tert-butylglycine, and a P4 N-terminal tert-butylcarbamide. By introducing modifications at all four positions, we synthesized 20 boceprevir-based MPro inhibitors including PF-07321332 and characterized their MPro inhibition potency in test tubes (in vitro) and 293T cells (in cellulo). Crystal structures of MPro bound with 10 inhibitors and cytotoxicity and antiviral potency of 4 inhibitors were characterized as well. Replacing the P1 site with a ß-(S-2-oxopyrrolidin-3-yl)-alanyl (Opal) residue and the warhead with an aldehyde leads to high in vitro potency. The original moieties at P2, P3 and the P4 N-terminal cap positions in boceprevir are better than other tested chemical moieties for high in vitro potency. In crystal structures, all inhibitors form a covalent adduct with the MPro active site cysteine. The P1 Opal residue, P2 dimethylcyclopropylproline and P4 N-terminal tert-butylcarbamide make strong hydrophobic interactions with MPro, explaining high in vitro potency of inhibitors that contain these moieties. A unique observation was made with an inhibitor that contains a P4 N-terminal isovaleramide. In its MPro complex structure, the P4 N-terminal isovaleramide is tucked deep in a small pocket of MPro that originally recognizes a P4 alanine side chain in a substrate. Although all inhibitors show high in vitro potency, they have drastically different in cellulo potency to inhibit ectopically expressed MPro in human 293T cells. In general, inhibitors with a P4 N-terminal carbamide or amide have low in cellulo potency. This trend is reversed when the P4 N-terminal cap is changed to a carbamate. The installation of a P3 O-tert-butyl-threonine improves in cellulo potency. Three molecules that contain a P4 N-terminal carbamate were advanced to cytotoxicity tests on 293T cells and antiviral potency tests on three SARS-CoV-2 variants. They all have relatively low cytotoxicity and high antiviral potency with EC50 values around 1 µM. A control compound with a nitrile warhead and a P4 N-terminal amide has undetectable antiviral potency. Based on all observations, we conclude that a P4 N-terminal carbamate in a boceprevir derivative is key for high antiviral potency against SARS-CoV-2.

Evaluation of SARS-CoV-2 Main Protease Inhibitors Using a Novel Cell-Based Assay.

As an essential enzyme of SARS-CoV-2, main protease (MPro) triggers acute toxicity to its human cell host, an effect that can be alleviated by an MPro inhibitor. Using this toxicity alleviation, we developed an effective method that allows a bulk analysis of the cellular potency of MPro inhibitors. This novel assay is advantageous over an antiviral assay in providing precise cellular MPro inhibition information to assess an MPro inhibitor. We used this assay to analyze 30 known MPro inhibitors. Contrary to their strong antiviral effects and up to 10 µM, 11a, calpain inhibitor II, calpain XII, ebselen, bepridil, chloroquine, and hydroxychloroquine showed relatively weak to undetectable cellular MPro inhibition potency implicating their roles in interfering with key steps other than just the MPro catalysis in the SARS-CoV-2 life cycle. Our results also revealed that MPI5, MPI6, MPI7, and MPI8 have high cellular and antiviral potency. As the one with the highest cellular and antiviral potency among all tested compounds, MPI8 has a remarkable cellular MPro inhibition IC50 value of 31 nM that matches closely to its strong antiviral effect with an EC50 value of 30 nM. Therefore, we cautiously suggest exploring MPI8 further for COVID-19 preclinical tests.

An Enhanced Hybrid Screening Approach to Identify Potent Inhibitors for the SARS-CoV-2 Main Protease From the NCI Compound Library.

The emergence and rapid spread of SARS-CoV-2, the pathogen of COVID-19, have caused a worldwide public health crisis. The SARS-CoV-2 main protease (Mpro) is an essential enzyme for the virus and therefore an appealing target for the development of antivirals to treat COVID-19 patients. Recently, many in silico screenings have been performed against the main protease to discover novel hits. However, the actual hit rate of virtual screening is often low, and most of the predicted compounds are false positive hits. In this study, we developed a refined virtual screening strategy that incorporated molecular docking and post-docking filtering based on parameters including molecular weight and surface area, aiming to achieve predictions with fewer false positive hits. We applied this strategy to the NCI library containing 284,176 compounds against Mpro. In vitro potency analyses validated several potent inhibitors and thus confirmed the feasibility of our virtual screening strategy. Overall, The study resulted in several potent hit Mpro inhibitors, in which two inhibitors have IC50 values below 1 µM, that are worth being further optimized and explored. Meanwhile, the refined virtual screen strategy is also applicable to improve general in silico screening hit rates and is useful to accelerate drug discovery for treating COVID-19 and other viral infections.

The Pyrrolysyl-tRNA Synthetase Activity can be Improved by a P188 Mutation that Stabilizes the Full-Length Enzyme.

The amber suppression-based noncanonical amino acid (ncAA) mutagenesis technique has been widely used in both basic and applied research. So far more than two hundred ncAAs have been genetically encoded by amber codon in both prokaryotes and eukaryotes using wild-type and engineered pyrrolysyl-tRNA synthetase (PylRS)-tRNAPyl (PylT) pairs. Methanosarcina mazei PylRS (MmPylRS) is arguably one of two most used PylRS variants. However, it contains an unstable N-terminal domain that is usually cleaved from the full-length protein during expression and therefore leads to a low enzyme activity. We discovered that the cleavage takes place after A189 and this cleavage is inhibited when MmPylRS is co-expressed with Ca. Methanomethylophilus alvus tRNAPyl (CmaPylT). In the presence of CmaPylT, MmPylRS is cleaved after an alternative site K110. MmPylRS is active toward CmaPylT. Its combined use with CmaPylT leads to enhanced incorporation of Nε-Boc-lysine (BocK) at amber codon. To prevent MmPylRS from cleavage after A189 in the presence of its cognate M. mazei tRNAPyl (MmPylT), we introduced mutations at P188. Our results indicated that the P188G mutation stabilizes MmPylRS. We showed that the P188G mutation in wild-type MmPylRS or its engineered variants allows enhanced incorporation of BocK and other noncanonical amino acids including Nε-acetyl-lysine when they are co-expressed with MmPylT.

Drug Repurposing for the SARS-CoV-2 Papain-Like Protease.

As the pathogen of COVID-19, SARS-CoV-2 encodes two essential cysteine proteases that process the pathogen's two large polypeptide products pp1a and pp1ab in the human cell host to form 15 functionally important, mature nonstructural proteins. One of the two enzymes is papain-like protease or PLPro . It possesses deubiquitination and deISGylation activities that suppress host innate immune responses toward SARS-CoV-2 infection. To repurpose drugs for PLPro , we experimentally screened libraries of 33 deubiquitinase and 37 cysteine protease inhibitors on their inhibition of PLPro . Our results showed that 15 deubiquitinase and 1 cysteine protease inhibitors exhibit strong inhibition of PLPro at 200 µM. More comprehensive characterizations revealed seven inhibitors GRL0617, SJB2-043, TCID, DUB-IN-1, DUB-IN-3, PR-619, and S130 with an IC50 value below 40 µM and four inhibitors GRL0617, SJB2-043, TCID, and PR-619 with an IC50 value below 10 µM. Among four inhibitors with an IC50 value below 10 µM, SJB2-043 is the most unique in that it does not fully inhibit PLPro but has a noteworthy IC50 value of 0.56 µM. SJB2-043 likely binds to an allosteric site of PLPro to convene its inhibition effect, which needs to be further investigated. As a pilot study, the current work indicates that COVID-19 drug repurposing by targeting PLPro holds promise, but in-depth analysis of repurposed drugs is necessary to avoid omitting critical allosteric inhibitors.

Bepridil is potent against SARS-CoV-2 in vitro.

Guided by a computational docking analysis, about 30 Food and Drug Administration/European Medicines Agency (FDA/EMA)-approved small-molecule medicines were characterized on their inhibition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro). Of these small molecules tested, six displayed a concentration that inhibits response by 50% (IC50) value below 100 µM in inhibiting Mpro, and, importantly, three, that is, pimozide, ebastine, and bepridil, are basic molecules that potentiate dual functions by both raising endosomal pH to interfere with SARS-CoV-2 entry into the human cell host and inhibiting Mpro in infected cells. A live virus-based modified microneutralization assay revealed that bepridil possesses significant anti-SARS-CoV-2 activity in both Vero E6 and A459/ACE2 cells in a dose-dependent manner with low micromolar effective concentration, 50% (EC50) values. Therefore, the current study urges serious considerations of using bepridil in COVID-19 clinical tests.

The P3 O - Tert -Butyl-Threonine is Key to High Cellular and Antiviral Potency for Aldehyde-Based SARS-CoV-2 Main Protease Inhibitors.

As an essential enzyme to SARS-CoV-2, main protease (M Pro ) is a viable target to develop antivirals for the treatment of COVID-19. By varying chemical compositions at both P2 and P3 sites and the N -terminal protection group, we synthesized a series of M Pro inhibitors that contain ß -(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 site. These inhibitors have a large variation of determined IC 50 values that range from 4.8 to 650 nM. The determined IC 50 values reveal that relatively small side chains at both P2 and P3 sites are favorable for achieving high in vitro M Pro inhibition potency, the P3 site is tolerable toward unnatural amino acids with two alkyl substituents on the α -carbon, and the inhibition potency is sensitive toward the N -terminal protection group. X-ray crystal structures of M Pro bound with 16 inhibitors were determined. All structures show similar binding patterns of inhibitors at the M Pro active site. A covalent interaction between the active site cysteine and a bound inhibitor was observed in all structures. In M Pro , large structural variations were observed on residues N142 and Q189. All inhibitors were also characterized on their inhibition of M Pro in 293T cells, which revealed their in cellulo potency that is drastically different from their in vitro enzyme inhibition potency. Inhibitors that showed high in cellulo potency all contain O - tert -butyl-threonine at the P3 site. Based on the current and a previous study, we conclude that O - tert -butyl-threonine at the P3 site is a key component to achieve high cellular and antiviral potency for peptidyl aldehyde inhibitors of M Pro . This finding will be critical to the development of novel antivirals to address the current global emergency of concerning the COVID-19 pandemic.

The N -Terminal Carbamate is Key to High Cellular and Antiviral Potency for Boceprevir-Based SARS-CoV-2 Main Protease Inhibitors.

Boceprevir is an HCV NSP3 inhibitor that has been explored as a repurposed drug for COVID-19. It inhibits the SARS-CoV-2 main protease (M Pro ) and contains an α-ketoamide warhead, a P1 ß-cyclobutylalanyl moiety, a P2 dimethylcyclopropylproline, a P3 tert -butyl-glycine, and a P4 N -terminal tert -butylcarbamide. By introducing modifications at all four positions, we synthesized 20 boceprevir-based M Pro inhibitors including PF-07321332 and characterized their M Pro inhibition potency in test tubes ( in vitro ) and human host cells ( in cellulo ). Crystal structures of M Pro bound with 10 inhibitors and antiviral potency of 4 inhibitors were characterized as well. Replacing the P1 site with a ß-(S-2-oxopyrrolidin-3-yl)-alanyl (opal) residue and the warhead with an aldehyde leads to high in vitro potency. The original moieties at P2, P3 and the P4 N -terminal cap positions in boceprevir are better than other tested chemical moieties for high in vitro potency. In crystal structures, all inhibitors form a covalent adduct with the M Pro active site cysteine. The P1 opal residue, P2 dimethylcyclopropylproline and P4 N -terminal tert -butylcarbamide make strong hydrophobic interactions with M Pro , explaining high in vitro potency of inhibitors that contain these moieties. A unique observation was made with an inhibitor that contains an P4 N -terminal isovaleramide. In its M Pro complex structure, the P4 N -terminal isovaleramide is tucked deep in a small pocket of M Pro that originally recognizes a P4 alanine side chain in a substrate. Although all inhibitors show high in vitro potency, they have drastically different in cellulo potency in inhibiting ectopically expressed M Pro in human 293T cells. All inhibitors including PF-07321332 with a P4 N -terminal carbamide or amide have low in cellulo potency. This trend is reversed when the P4 N -terminal cap is changed to a carbamate. The installation of a P3 O-tert -butyl-threonine improves in cellulo potency. Three molecules that contain a P4 N -terminal carbamate were advanced to antiviral tests on three SARS-CoV-2 variants. They all have high potency with EC 50 values around 1 µM. A control compound with a nitrile warhead and a P4 N -terminal amide has undetectable antiviral potency. Based on all observations, we conclude that a P4 N -terminal carbamate in a boceprevir derivative is key for high antiviral potency against SARS-CoV-2.

Bepridil is potent against SARS-CoV-2 In Vitro.

Guided by a computational docking analysis, about 30 FDA/EMA-approved small molecule medicines were characterized on their inhibition of the SARS-CoV-2 main protease (MPro). Of these tested small molecule medicines, six displayed an IC50 value in inhibiting MPro below 100 µM. Three medicines pimozide, ebastine, and bepridil are basic small molecules. Their uses in COVID-19 patients potentiate dual functions by both raising endosomal pH to slow SARS-CoV-2 entry into the human cell host and inhibiting MPro in infected cells. A live virus-based microneutralization assay showed that bepridil inhibited cytopathogenic effect induced by SARS-CoV-2 in Vero E6 cells completely at and dose-dependently below 5 µM and in A549 cells completely at and dose-dependently below 6.25 µM. Therefore, the current study urges serious considerations of using bepridil in COVID-19 clinical tests.

How Chi Sequence Modifies RecBCD Single-Stranded DNA Translocase Activity.

E. coli RecBCD initiates homologous repair as well as degrades foreign DNA. Recognition of chi sequence (5'-GCTGGTGG-3') switches RecBCD from a destructive, nucleolytic mode into a repair-active one that promotes RecA-mediated recombination. RecBCD includes a 3'-to-5' single-stranded DNA (ssDNA) translocase in RecB subunit, a 5'-to-3' translocase in RecD, and a secondary translocase activity associated with RecBC. To understand how chi specifically affects each translocase activity, we directly visualized individual RecBCD translocating along DNA substrates containing a ssDNA gap of different polarities, with or without chi. Disappearance of RecBCD from the ssDNA signals the loss of the ssDNA translocase activity. For substrates containing a ssDNA gap that RecBCD encounters in the 3'-to-5' polarity (3'-to-5' ssDNA), wild-type RecBCD disappears from the DNA substrates with similarly high percentage, either with chi or without. This suggests that (1) the 3'-to-5' translocase in RecB is unaffected by chi and (2) it is low in processivity. With substrates containing a ssDNA gap that RecBCD encounters in the 5'-to-3' polarity (5'-to-3' ssDNA), we found that the leaving percentage increases significantly with chi, implying inactivation of the 5'-to-3' translocase of RecD upon chi recognition. Surprisingly, the RecD defective mutant RecBCDK177Q showed only ≈50 % leaving on 5'-to-3' ssDNA, directly revealing the presence of RecBC secondary translocase and its activity is unaffected by chi. Multiple ssDNA translocases within the RecBCD complex both before and after chi ensures processive unwinding of DNA substrates required for efficient recombination events.

Stable Nuclei of Nucleoprotein Filament and High ssDNA Binding Affinity Contribute to Enhanced RecA E38K Recombinase Activity.

RecA plays central roles in the homologous recombination to repair double-stranded DNA break damage in E. coli. A previously identified recA strain surviving high doses of UV radiation includes a dominant RecA E38K mutation. Using single-molecule experiments, we showed that the RecA E38K variant protein assembles nucleoprotein filaments more rapidly than the wild-type RecA. We also used a single-molecule fluorescence resonance energy transfer (smFRET) experiment to compare the nucleation cluster dynamics of wild-type RecA and RecA E38K mutants on various short ssDNA substrates. At shorter ssDNA, nucleation clusters of RecA E38K form dynamically, while only few were seen in wild-type RecA. RecA E38K also forms stable nuclei by specifically lowering the dissociation rate constant, k d . These observations provide evidence that greater nuclei stability and higher ssDNA binding affinity contribute to the observed enhanced recombination activity of the RecA E38K mutant. Given that assembly of RecA nucleoprotein filaments is the first committed step in recombinational repair processes, enhancement at this step gives rise to a more efficient recombinase.